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6-Phosphogluconate Dehydrogenase and
Glucose-6-Phosphate Dehydrogenase Form a
Supramolecular Complex in Human
Neutrophils That Undergoes Retrograde
Trafficking during Pregnancy
Andrei L. Kindzelskii, Tatsuya Ueki, Hitoshi Michibata,
Tinnakorn Chaiworapongsa, Roberto Romero and Howard
R. Petty
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:6373-6381; ;
doi: 10.4049/jimmunol.172.10.6373
http://www.jimmunol.org/content/172/10/6373
The Journal of Immunology
6-Phosphogluconate Dehydrogenase and Glucose-6-Phosphate
Dehydrogenase Form a Supramolecular Complex in Human
Neutrophils That Undergoes Retrograde Trafficking during
Pregnancy1
Andrei L. Kindzelskii,* Tatsuya Ueki,‡ Hitoshi Michibata,‡ Tinnakorn Chaiworapongsa,§¶
Roberto Romero,§¶ and Howard R. Petty2*†
S
everal lines of evidence suggest that a mother’s inflammatory response is modified by pregnancy. In vitro studies
have indicated that normal maternal neutrophils and/or
macrophages display reduced chemotaxis, adherence, reactive oxygen metabolite (ROM)3 release, phagocytosis, and microbial killing (1–7). In vivo studies and clinical observations also suggest
that pregnancy alters inflammatory responses. First, host resistance
to infectious disease may be compromised during pregnancy. For
example, in humans and/or animal models, host resistance to Neis-
Departments of *Ophthalmology and Visual Sciences, and †Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48105; ‡Marine
Biological Laboratory, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Perinatology Research Branch, National Institute of Child Health and
Human Development, Bethesda, MD 20892; and ¶Hutzel Hospital, Detroit, MI 48201
Received for publication December 15, 2003. Accepted for publication March
12, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the National Institute of Allergy and Infectious Diseases and National Multiple Sclerosis Society (to H.R.P.) and the intramural program
of National Institute of Child Health and Human Development (to R.R.).
2
Address correspondence and reprint requests to Dr. Howard R. Petty, Department of
Ophthalmology and Visual Sciences, University of Michigan Medical School, 1000
Wall Street, Ann Arbor, MI 48105. E-mail address: [email protected]
3
Abbreviations used in this paper: ROM, reactive oxygen metabolite; RNI, reactive
nitrogen intermediate; HMS, hexose monophosphate shunt; G-6-P, glucose-6-phosphate; G-6-PDase, glucose-6-phosphate dehydrogenase; 6-PG, 6-phosphogluconolactone; 6-PGDase, 6-phosphogluconate dehydrogenase; MTOC, microtubule-organizing center; TRITC, tetramethylrhodamine isothiocyanate; LDH, lactate
dehydrogenase; RET, resonance energy transfer; PFK, phosphofructokinase.
Copyright © 2004 by The American Association of Immunologists, Inc.
seria gonorrhoeae, Listeria monocytogenes, Toxoplasma gondii,
and Plasmodium berghii are reduced during pregnancy (8 –11).
Poliomyelitis, influenza, malaria, pneumonia, periodontal disease,
acute pyelonephritis, and other infectious diseases have also been
reported to have increased incidence or severity during pregnancy
(8 –12). Second, autoimmune diseases with a substantial inflammatory component improve during pregnancy, but relapse after
delivery. It has been known for some time, both qualitatively and
quantitatively, that ⬃70% of women with arthritis, multiple sclerosis, and uveitis go into remission during pregnancy (e.g., Refs.
13–15). Thus, it would seem possible that functional changes in
the neutrophils and monocytes of pregnant women may underlie
their diminished proinflammatory capacity.
ROM and reactive nitrogen intermediate (RNI) production are
key factors contributing to host defense during infectious disease
and in tissue damage during autoimmune disease. These molecules
also act as paracrine and autocrine messengers (16). ROM production begins with the synthesis of superoxide, which is produced
by the NADPH oxidase according to the following:
1/2NADPH ⫹ O2 3 1/2NADP ⫹ ⫹ 1/2H⫹ ⫹ O2⫺
(1)
RNI production begins with the synthesis of NO:
L-arginine
⫹ NADPH ⫹ H⫹ ⫹ O2 3 NG-hydroxy-L-arginine ⫹
NADP⫹ ⫹ H2O and 2NG-hydroxy-L-arginine ⫹ NADPH ⫹
H⫹ ⫹ O2 3 2L-citrulline ⫹ NADP⫹ ⫹ 2H2O ⫹ 2NO
(2)
0022-1767/04/$02.00
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Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite
(ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH
is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate
dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence
microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in
neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant
women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the
distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubuleorganizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme’s intracellular
distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant
and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that
6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH
production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing
ROM and NO production in comparison with fully activated neutrophils from nonpregnant women. The Journal of Immunology,
2004, 172: 6373– 6381.
