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Inflammation Program and Departments of
Medicine and
Microbiology, Veterans Administration Medical Center and University of Iowa, Iowa City, IA 52246
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In unstimulated cells the NADPH oxidase is unassembled and inactive, with its seven protein components segregated into membrane and cytosolic locations (reviewed in Ref. 12). Once phagocytes are activated, however, oxidase components p47phox (13, 14, 15), p67phox (13, 16), and p40phox (17) translocate en bloc from the cytosol (18, 19) and associate with membrane-bound flavocytochrome b (4, 7, 20). A low m.w. GTPase, Rac2, translocates independently to the assembling oxidase (21, 22, 23). Presumably, the intricate assembly event involving cytosolic components regulates the onset of O2- generation by limiting the potential for inadvertent generation of damaging ROS in host tissues. To date, our understanding of NADPH oxidase activation and assembly has been determined in part from studies using PMN treated with soluble stimuli such as PMA, FMLP, calcium ionophores, or analysis using the broken cell NADPH oxidase reconstitution assay (24). The applicability of conclusions derived from these studies to NADPH oxidase function during phagocytosis of microorganisms remains undetermined. Albeit more related to its role in host defense against bacteria, characterization of oxidase assembly and activation in PMN during phagocytosis is relatively limited.
To this end, we have characterized activation and assembly of the NADPH-dependent oxidase during phagocytosis of opsonized Neisseria meningitidis serogroup B (NMB), an encapsulated Gram-negative diplococcus that can cause severe septicemia in certain individuals (reviewed in Ref. 25), and have observed phenomena not previously reported when soluble agonists such as PMA and FMLP were used. In this study we demonstrate that termination of NADPH oxidase activity parallels increased phosphorylation of membrane-associated p47phox, an event coupled with the loss of p47/67phox from phagosomes. Collectively, these data provide evidence that inactivation of the NADPH oxidase is regulated at the level of enzyme disassembly.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2',7'-Dichlorodihydrofluorescein diacetate (DCF) and Texas Red-conjugated secondary Abs were obtained from Molecular Probes (Eugene, OR), and superoxide dismutase (SOD) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). [32P]Orthophosphatidic acid (10 mCi/ml) was purchased from DuPont-NEN (Boston, MA). Heat-killed, formalin-fixed, protein-A bearing Staphylococcus aureus (Pansorbin cells) were purchased from Calbiochem (San Diego, CA). RPMI 1640 was purchased from Life Technologies (Gaithersburg, MD). FITC-conjugated goat anti-mouse and donkey anti-rabbit IgGs were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Dr. Michio Nakamura provided the flavocytochrome b-specific mAb 7D5. Drs. Algirdas J. Jesaitis, James B. Burritt, and Mark T. Quinn provided gp91phox- and p22phox-specific mAbs 54.1 and 44.1, respectively. All other reagents were purchased from Sigma (St. Louis, MO) unless specified otherwise.
Neutrophil isolation
Heparinized venous blood was obtained from healthy individuals and from an individual with X-linked CGD in accordance with a protocol approved by the institutional review board for human subjects at the University of Iowa. PMN were isolated using dextran sedimentation and Hypaque-Ficoll density-gradient separation followed by hypotonic lysis of erythrocytes as described previously (26). Purified PMN were resuspended in Dulbecco’s PBS containing 10 mM D-glucose (DPBS/g), DPBS/g containing 25 µM DCF, HBSS supplemented with 10 mM D-glucose (HBSS/g), RPMI 1640 containing 20 mM HEPES, pH 7.2 (HEPES/RPMI), or phosphate-free loading buffer (LB; see below) and kept on ice until use.