6374
Materials and Methods
Patients
Peripheral blood samples were obtained from nonpregnant women and
pregnant women after written informed consent was provided. The collection of specimens for the study of inflammatory mechanisms was approved
by the Institutional Review Board. The nonpregnant group consisted of
women in the secretory phase of the menstrual cycle who were not taking
oral contraceptives and who had no history of acute or chronic inflammatory conditions (such as asthma or recent infections). Women with normal
pregnancies had no medical or obstetric complications, and their pregnancies ranged in gestational age from 20 wk to term. Eligible patients were
approached at the Detroit Medical Center/Wayne State University
(Detroit, MI).
by a similar procedure (26, 27). Cells were washed, fixed with 2% paraformaldehyde, and then incubated for 30 min. Samples were washed to
stop the reaction. The samples were transilluminated using a 590 long-pass
optical filter (Omega Optical, Brattleboro, VT) to enhance the contrast of
the reaction product relative to background.
6-PGDase activity was also evaluated using quantitative microphotometry. To evaluate 6-PGDase kinetics, samples were incubated with reagent
while being observed microscopically. In these experiments the iris was
reduced in size to minimize the illuminated region, thus reducing stray
light.
Immunofluorescence staining
Neutrophils were placed on glass coverslips, incubated with reagents as
described below, and then fixed with Naftalin’s protocol (22). Briefly, cells
were fixed with 2% paraformaldehyde, permeabilized with 1% Brij-58, and
fixed with 2% paraformaldehyde at room temperature for 20 min. Cells
were washed with HBSS, labeled with 1 ␮g of FITC and/or TRITC-conjugated Abs at 4°C for 30 min, and then washed again with HBSS at room
temperature.
Fluorescence microscopy
Cells were observed using an Axiovert fluorescence microscope (Carl
Zeiss, New York, NY) with mercury illumination interfaced to a computer
using Scion image-processing software (28). A narrow bandpass discriminating filter set (Omega Optical) was used with excitation at 485/22 nm
and emission at 530/30 nm for FITC, and an excitation of 540/20 nm and
emission at 590/30 nm for TRITC. Long-pass dichroic mirrors of 510 and
560 nm were used for FITC and TRITC, respectively. For resonance energy transfer (RET) imaging, 485/22- and 590/30-nm optical filters were
used for excitation and emission, respectively, in conjunction with a
510-nm dichroic mirror. The fluorescence images were collected with an
intensified charge-coupled device camera (Princeton Instruments,
Princeton, NJ).
NAD(P)H oscillations
NAD(P)H autofluorescence oscillations were detected as previously described (23). Briefly, a 365WB50 excitation filter, a 400-nm long-pass
dichroic mirror, and a 450AF58 emission filter were used. A cooled highsensitivity photomultiplier tube in a D104 detection system (Photon Technology, Lawrenceville, NJ) attached to a Zeiss microscope was used. Data
were analyzed using Felix software (Photon Technology).
Single-cell emission spectrophotometry
Energy transfer was also examined by means of microscope spectrophotometer apparatus (29, 30). Fluorescence emission spectra were collected
from single cells by a Peltier-cooled IMAX camera with a liquid nitrogencooled intensifier (Princeton Instruments) attached to a modified Zeiss Axiovert fluorescence microscope. Microspectrophotometry used a 485/
22-nm narrow bandpass discriminating filter for excitation, a 510-nm longpass dichroic mirror, and a 520-nm long-pass emission filter. Winspec
software (Princeton Instruments) was used to analyze spectrophotometric data.
Cell preparation
Detection of ROM and NO production
Neutrophils were isolated from blood samples using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation (23). Neutrophil viability was ⬎95% as assessed by trypan blue exclusion. Cells were
suspended in HBSS (Life Technologies, Grand Island, NY).
Pericellular release of ROMs from single cells was monitored as described
(31). Briefly, adherent neutrophils were surrounded in 2% gelatin containing 100 ng/ml dihydrotetramethylrosamine (Molecular Probes). ROMs, especially H2O2, released by the cells entered the gelatin matrix, where they
oxidized dihydrotetramethylrosamine to tetramethylrosamine, which was
detected by fluorescence microscopy. NO production was monitored using
diaminofluorescein-2 as previously described (32).
Reagents and Abs
Colchicine, LPS (serotype 026:B6), melatonin, and FMLP were obtained
from Sigma-Aldrich. FITC and tetramethylrhodamine isothiocyanate
(TRITC) were obtained from Molecular Probes (Eugene, OR). Rabbit antiG-6-PDase and goat anti-lactate dehydrogenase (LDH) polyclonal Abs
were obtained from Chemicon International (Temecula, CA). Anti-␥-tubulin was obtained from R&D Systems (Minneapolis, MN). Anti-6-PGDase
(S4D5) was prepared as previously described (24). FITC- or TRITC-conjugated Abs were prepared as described (25).