Growth and opsonization of NMB
For each experiment, N. meningitidis were plated for overnight growth at 37°C with 5.0% CO2 on gonorrhea culture medium-based agar (Difco, Detroit, MI) and then collected into the appropriate buffer (HBSS/g, DPBS/g, LB, or HEPES/RPMI) via a glass spreading rod the following day for immediate use. Cell density was determined spectrophotometrically at 550 nm from a predetermined growth curve, and Neisseria were then opsonized with 100% normal human serum (NHS) for 30 min at 37°C and subsequently washed in the appropriate buffer to remove excess serum. Opsonized NMB were then resuspended in assay buffer to the indicated cell density and kept on ice until use. Zymosan were also opsonized with 100% NHS as described for NMB.
Neutrophil assays for ROS
To measure the intracellular generation of ROS, PMN were resuspended in DPBS/g containing 25 µM DCF to 107 cells/ml and then equilibrated for 45 min at room temperature with gentle agitation. PMN (106), 8.0 µg of SOD, 3 x 107 NMB, 5 x 107 opsonized zymosan (OPZ), or 0.2 µg of PMA were dissolved or resuspended in DPBS/g containing 25 µM DCF and then added to NHS-coated wells of a chilled 96-well microtiter plate. In all experiments, free NHS was removed from plates and vials following coating by two sequential washes in the appropriate assay buffer. Cold DPBS/g containing 25 µM DCF was added to each well so that the final volume was 200 µl. Exogenous DCF was added with the buffer to maximize fluorescent signal derived from NMB- or OPZ-stimulated PMN. The plate was centrifuged at 400 x g for 5 min at 4°C to synchronize phagocytosis, and generation of ROS was monitored continuously at 37°C for 90 min using a BMG FLUOstar 403 microplate spectrofluorometer (BMG Lab Technologies, Durham, NC) without agitation.
Alternatively, O2- generation
was determined by the reduction of ferricytochrome c as
described previously (27), but with several modifications.
Briefly, PMN were adjusted to 107 cells/ml in
HBSS/g, and 100 µl was aliquoted to wells of a chilled 96-well
microtiter plate precoated with 100% NHS for 
1 h at 37°C.
Opsonized N. meningitidis (3 x
107) in HBSS/g were added to each well, and the
plate was centrifuged at 400 x g for 5 min at 4°C to
synchronize phagocytosis. Subsequently, the plate was warmed to 37°C
using a microplate spectrophotometer (Bio-Rad, Hercules, CA), and the
rate of O2- generation was
measured by the SOD-inhibitable reduction of ferricytochrome
c at 550 nm with plate agitation. Although all PMN were
stimulated simultaneously with NMB, ferricytochrome c was
added to individual wells at each 10-min interval. For example, the
rate of O2- produced 30 min
after NMB stimulation was determined by adding ferricytochrome
c at 30 min and measuring
O2- generation between 30 and
40 min. For comparison, O2-
generation by cells stimulated with 1 µg/ml PMA was measured for the
first 10 min. We also observed that after 90 min of exposure to NMB,
PMN still generated O2- in
response to PMA.