6-PGDase cytochemistry
6-PGDase activity was studied using cytochemical methods (26, 27).
Briefly, the 6-PGDase incubation medium consisted of phosphate buffer
(pH 7.4), 32% (w/v) polyvinyl alcohol, 2 mM 6-phosphogluconate (VWR,
Batavia, IL), 0.4 mM NADP⫹ (Calibiochem, San Diego, CA), 2.5 mM
MgCl2, 2.5 mM NaN3, 0.16 mM 1-methoxphenazine methosulfate (SigmaAldrich), and 2.5 mM Nitro BT (VWR). G-6-PDase and LDH were stained
Results
6-PGDase is found at the MTOC in neutrophils from normal
pregnant women, but not nonpregnant women
We have recently reported that G-6-PDase undergoes retrograde
trafficking in neutrophils from pregnant women, whereas anterograde trafficking is found in cells from nonpregnant women (23).
Because 6-PGDase is another key NADPH-producing component
of the HMS, we hypothesized that 6-PGDase undergoes specific
translocation to the MTOC of neutrophils from pregnant women,
thus leading to a complete spatial polarization of glycolysis and the
NADPH-producing steps of the HMS. To test this hypothesis, we
examined the intracellular distribution of 6-PGDase in neutrophils
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The superoxide and NO produced yield additional downstream
ROMs and RNIs.
ROM and RNI production require electrons in the form of
NADPH, as illustrated in Equations 1 and 2. NADPH production,
in turn, requires the hexose monophosphate shunt (HMS) and glucose. For example, neutrophils do not produce superoxide anions
in the absence of glucose (17–19). To drive the HMS, glucose
transport, a rate-controlling step in metabolism (20), is accelerated
by neutrophil activation (21). One process that increases glucose
transport is hexokinase translocation to the plasma membrane (22),
where it catalyzes the formation of glucose-6-phosphate (G-6-P).
G-6-P is metabolized by the HMS, a cell’s primary NADPH
source, and by glycolysis. The first step of the HMS is mediated by
glucose-6-phosphate dehydrogenase (G-6-PDase), which converts
G-6-P into 6-phosphogluconolactone (6-PG) with the release of
NADPH. 6-Phosphogluconate dehydrogenase (6-PGDase) converts 6-PG into ribose 5-phosphate and NADPH. We have recently
discovered that the intracellular trafficking of G-6-PDase regulates
the HMS and, in turn, ROM production (23). In nonpregnant individuals, G-6-PDase is located at the cell periphery where G-6-P
is produced by hexokinase and is readily available. However, in
pregnant women, G-6-PDase undergoes retrograde transport on
microtubules to a cell’s microtubule-organizing center (MTOC)
(23). In this location, G-6-P is less available to G-6-PDase, because it is metabolized by glycolytic enzymes at the cell periphery.
In the present study, we extend these previous observations by
showing that 6-PGDase undergoes similar retrograde trafficking
during pregnancy. Moreover, 6-PGDase and G-6-PDase appear to
form a complex within cells, which may account for their parallel
trafficking, and the accompanying reduction in ROM and NO
release.
METABOLIC REGULATION IN PREGNANCY
The Journal of Immunology
6375
from nonpregnant and pregnant women with and without in vitro
stimulation with LPS using immunofluorescence microscopy. As
illustrated in Fig. 1, the anti-6-PGDase label is found primarily at
the periphery of neutrophils from nonpregnant women (Fig. 1a).
Untreated neutrophils, whether spherical or polarized in shape,
show this intracellular distribution of 6-PGDase (data not shown).
As HMS activation may alter the trafficking of its constituent enzymes, we evaluated the effect of LPS, a reagent known to stimulate cells, on the intracellular distribution of 6-PGDase. Indistinguishable results were obtained when neutrophils were exposed to
LPS (50 ng/ml) for 20 min (Fig. 1, a and b). We next examined the
intracellular location of 6-PGDase in cells from pregnant women.
In contrast to neutrophils from nonpregnant women, 6-PGDase is
found in the vicinity of the MTOC in cells from pregnant women
(Fig. 1, g and h) in the presence and absence of LPS exposure. As
a positive control, similar changes were noted for G-6-PDase (Fig.
1, c, d, i, and j). As a negative control, the intracellular distribution
of LDH in neutrophils from pregnant and nonpregnant women in
the presence and absence of LPS stimulation was evaluated (Fig.