Phosphorylation and immunoprecipitation of p47phox
PMN (1–2 x 108) were resuspended in 1 ml of LB (10 mM Na-HEPES, 138 mM NaCl, 2.7 mM KCl, and 7.5 mM D-glucose, pH 7.5), with 0.5 mCi/ml [32P]orthophosphate and rotated at room temperature for 60 min. Following loading, PMN were washed twice in LB, and 107 cells (50 µl) were transferred to NHS-precoated glass scintillation vials containing 150 µl of LB and incubated for 10–15 min at 25°C. Subsequently, vials were iced immediately for 10 min, and 3 x 108 chilled NMB or 5 x 107 chilled OPZ were pipetted onto the cells. Vials were immediately centrifuged at 400 x g for 5 min at 4°C and then transferred to 37°C. At the indicated time points, cells were solubilized with an equal volume of lysis buffer (3% Triton X-100, 0.25 mg/ml leupeptin and pepstatin A, 2 mM PMSF, 3 mM sodium orthovanadate, and 1.0% mixed alkyltrimethylammonium bromide in Tris-buffered saline, pH 7.5) for 30 min on ice. Insoluble material was removed by centrifugation at 13,000 x g for 60 s at room temperature, and supernatants were incubated with preimmune rabbit serum and Pansorbin cells as described previously (28). Precleared supernatants were diluted to 1.0 ml with 50 mM Tris-Cl and 190 mM NaCl containing 2.5% Triton X-100, and p47phox was precipitated using polyclonal Ab to p47phox and protein A-Sepharose CL-4b (Pharmacia Biotech, Piscataway, NJ) as described previously (29). Protein A-immune complexes were washed twice in Tris-buffered saline containing 0.5% Triton X-100 and then boiled in SDS-sample buffer without reducing agent and resolved with 5–20% SDS-PAGE. Dried gels were subjected to autoradiography using Kodak XAR films (Eastman Kodak, Rochester, NY), and the [32P]orthophosphate incorporated into p47phox was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
To study subcellular fractions from NMB-stimulated PMN, PMN were loaded
with 0.5 mCi/ml [32P]orthophosphate and used
for synchronized phagocytosis assays as described above, but with the
following changes. PMN (3 x 107)
distributed among three separate NHS-coated vials were processed for
each time point. In addition, 109 NMB were added
to each vial so that the final NMB to PMN ratio was 100:1. PMN
containing ingested NMB were recovered from each time point by
aspiration and scraping in a total of 2 ml of LB. Cells were
centrifuged at 100 x g for 5 min to pellet only PMN
and then were resuspended in 1 ml of relaxation buffer
(30) containing 1 mM sodium orthovanadate. PMN were lysed
by nitrogen bomb cavitation as described previously (30).
Because phagosomes and plasma membrane vesicles could not be
satisfactorily separated, a crude plasma membrane/phagosome-enriched
fraction was recovered by sequential centrifugation at 100 x
g for 5 min, to remove unbroken cells, and then at
250,000 x g for 10 min. Following the 250,000 x
g spin, supernatant was collected as the cytosol-derived
fraction. The crude plasma membrane/phagosome-enriched fraction was
washed in relaxation buffer, which included brief sonication using a
low setting, and recentrifuged twice. This pellet was resuspended in
RIPA lysis buffer (10 mM sodium phosphate, pH 7.2, containing 1%
Triton X-100, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 150
mM NaCl, 1 mM EDTA, 20 mM NaF, 200 µM sodium orthovanadate, 0.4 U/ml
aprotinin, and 2 mM PMSF), precipitated with Ab specific for
p47phox, and subjected to SDS-PAGE and
autoradiography as described above. Unlabeled samples prepared in
parallel were immunoblotted using biotinylated Ab specific for
p47phox coupled with enhanced chemiluminescence
detection (Super Signal Substrate, Pierce, Rockford, IL). Immunoblots
were quantitated by densitometry using an IS-1000 Digital Imaging
System (
Innotech, San Leandro, CA). The sp. act. of the
membrane-associated p47phox was calculated by
determining the ratio of 32P counts obtained from
the PhosphorImager per unit of p47phox protein
as measured by densitometry.
Alternatively, OPZ phagosomes and a plasma membrane-enriched fraction
from OPZ-stimulated cells were isolated using two separate Percoll
gradients. Following nitrogen cavitation, cells were centrifuged at
300 x g for 10 min to pellet unbroken PMN, nuclei, and
OPZ phagosomes. Plasma membrane-enriched fractions and a
cytosol-derived fraction were isolated from the postnuclear supernatant
using a Percoll step gradient as described by Borregaard et al.