1, e, f, k, and l). The LDH distribution was not effected by pregnancy or LPS stimulation. Similar negative controls with other
metabolic enzymes including phosphofructokinase (PFK) and
pyruvate kinase have been reported previously (23). Thus, 6-PGDase undergoes differential trafficking in cells from pregnant and
nonpregnant women.
Cytochemical evaluation of 6-PGDase activity in neutrophils
from pregnant and nonpregnant women
Our immunofluorescence microscopy experiments demonstrate a
dramatic translocation of the 6-PGDase Ag in neutrophils from
pregnant women. To establish that the distribution of 6-PGDase
functional activity parallels its antigenic localization, we have also
used immunohistochemical techniques to localize 6-PGDase activity in cells. The method of Van Noorden and Butcher (26, 27)
has been used to localize the reaction product of 6-PGDase in
neutrophils. Fig. 2 shows micrographs of neutrophils from preg-
FIGURE 2. Localization of enzyme activities in neutrophils. Representative cells from nonpregnant women (top row; a–f) and pregnant women (bottom
row; g–l) are shown. Neutrophils were studied with and without prior incubation with LPS (50 ng/ml, 20 min). Cells were fixed then stained for 6-PGDase
(a, b, g, and h), G-6-PDase (c, d, i, and j), and LDH (e, f, k, and l) activity (26, 27). 6-PGDase enzymatic activity is peripherally located in cells from
nonpregnant women (a and b), but was found near the center of cells from pregnant women (g and h). Thus, 6-PGDase activity, in addition to its antigenic
epitopes, undergoes differential trafficking in cells from nonpregnant and pregnant women. Positive and negative controls were performed using G-6-PDase
activity and LDH activity, as in Fig. 1. LPS did not affect the intracellular distributions of 6-PGDase, G-6-PDase, and LDH. Thus, G-6-PDase activity
parallels the distribution of G-6-PDase Ag identified in the immunofluorescence experiments above. Magnification, ⫻760. a– d and g–j: n ⫽ 12; e, f, k,
and l: n ⫽ 5.
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FIGURE 1. Immunofluorescence microscopy of 6-PGDase, G-6-PDase, and LDH in neutrophils. Representative cells from nonpregnant women (top
row; a–f) and pregnant women (bottom row; g–l) are shown. Neutrophils were stained with and without prior incubation with LPS (50 ng/ml, 20 min). Cells
were fixed as described (22), and then stained with polyclonal Abs directed against 6-PGDase (a, b, g, and h), G-6-PDase (c, d, i, and j), and LDH (e, f,
k, and l). Anti-6-PGDase is peripherally located in cells from nonpregnant women (a and b), but was found at the MTOC in cells from pregnant women
(g and h). Thus, 6-PGDase undergoes differential trafficking in cells from nonpregnant and pregnant women. As a positive control, similar trafficking of
G-6-PDase was observed (c, d, i, and j). As a negative control, LDH did not undergo intracellular redistribution during pregnancy (e, f, k, and l). Addition
of LPS, which activates the HMS, did not affect the intracellular distributions of 6-PGDase, G-6-PDase, and LDH. Magnification, ⫻760. a– d and g–j: n ⫽
16; e, f, k, and l: n ⫽ 5, where n is the number of patients contributing cells for these in vitro analyses.
6376
METABOLIC REGULATION IN PREGNANCY
Colchicine disrupts 6-PGDase trafficking in pregnancy
neutrophils
FIGURE 3. Quantitative microphotometry of 6-PGDase reaction product formation. Neutrophils from nonpregnant (NP) and pregnant (P)
women were fixed and processed as described in Fig. 2. In this case, cells
were observed immediately by optical microscopy. The iris was adjusted to
illuminate an area just slightly larger than the cell. As the reaction product
was formed, the cells became more opaque at 590 nm, thereby reducing the
observed intensity. The slopes of the cells from nonpregnant and pregnant
women were identical, thus suggesting that the rates of substrate conversion are indistinguishable. n ⫽ 3.