(30). The pellet resulting from the 300 x
g centrifugation was resuspended in 1 ml of relaxation
buffer. Opsonized zymosan phagosomes were isolated by centrifugation
through an 8-ml self-forming Percoll gradient with an initial density
of 1.065 g/ml at 20,000 x g for 15 min. Phagosomes
were collected from the gradient near the buffer/Percoll interface
(
1.037 g/ml); unbroken cells and debris sedimented near the bottom
of the gradient (1.09 g/ml). Opsonized zymosan phagosomes phagosomes
and plasma membrane-enriched fractions were each washed twice in
relaxation buffer containing 1 mM sodium orthovanadate and then
combined. Although phagosomes and plasma membrane vesicles could be
readily isolated from OPZ-stimulated PMN, these fractions were pooled
for two reasons: 1) to recover any phagosomal membrane stripped from
OPZ during nitrogen bomb cavitation, and 2) to mimic studies of NMB
phagosomes that were isolated as crude plasma- and phagosome-enriched
fractions. In this way we could make pertinent comparisons between
membranes derived from OPZ- and NMB-stimulated PMN. The pooled
fractions were solubilized in RIPA buffer and precipitated with Ab
specific for p47phox as described above.
Immunoprecipitates were processed for either autoradiography or
immunoblotting.
Translocation of NADPH oxidase components assessed by fluorescence microscopy
Synchronized phagocytosis, staining, and fluorescence microscopy were performed as described previously (31, 32). Briefly, 106 PMN in 2 ml of HEPES/RPMI 1640 were allowed to adhere to glass coverslips (precoated with 10–100% NHS for 1 h at 37°C) in 35-mm dishes for 15 min at 37°C. Subsequently, cells were chilled to 4°C, 107 cold NMB or 5 x 106 OPZ was added, and dishes were immediately centrifuged at 500 x g for 2 min at 10–12°C to synchronize phagocytosis. Dishes were then incubated at 37°C for 0–90 min, at which time cells were washed with DPBS to remove unbound bacteria or OPZ and then fixed with 10% formalin for 15 min. Cells were permeabilized with acetone for 5 min at -20°C, washed, and then blocked overnight in DPBS containing 5 mg/ml BSA, 10% normal goat serum, and 0.02% sodium azide. Flavocytochrome b was detected using a combination of mAbs, 7D5, 54.1, and 44.1. P47phox and p67phox were detected as previously described (31, 32). Opsonized N. meningitidis was detected using mouse mAb 2C3 (a gift from Dr. Peter Rice, Boston University, Boston, MA), which reacts with outer membrane protein H8, or using rabbit polyclonal antiserum specific for NMB. Primary Abs were detected with secondary Abs conjugated to FITC or Texas Red. The specificity of staining was assessed by analysis in the absence of primary Abs.
Fluorescence was visualized using a Zeiss Axioplan2 epifluorescence microscope (Carl Zeiss, Thornwood, NY), a Zeiss LSM 510 laser-scanning confocal microscope (Carl Zeiss) at the Veterans Administration Medical Center, or a Bio-Rad 1024 laser-scanning confocal microscope (Bio-Rad, Hercules, CA) at the University of Iowa Central Microscopy Research Facility.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the kinetics of ROS produced by PMN in response to NMB,
we monitored the generation of both intracellular and extracellular ROS
over a 90-min period following synchronized phagocytosis (Fig. 1
A). For reference, we
compared the PMN response to NMB (30:1, NMB:PMN ratio) with those to
OPZ (5:1, OPZ:PMN ratio) and PMA (1 µg/ml; Fig. 1, A and
B). The data obtained from these analyses are displayed as
direct kinetic plots (Fig. 1A) and as maximum rates
calculated from each 5-min interval over the 90-min time course (Fig. 1B). Although the PMN response to NMB was reduced compared
with that elicited by OPZ, maximal rates for both occurred within
20–30 min after the assay was initiated (Fig. 1, A and
B, inset). The delayed response of PMN to all
three agonists appeared to be partly the result of the time necessary
for the microtiter plate to warm from 4 to 37°C (40 min), and peak
activities for either OPZ or NMB occurred at 25.0–27.5°C during
warming as indicated by arrows (Fig. 1B, inset).
Peak activity was subsequently followed by an abrupt decline in the
rate of ROS generation, which returned to near-basal levels by 70–90
min (Fig. 1B, inset). The reduction in
fluorescence following peak activity was not a result of insufficient
quantities of substrate (DCF), as PMA-stimulated PMN produced a
response 5- and 3-fold greater than those elicited by NMB and OPZ,
respectively (Fig. 1B). Although similar kinetic profiles of
ROS generation were elicited by all agonists, the rate of ROS
generation by PMA-stimulated PMN was still greater than the maximal
rates observed with NMB-stimulated PMN 50 min after peak activity
(Fig. 1B). Therefore, diminishing fluorescence reflected
a termination of the respiratory burst in all stimulated cells.
Superoxide dismutase had little effect on ROS generated by either NMB-
or OPZ-stimulated PMN in the fluorescence assays, inhibiting
fluorescence by only 9.4 ± 0.7 and 13.0 ± 4.6%,
respectively, but eliminated 44.2 ± 3.5% of that generated by
PMA-stimulated PMN (data not shown). The inhibitory effect of SOD
suggested that most of the ROS were generated within NMB- and
OPZ-stimulated PMN and presumably within phagosomes, rather than at the
PMN plasma membrane. The specificity of this assay to measure only
NADPH oxidase-derived activity was confirmed by experiments using NMB-,
OPZ-, and PMA-stimulated PMN isolated from an individual with X-linked
CGD (data not shown). We also monitored the generation of
O2- during phagocytosis over
the same time period by the reduction of ferricytochrome c
(Fig. 1C). Although the ferricytochrome c assay
measured mainly O2- escaping
forming phagosomes, ROS diffusing out of the cell, or that produced at
the plasma membrane, the kinetics of
O2- production were comparable
to those of the DCF assay (Fig. 1C)
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Phosphorylation of p47phox has been
previously shown to correlate with activation of the respiratory burst
in PMN (33, 34, 35, 36, 37, 38). To investigate the mechanism of NMB- and
OPZ-dependent ROS generation, we monitored the phosphorylation of
p47phox during synchronized phagocytosis.
P47phox from unstimulated PMN had little
phosphate incorporated over the 90-min period studied, and
correspondingly, these PMN did not generate ROS (compare Fig. 1
B, filled circles, with Fig. 2, A and B,
bottom panel and filled circles). By contrast,
p47phox precipitated from PMN stimulated with
either NMB or OPZ was phosphorylated in a time-dependent manner, and
this phosphorylation correlated with activation of the NADPH oxidase
(compare Figs. 1 and 2). However, termination of the respiratory burst
was not reflected by decreased phosphorylation of
p47phox using this assay (compare Figs. 1B, inset, and 2B); i.e., even after
oxidase activity had terminated, the levels of phosphorylated
p47phox in those PMN were still increased
compared with those in unstimulated cells despite similarities in
oxidant generation (little or none).
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, A and B). As
shown in Fig. 3, A and B, the bulk of
phosphorylated p47phox was recovered from the
cytosolic fraction. Although only a small fraction of the total
cellular pool of p47phox becomes membrane
associated during oxidase activation (18, 39), we
determined by immunoblotting that 5.3 ± 2.2 and 7.8 ± 2.3%
of the total p47phox precipitated from cytosol
and membrane fractions had translocated to membrane after 30 min of
phagocytosis of NMB and OPZ, respectively (Fig. 3, A and
B). In contrast to the coupling of phosphorylation of
p47phox with respiratory burst activity,
termination of the respiratory burst did not reflect decreased
phosphorylation of membrane-associated
p47phox. In fact, the sp. act. of
membrane-associated p47phox phosphoprotein had
increased slightly by 90 min when oxidase activity was at a nadir, from
3.5 ± 0.8 and 2.7 ± 0.2 at 30 min for NMB- and
OPZ-stimulated PMN, respectively, to 5.8 ± 1.8 and 3.7 ±
0.1 (Fig. 3, A and B). These data demonstrate
that there was additional phosphorylation of membrane-associated
p47phox coincident with termination of
respiratory burst activity.