6-PGDase traffics to the MTOC in cells from pregnant women
The fluorescence micrographs of Figs. 1g and 4g suggest that
6-PGDase undergoes retrograde motion in neutrophils from pregnant women, in contrast to the anterograde distribution within cells
from nonpregnant individuals. To further test this concept, cells
were labeled using direct immunofluorescence with FITC-conjugated anti-␥-tubulin and TRITC-conjugated anti-6-PGDase. ␥-Tubulin is specific for MTOCs, which include the centrosome of
interphase cells, polar bodies of mitotic cells, and basal bodies of
flagella (33). Representative micrographs of neutrophils from
pregnant and nonpregnant women that were fixed, extracted, and
stained with FITC-anti-␥-tubulin and TRITC-anti-6-PGDase are
shown in Fig. 5. Not surprisingly, anti-␥-tubulin decorated the centrosome (Fig. 5, b and e). When cells from pregnant women were
stained with anti-␥-tubulin and anti-6-PGDase, the FITC-anti-␥tubulin and TRITC-anti-6-PGDase patterns overlapped substantially (Fig. 5, d with e), which was not observed using cells from
FIGURE 4. Effect of colchicine on the intracellular distribution of 6-PGDase, G-6-PDase, and LDH in neutrophils. Representative cells from nonpregnant women (top row; a–f) and pregnant women (bottom row; g–l) are shown. Neutrophils were stained without (a, c, e, g, i, and k) and with (b, d, f, h,
j, and l) prior incubation with colchicine (50 ␮g/ml for 30 min at 37°C) to disrupt microtubules. Cells were stained with polyclonal Abs directed against
6-PGDase (a, b, g, and h), G-6-PDase (c, d, i, and j), and LDH (e, f, k, and l). Although anti-6-PGDase was found at the MTOC in cells from pregnant
women (g), addition of colchicine promoted a more uniform distribution of this Ag (h). Colchicine had no discernible effect on cells from nonpregnant
women (a and b). Similar effects were noted for G-6-PDase (c, d, i, and j). Colchicine did not affect LDH location (e, f, k, and l). Magnification, ⫻760.
a– d and g–j: n ⫽ 8; e, f, k, and l: n ⫽ 4.
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nant and nonpregnant individuals stained for 6-PGDase (a, b, g,
and h) and G-6-PDase (c, d, i, and j) activity as described in Materials and Methods. The 6-PGDase and G-6-PDase reaction products were enriched at a cell’s periphery (Fig. 2, a– d). Moreover,
they were unaffected by LPS-mediated stimulation (50 ng/ml LPS
for 40 min at 37°C) (Fig. 2, b and d). In contrast, 6-PGDase and
G-6-PDase activity were centrally located in cells from pregnant
women (Fig. 2, g–j) with or without treatment with LPS. Furthermore, staining could not be observed during negative control experiments using the omission of substrate (6-PG or NADP⫹).
These findings parallel those obtained using immunofluorescence
microscopy (Fig. 1). Although 6-PGDase and G-6-PDase activities
underwent substantial reorganization in neutrophils from pregnant
women, LDH activity did not (Fig. 2, e, f, k, and l).
These findings illustrate the fact that 6-PGDase remains active
after translocation to the MTOC. In situ kinetic quantitative microphotometry studies indicate that the rates of product deposition
in these experiments do not differ in cells from pregnant and nonpregnant women (Fig. 3). Thus, G-6-PDase remains functional
during pregnancy, but its activity is restricted to the cell’s center.
The ability of 6-PGDase to undergo either anterograde or retrograde distribution in cells under differing physiological conditions
suggests that a component of the cytoskeleton is capable of actively translocating 6-PGDase within a cell. To test this concept,
immunofluorescence localization of metabolic enzymes was performed on cells from pregnant and nonpregnant women in the
presence and absence of colchicine, a microtubule-disrupting drug.
Colchicine (50 ␮g/ml for 30 min at 37°C) had no effect on the
intracellular distributions of 6-PGDase, G-6-PDase, and LDH of
neutrophils from nonpregnant women (Fig. 4, a–f). However,
when cells from pregnant women were treated with colchicine, the
intracellular distribution of 6-PGDase became more normalized
with staining associated with the MTOC, cytoplasm, and cell periphery (Fig. 4h). As a positive control, similar changes were noted
for G-6-PDase (Fig. 4, c, d, i, and j). As a negative control, the
intracellular distribution of LDH in neutrophils from pregnant and
nonpregnant women in the presence and absence of colchicine was
evaluated (Fig. 4, e, f, k, and l). The LDH distribution was not
effected by colchicine. The ability of colchicine to disrupt the intracellular distribution of 6-PGDase suggests that its localization
within pregnancy neutrophils is dependent upon microtubules.
The Journal of Immunology
6377
nonpregnant women (a and b). Although the similarity of the staining patterns in Fig. 5, d and e, suggests an association of ␥-tubulin
and 6-PGDase, it cannot assess the molecular proximity of these
proteins. To detect molecular proximity of ␥-tubulin and 6-PGDase, the technique of RET was used. Fluorescent labels must be
within ⬃7 nm to obtain positive RET signals. We chose anti-6PGDase as the acceptor label, because 6-PGDase is more abundant
than ␥-tubulin, thereby maximizing the RET signal. RET was studied using RET microscopy to determine the spatial locations of
molecular proximity within cells, and emission microspectrophotometry to quantitatively measure the spectral intensities. As expected, RET was not detected between these labels on neutrophils
from nonpregnant women using optical imaging (Fig. 5c) or spectrophotometry (Fig. 6e). However, RET imaging of pregnancy
neutrophils indicates that RET is present at the MTOC (Fig. 5f),
which is confirmed by spectrophotometry studies (Fig. 6g). These
results indicate that 6-PGDase traffics to the MTOC in cells from
pregnant women. Furthermore, 6-PGDase is within molecular
proximity (⬃7 nm) of ␥-tubulin in neutrophils from pregnant
women, but not in cells from nonpregnant women.