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Recovering sufficient phosphorylated
p47phox from these assays required enormous
quantities of NMB phagosomes, and we routinely used 100 NMB/PMN.
Therefore, only early phagocytic events were synchronized, as PMN were
continually ingesting NMB at up to 90 min after activation (data not
shown). Despite this technical complication, the results shown in Fig. 3 suggested that less p47phox was membrane
associated at 90 min after stimulation compared with that at 30 min
(3.8 ± 2.7 vs 5.3 ± 2.2%, respectively, for NMB, and
2.9 ± 1.2 vs 7.8 ± 2.3%, respectively, for OPZ). To
examine this event more precisely we monitored phagosomal association
of p47/p67phox following synchronized
phagocytosis using immunofluorescence microscopy (32).
This assay allows precise control of phagocytosis using low NMB and OPZ
to PMN ratios, 10:1 and 5:1, respectively. Opsonized N.
meningitidis and OPZ phagosomes were enriched for flavocytochrome
b immediately upon ingestion as judged using mAbs specific
for gp91phox and p22phox
(Fig. 4, A and B).
Although NMB were difficult to identify at early time points due to the
large number of flavocytochrome b-laden granules, it was
obvious that by 60 min these phagosomes, and also those containing OPZ,
remained enriched for flavocytochrome b (Fig. 4, A and B). We then monitored the NMB phagosomal
association of p47phox and
p67phox as an indicator of NADPH oxidase
assembly (12, 18, 32, 40). Immediately after
centrifugation at 10°C, the majority of PMN-bound NMB were not
stained using Abs specific for p47phox and
p67phox, suggesting that NMB phagosomes had not
yet formed, and the oxidase was unassembled (Fig. 5, A and B).
Subsequently, phagosomal association of
p47/67phox occurred rapidly as NMB were
ingested, and translocation peaked at 10–15 min (e.g., by 10 min
62.4 ± 4.0 and 68.9 ± 10.3% of the NMB associated with PMN
were p47phox and p67phoxpositive, respectively; Figs. 5, A and B,
and 6). As with peak ROS-generating activity, peak phagosomal
association of p47/67phox with NMB phagosomes
occurred at 26–27.5°C (indicated with an arrow in Fig. 6), indicating a correlation between
translocation (assembly) and induction of the respiratory burst by NMB
(compare peaks in Figs. 1B, inset, and 6).
Although a large percentage of NMB phagosomes remained
p47phox and p67phox
positive at 30 min (42.6 ± 10.1 and 46.4 ± 5.2%,
respectively), by 90 min this was reduced significantly to 12.2 ±
3.9 and 13.7 ± 3.7% (p = 0.0006 and
0.005 for 10 vs 90 min; Figs. 5, A and B,
and 6).
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, C and D). As we have
shown previously (32), p47phox and
p67phox are recruited to forming OPZ phagosomes
rapidly upon initiation of ingestion, with peak association of
p47phox and p67phox with
OPZ phagosomes between 5 and 15 min (68.5 ± 1.4 to 92.3 ±
2.5%; Figs. 5, C and D, and 6). As we observed
with NMB phagosomes, the quantity of
p47/67phox-positive OPZ phagosomes decreased
significantly over time, and by 90 min, there was little
p47/67phox associated with OPZ phagosomes (Figs. 5, C and D, and 6). Thus, dissociation of
p47/67phox from PMN phagosomal membranes
appeared to be a general phenomenon of late phagocytosis. Furthermore,
the point at which phagosomal association of
p47/67phox either peaked or was at a nadir
corresponded to the highest and lowest levels of ROS generation,
respectively (compare Figs. 1 and 6). These results were consistent
with our immunoblotting data using the phosphorylation assay, although
kinetics in those assays were skewed due to the large particulate load
(compare 10 and 60 min in Fig. 6 with 30 and 90 min in Fig. 3, A and B). Analysis by immunofluorescence
microscopy was more selective in that it was restricted to localization
of the assembled oxidase on phagosomes, whereas data from the
phosphorylation assays reflected the sum of reactivity at the plasma
membrane and phagosomal membrane. Taken together, these data suggest
that termination of the respiratory burst reflects dissociation of
p47/67phox from phagosomal membranes. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the kinetics of ROS generation, phosphorylation of p47phox, and translocation of p47/67phox in response to NMB and OPZ to understand activation, assembly, and subsequent termination of NADPH oxidase activity during phagocytosis. Using NMB- or OPZ-stimulated PMN, we observed that respiratory burst activity peaked following phagocytosis and subsequently declined. These kinetics were also observed in PMA-stimulated PMN, although the magnitude of the response was much greater. The ability of PMA-stimulated PMN to generate significantly more ROS than either OPZ- or NMB-stimulated PMN demonstrates that the decline in the generation of ROS following peak respiratory burst activity during phagocytosis of NMB or OPZ reflected neither a limitation of available substrate nor the inability of PMN to produce ROS, but rather the termination of NADPH oxidase activity. A general mechanism for termination of NADPH oxidase activity has not been previously identified, although it may reflect receptor internalization in the case of FMLP-stimulated PMN (57, 58). Our data suggest that termination of the respiratory burst correlates with the removal of p47/67phox from phagosomes and/or the plasma membrane.
Initial phosphorylation of p47phox coincided
with the generation of ROS by NMB and OPZ-stimulated PMN (compare peaks
in Figs. 1B and 2B). Moreover, initial
p47phox phosphorylation kinetics using either
NMB or OPZ-stimulated PMN closely mirrored the kinetics of oxidase
assembly at phagosomes for the first 10–15 min (compare Figs. 2B and 6). Thus, our findings with NMB or
OPZ-stimulated PMN agree with previous reports suggesting that NADPH
oxidase activation and assembly are linked to the phosphorylation of
p47phox (33, 38, 44, 46, 47, 53, 59, 60, 61). Recent biochemical evidence suggests that domains of
p47phox necessary for flavocytochrome
b and/or p67phox binding during NADPH
oxidase activation are sequestered when the enzyme is inactive
(54, 60, 62, 63), and in vitro studies demonstrate that
p47phox changes conformation when phosphorylated
and functionally competent (53, 54, 60). During
phagocytosis, phosphorylation of p47phox may be
a permissive event, necessary only to expose domains and/or impart
conformational change to allow association with
p67phox and subsequently with flavocytochrome
b. Precise quantitation of the subcellular distribution of
phosphorylated p47phox is difficult, because
translocated p47phox may undergo additional
modification, such as dephosphorylation and/or hyperphosphorylation,
which would change the sp. act. of membrane-associated species relative
to those remaining in or having just returned to the cytosolic
compartment. However, it was possible to compare the sp. act. of
translocated p47phox phosphoprotein at different
times during phagocytosis (Fig. 3, A and B). If
dephosphorylation were a mechanism for releasing membrane-associated
p47phox, it was not apparent in our assays,
because no decrease in p47phox phosphorylation
was observed in NMB- or OPZ-stimulated PMN by 90 min, a time by which
ROS generation was minimal.