The ability of colchicine to break up microtubules and normalize the intracellular distribution of 6-PGDase suggests that it
should also reduce the amount of RET between 6-PGDase and
␥-tubulin. Colchicine had no effect on the emission properties of
cells labeled with only donor or acceptor labels (Fig. 6, a– d).
Neutrophils from pregnant and nonpregnant individuals were
treated with 50 ␮g/ml colchicine for 30 min at 37°C. Although
colchicine treatment had no effect on cells from nonpregnant
women (Fig. 6, e and f), colchicine exposure significantly reduced
RET between 6-PGDase and ␥-tubulin in cells from pregnant
women (g vs h).
FIGURE 6. RET emission spectrophotometry studies of 6-PGDase and
␥-tubulin in neutrophils. Emission spectra were recorded for cells labeled
with FITC only (a and b), TRITC only (c and d), both labels associated
with cells from nonpregnant women (e and f), and both labels for cells from
pregnant women (g and h). In some experiments, cells were incubated with
colchicine (50 ␮g/ml for 30 min at 37°C) (b, d, f, and h). The fluorescence
emission spectra of FITC (a and b) and TRITC (c and d) were not affected
by colchicine. RET was not observed for cells from nonpregnant women (e
and f). However, RET was observed for cells from pregnant women in both
the absence (g) and presence (h) of colchicine. n ⫽ 7.
hibited peripheral staining of 6-PGDase and G-6-PDase (Fig. 7, a
and b). Importantly, RET imaging showed that these two enzymes
exhibited molecular proximity (Fig. 7c). RET is also indicated by
the emission spectroscopy results of Fig. 8e in comparison with
that of a. When neutrophils from pregnant women were evaluated,
RET between these two HMS enzymes were found at the MTOC
6-PGDase and G-6-PDase form a supramolecular complex in
neutrophils
The parallel trafficking of 6-PGDase and G-6-PDase and their sensitivities to colchicine suggest that they may form a molecular
complex on microtubules. RET experiments were performed to
test this idea. Neutrophils from pregnant and nonpregnant women
were stained with FITC-anti-G-6-PDase and TRITC-anti6-PGDase as described above. Cells from nonpregnant women ex-
FIGURE 7. 6-PGDase and G-6-PDase exhibit RET in neutrophils from
nonpregnant (a– c) and pregnant (d–f) women. Cells were fixed then
stained with FITC-conjugated anti-G-6-PDase and TRITC-conjugated anti6-PGDase. Although 6-PGDase and G-6-PDase are found at the periphery of nonpregnancy neutrophils (a and b) and at the MTOC of pregnancy cells (d and e), both types of cells exhibit RET (c and f).
Magnification, ⫻820. n ⫽ 8.
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FIGURE 5. 6-PGDase trafficks to the centrosome in pregnancy neutrophils. Cells from nonpregnant (a– c) and pregnant (d–f) women were studied using direct immunofluorescence. Cells were fixed and then stained
with FITC-conjugated anti-␥-tubulin and TRITC-conjugated anti6-PGDase. Although ␥-tubulin and G-6-PDase did not colocalize (a and b)
or exhibit RET (c) in neutrophils from nonpregnant women, both colocalization (d and e) and RET (f) were found for these proteins in cells from
pregnant women. Thus, G-6-PDase accumulates in the region of the centrosome of neutrophils from pregnant, but not nonpregnant women. Magnification, ⫻820. n ⫽ 5.
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METABOLIC REGULATION IN PREGNANCY
hibit molecular proximity in cells from both pregnant and nonpregnant women.
We next sought to better understand the nature of the 6-PGDase
and G-6-PDase complexes. For example, 6-PGDase and G-6PDase could simply be brought together, because they both bind to
microtubules or they could be assembled independently of microtubule structures. To ascertain the role of microtubules in 6-PGDase/G-6-PDase complex assembly, cells were treated with colchicine at 50 ␮g/ml for 30 min at 37°C. RET was observed using
emission spectrophotometry (Fig. 8, f and h) and microscopic imaging (Fig. 10, c and f) in both the presence and absence of colchicine, which suggests that their proximity is not colchicine
sensitive.
Pregnancy alters the dynamic production of NAD(P)H
(Fig. 7f). This was confirmed with emission spectroscopy experiments (Fig. 8g). However, RET was not observed between FITCanti-G-6-PDase and TRITC-anti-LDH for neutrophils from nonpregnant and pregnant women (Fig. 9, c and f). Similarly, RET was
not observed in neutrophils from nonpregnant and pregnant
women when labeled with anti-6-PGDase and anti-hexokinase reagents (data not shown). These findings suggest that some specificity is observed in the molecular proximity relationships formed
among metabolic enzymes. Hence, 6-PGDase and G-6-PDase ex-
FIGURE 9. 6-PGDase and LDH do not exhibit RET in both neutrophils
from nonpregnant (a– c) and pregnant (d–f) women. Cells were fixed and
then stained with FITC-conjugated anti-G-6-PDase and TRITC-conjugated
anti-LDH. Although 6-PGDase and LDH are found at the periphery of
nonpregnancy neutrophils (a and b), they do not exhibit RET, which suggests that they are not in close proximity. There different localizations in
pregnancy cells is consistent with the absence of RET. Magnification,
⫻820. n ⫽ 3.
FIGURE 10. RET between 6-PGDase and G-6-PDase is observed in
neutrophils from pregnant women in the presence of colchicine. Cells were
fixed and then stained with FITC-conjugated anti-␥-tubulin and TRITCconjugated anti-6-PGDase. In the absence of colchicine, MTOC labeling
was observed (a– c). As suggested by the spectrophotometry results of Fig.
7h, RET is observed in the presence of colchicine (f), although both enzymes have undergone dramatic redistributions due to colchicine treatment. Magnification, ⫻820. n ⫽ 4.
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FIGURE 8. RET spectrophotometry studies of 6-PGDase/G-6-PDase
complexes in neutrophils. Control studies with FITC only (a and b) and
TRITC only (c and d) are shown. Experiments were performed by incubating cells without (a, c, e, and g) or with colchicine (50 ␮g/ml for 30 min
at 37°C) (b, d, f, and h). RET was observed for cells from nonpregnant
women in both the absence and presence of colchicine (e and f). However, RET was not intense (see arrows). RET was also observed for
cells from pregnant women in both the absence (g) and presence (h) of
colchicine. n ⫽ 4.
Because 6-PGDase produces NADPH, we considered that 6-PGDase translocation may affect the nature of NADPH production.
Previous studies have shown that the metabolism of neutrophils
oscillates in time, and that the amplitudes and frequencies of these
oscillations vary with exposure to activating stimuli (30, 34). Both
experimental and theoretical studies indicate that NADPH oscillations are demodulated by living cells to yield oscillations in
ROM production (34, 35). Fig. 11 shows real-time microfluorometry experiments of NADH ⫹ NADPH (NAD(P)H) autofluorescence in living cells as a function of time. Adherent neutrophils
from nonpregnant and pregnant women were continuously analyzed during additions of FMLP and melatonin. Untreated cells
from nonpregnant individuals displayed low-amplitude oscillations with a 20-s period. The period of these oscillations is reduced
to ⬃10 s after addition of 0.5 ␮M FMLP. Melatonin promotes
electron trafficking between the NADPH oxidase and myeloperoxidase and increases the amplitude of NAD(P)H oscillations (35).
In contrast to IFN-␥, which requires 1–2 h to increase metabolic
amplitudes, melatonin acts immediately and is therefore the preferred reagent in these real-time studies. When melatonin is added
at 150 ␮g/ml, the oscillations are dramatically increased in amplitude. In contrast to the variable level of metabolic stimulation seen
in cells from nonpregnant women, an intermediate behavior is observed for cells from pregnant women under all conditions; FMLP
and melatonin had no effect on metabolic oscillations (Fig. 11).
Thus, cell metabolism is unresponsive to different types of activating stimuli, which parallels a previous report from our laboratory using other activating agents (23).
The Journal of Immunology
6379
12a, trace 4). In contrast, ROM production was at an intermediate
value for ⬃75% of pregnancy neutrophils under all conditions
(Fig. 12c) (see also Ref. 23). NO production was also evaluated
(Fig. 12, b and d). Neutrophils from nonpregnant women display
low levels of NO release (Fig. 12b, trace 1), which can be increased by incubation with IFN-␥ (trace 2) or LPS (trace 3) and
further increased by their combination (trace 4), as described
above. Neutrophils from pregnant women displayed an intermediate slope that was not influenced by IFN-␥, LPS, or both of these
reagents (Fig. 12d). Because both ROM and RNI production are
powered by the same metabolic apparatus, it is not surprising that
parallel observations concerning ROM and NO production were
obtained.