Based on these findings, one can conclude that termination of the
respiratory burst is not coincident with dephosphorylation of
membrane-associated p47phox. In contrast, the
data suggest that additional phosphorylation of membrane-associated
p47phox or a change in specific phosphorylated
residues could mediate its release to the cytosol and concomitant
termination of oxidase activity. The results shown in Fig. 3, viz.,
increased p47phox phosphorylation over time
concurrent with decreased membrane-associated
p47phox immunoreactivity, support the
interpretation that hyperphosphorylation of membrane-associated
p47phox precedes its dissociation from the
membrane. Thus, our data suggest that additional phosphorylation of
membrane-associated p47phox correlates with the
loss of p47/67phox from phagosomes during
termination of the respiratory burst. Studies to identify and
characterize specific phosphorylated species of
p47phox from cytosol and phagosomes are
necessary to prove this hypothesis and are ongoing.
Translocation of p47/67phox to the plasmalemmal
and/or phagosomal membrane is necessary for assembly of the NADPH
oxidase and initiation of oxidase activity, although features of
oxidase termination are undefined. Our results show that association of
p47/67phox with NMB- and OPZ-containing
phagosomes coincided with the onset of respiratory burst activity
(compare Figs. 1B, inset, and 6) and with the
phosphorylation of p47phox (compare Figs. 2B and 6). In addition, this correlation held as well during
termination of the respiratory burst, as little
p47/67phox were phagosome associated when burst
activity was minimal; e.g., 68.7 ± 10.3% of NMB phagosomes were
p67phox positive at 10 min compared with
13.7 ± 3.7% at 90 min (Figs. 5 and 6). These data indicate that
termination of the respiratory burst is coupled to the loss of
p47/67phox from the membrane-associated oxidase
by either dissociation or limited proteolysis. Phagosomes remained
enriched for flavocytochrome b 90 min after PMN stimulation,
suggesting that termination of the respiratory burst and release of
p47/67phox from phagosomes are not related to
selective partitioning of flavocytochrome b from the
phagosomal membrane. It should also be noted that colocalization of
p47/67phox with flavocytochrome b was
restricted to the phagosomal membrane; hence, the cytosolic complex did
not colocalize with flavocytochrome b-laden granules during
activation (Fig. 4B). Therefore,
p47/67phox were targeted specifically to
flavocytochrome b docking sites at phagosomes containing NMB
or OPZ. The mechanism for specific targeting is unknown, but our
previous studies (32) suggest that flavocytochrome b is
insufficient for such targeting.
Many clinically important sequelae of inflammation reflect unbridled PMN activation, supporting the need to understand better the determinants of the termination of cell activation as well as the features important in its initiation. Although this study extends our understanding of the features of oxidase assembly and activation during phagocytosis, many questions remain unanswered. Our data implicate loss of p47/67phox from the phagosome as well as additional phosphorylation of plasma membrane/phagosome-associated p47phox as features of oxidase termination. Additional studies are necessary to identify the exact mechanism(s) for release of p47/67phox from phagosomal membranes and for termination of NADPH oxidase activity during phagocytosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William M. Nauseef, Department of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52246. E-mail address:
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; ROS, reactive oxygen species; O2-, superoxide; CGD, chronic granulomatous disease; NMB, opsonized Neisseria meningitidis; DCF, 2',7'-dichlorodihydrofluorescein diacetate; SOD, superoxide dismutase; LB, loading buffer; NHS, normal human serum; OPZ, opsonized zymosan.
Received for publication May 18, 1999. Accepted for publication September 22, 1999.
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-RII and Fc--RIII induces actin polymerization by a pertussis toxin-insensitive transduction pathway. J. Immunol. 146:4282.[Abstract]
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; 30:1 NMB:PMN ratio), OPZ
(
; 5:1 OPZ:PMN ratio), PMA (
; 1 µg/ml), or without agonist
(•) following synchronized phagocytosis, and rates of ROS generation
were determined every 5–10 min as indicated. Results are expressed as
the mean ± SD of two to four separate trials from a representative
experiment performed at least four times. Arrows indicate
) and p47phox
(
) were quantitated using confocal laser-scanning microscopy or
indirect immunofluorescence microscopy, respectively. The arrow
indicates