Discussion
Pregnancy affects the production of oxidants
The effect of pregnancy on ROM and RNI production was evaluated. In the first series of experiments, we confirmed our recent
observation that pregnancy neutrophils cannot be properly activated (23). Fig. 12 shows the rates of ROM and NO production by
neutrophils from pregnant and nonpregnant women. In cells from
nonpregnant women, low basal rates of ROM production are observed (Fig. 12a, trace 1), which are increased by exposure to
IFN-␥ (10 ␮g/ml for 1 h at 37°C) or to LPS (50 ng/ml for 20 min)
(trace 3). Maximal levels of the ROM production rate are observed
when IFN-␥ pretreatment is combined with LPS addition (Fig.
FIGURE 12. Rates of ROM (a and c) and NO (b and d) release from
neutrophils of nonpregnant (a and b) and pregnant (c and d) women. ROM
and NO release were measured for morphologically polarized cells in a
matrix containing either dihydrotetramethylrhodamine (left-hand side) or
diaminofluorescein-2 (right-hand side), respectively. The traces labeled
1– 4 in each of the four panels are as follows: 1, control; 2, IFN-␥; 3, LPS;
and 4, IFN-␥ plus LPS. Although cells from nonpregnant women underwent
normal activation, cells from pregnant women displayed an intermediate level
of ROM and NO production that was unaffected by stimulants. n ⬎ 10.
Pregnancy is a unique immunological state characterized by
changes in both the adaptive and innate immunological responses
(36, 37). One key element of both the innate and adaptive responses is the ability of leukocytes to generate ROMs and RNIs in
response to opsonized and unopsonized pathogens. Neutrophils
from pregnant women have been reported to have depressed ROM
production (e.g., Ref. 2) and enhanced ROM production (38). A
recent study from this laboratory (23) has suggested that the basal
level of ROM production by pregnancy neutrophils is enhanced
relative to unstimulated cells from nonpregnant women, whereas
they cannot undergo activation to the same level as neutrophils
from nonpregnant women. Thus, pregnancy neutrophils have an
intermediate level of ROM production. A key factor limiting ROM
production appears to be the translocation of G-6-PDase from the
cell periphery to the MTOC in pregnancy neutrophils (23). We
have proposed that the availability of the substrate NADPH is a
key element in regulating NADPH and subsequent ROM production (e.g., Refs. 23, 34, 35, 39). Thus, NADPH oxidase assembly
provides for a course regulation of its activity, whereas NADPH
availability, including the spatial and temporal components of its
production, provide a fine-tuning mechanism.
Our colocalization and RET experiments have shown that 6-PGDase accumulates at the MTOC and is in close physical proximity
with ␥-tubulin in neutrophils from pregnant women, but not nonpregnant women. This suggests that 6-PGDase undergoes retrograde trafficking during pregnancy and anterograde motion in cells
from nonpregnant individuals, which parallels our recent work on
G-6-PDase (23). Furthermore, the molecular proximity of 6-PGDase to cytoskeletal components is consistent with the ability of
hexokinase, aldolase, PFK, GAPDH, and pyruvate kinase, to bind
to microfilaments and/or microtubules (40 – 45). Thus, the intracellular trafficking of HMS and other cytoskeleton-associated metabolic enzymes allows neutrophils to vary HMS activity relative to
glycolysis.
Enzyme-enzyme interactions have been demonstrated in several
metabolic pathways, such as glycolysis and the tricarboxylic acid
cycle. The formation of enzyme complexes allows the products of
one enzyme to be directly passed to the next enzyme of the pathway without being released into the aqueous phase, thereby increasing efficiency. The first two steps of the HMS, which are
catalyzed by G-6-PDase and 6-PGDase, constitute the primary
source of NADPH production in cells. The present study has demonstrated RET between 6-PGDase and G-6-PDase, thus indicating
that these enzymes are within ⬃7 nm of each other. The proximity
of these two enzymes suggests that they form a supramolecular
complex or metabolon within cells. This suggestion is in agreement with a previous biochemical study in plant and yeast systems
using radiolabeled substrates that showed substrate channeling in
the HMS (46). Hence, supramolecular complex formation explains
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FIGURE 11. NAD(P)H dynamics in neutrophils from nonpregnant
(NP; upper trace) and pregnant women (P; lower trace). The autofluorescence intensity is given at the ordinate in counts per second, whereas time
in seconds is listed at the abscissa. In the upper trace, a neutrophil from a
nonpregnant individual was observed over time. The addition of 0.5 ␮M
FMLP increased the frequency of metabolic oscillations. Subsequent addition of 150 ␮g/ml melatonin increased the amplitude of these oscillations. In contrast, the NAD(P)H oscillations of neutrophils from pregnant
women were not affected by the addition of these two reagents. Thus,
pregnancy neutrophils exhibit an intermediate level of metabolic activity
that was refractory to stimulation.
6380
physiological pathway could be understood, it might be possible to
use this information as a route to develop novel anti-inflammatory
compounds that provide similar functional changes in leukocytes.
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