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This information is current as of June 16, 2017. 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 References Subscription Permissions Email Alerts This article cites 47 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/172/10/6373.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. 6378 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. References 1. Crouch, S. P. M., I. P. Crocker, and J. Fletcher. 1995. The effect of pregnancy on polymorphonuclear leukocyte function. J. Immunol. 155:5436. 2. Cotton, D. J., B. Seligmann, B. O’Brian, and J. I. Gallin. 1983. Selective defect in human neutrophil superoxide anion generation elicited by the chemoattractant N-formylmethionylleucylphenylalanine in pregnancy. J. Infect. Dis. 148:194. 3. Benjamin, J. L., and J. S. Remington. 1984. The adverse effect of pregnancy on macrophage activation. Cell. Immunol. 85:94. 4. El-Maallem, H., and J. Fletcher. 1980. Impaired neutrophil function and myeloperoxidase deficiency in pregnancy. Br. J. Hematol. 44:375. 5. Bjoksten, B., T. Soderstrom, M. G. Damber, B. Von Schoultz, and T. Stigbrand. 1978. Polymorphonuclear leucocyte function during pregnancy. Scand. J. Immunol. 8:257. 6. Krause, P. J., C. J. Ingardia, L. T. Pontius, H. L. Malech, T. M. Lobello, and E. G. Maderazo. 1987. Host defense during pregnancy: neutrophil chemotaxis and adherence. Am. J. Obstet. Gynecol. 157:274. 7. Persellin, R. H., and L. L. Thoi. 1979. Human polymorphonuclear leukocyte phagocytosis in pregnancy: development of inhibition during gestation and recovery in the postpartum period. Am. J. Obstet. Gynecol. 134:250. 8. Brabin, B. J. 1985. Epidemiology of infection in pregnancy. Rev. Infect. Dis. 7:579. 9. Larsen, B., and R. P. Galask. 1978. Host-parasite interactions during pregnancy. Obstet. Gynecol. Survey 33:297. 10. Reinhardt, M. C. 1979. Effects of parasitic infections in pregnant women. Ciba Found. Symp. 77:149. 11. Luft, B. J., and J. S. Remington. 1982. Effect of pregnancy on resistance to Listeria monocytogenes and Toxoplasma gondii infections in mice. Infect. Immun. 38:1164. 12. Cunningham, F. G., K. J. Leveno, G. D. V. Hankins, and P. J. Whalley. 1984. Respiratory insufficiency associated with pyelonephritis during pregnancy. Obstet. Gynecol. 63:121. 13. Confavreux, C., M. Hutchinson, M. M. Hours, P. Cortinovis-Tourniaire, and T. Moreau,. 1998. Rate of pregnancy-related relapse in multiple sclerosis. N. Engl. J. 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Oxygen metabolism in cloned macrophage cell lines: glucose dependence of superoxide production, metabolic and spectral analysis. J. Immunol. 132:857. 20. Ozcan, S., and M. Johnston. 2000. Function and regulation of yeast hexose transporters. Microbiol. Mol. Biol. Rev. 63:554. 21. Tan, A. S., N. Ahmed, and M. V. Berridge. 1998. Acute regulation of glucose transport after activation of human peripheral blood neutrophils by phorbol myristate acetate, fMLP, and granulocyte-macrophage colony-stimulation factor. Blood 91:649. 22. Pedley, K. C., G. E. Jones, M. Magnani, R. J. Rist, and R. J. Naftalin. 1993. Direct observation of hexokinase translocation in stimulated macrophages. Biochem. J. 291:515. 23. Kindzelskii, A. L., J.-B. Huang, T. Chaiworapongsa, Y. M. Kim, R. Romero, and H. R. Petty. 2002. Pregnancy alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism and oxidant release of maternal neutrophils. J. Clin. Invest. 110:1801. 24. Uyama, T., T. Kinoshita, H. Takahashi, N. Satoh, K. Kanamori, and H. Michibata. 1998. 6-Phosphogluconate dehydrogenase is a 45-kDa antigen recognized by S4D5, a monoclonal antibody specific to vanadocytes in the vanadium-rich ascidian Ascidia sydneiensis samea. J. Biochem. 124:377. 25. Kindzelskii, A. L., M. M. Eszes, R. F. Todd III, and H. R. Petty. 1997. Proximity oscillations of complement receptor type 4 and urokinase receptors on migrating neutrophils are linked with signal transduction/metabolic oscillations. Biophys. J. 73:1777. 26. Van Noorden, C. J. F., and R. G. Butcher. 1984. Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts. J. Histochem. Cytochem. 32:998. 27. Butcher, R. G., and C. J. F. Van Noorden. 1985. Reaction rate studies of glucose6-phosphate dehydrogenase activity in section of rat liver using four tetrazolium salts. Histochem. J. 17:993. 28. Petty, H. R., and A. L. Kindzelskii. 2001. Dissipative metabolic patterns respond during neutrophil transmembrane signaling. Proc. Natl. Acad. Sci. USA 98:3145. 29. Petty, H. R., R. G. Worth, and A. L. Kindzelskii. 2000. Imaging sustained dissipative patterns in the metabolism of individual living cells. Phys. Rev. Lett. 84:2754. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 why 6-PG formed by G-6-PDase does not equilibrate with 6-PG in the aqueous phase. However, it is not clear whether the molecular proximity of 6-PGDase and G-6-PDase was a result of enzymeenzyme complex formation of simply the fact that both enzymes clustered together at the MTOC of pregnancy neutrophils or at the periphery of cells from nonpregnant women. In other words, substrate channeling could be due to enzyme-microtubule interactions that lead to the molecular proximity of 6-PGDase and G-6-PDase. When cells from pregnant women were incubated with colchicine, 6-PGDase and G-6-PDase underwent dramatic redistribution within cells, but a substantial amount of RET remained, indicating that these enzymes were in the molecular proximity of each other independently of microtubules. Thus, our studies provide new structural evidence for the formation of supramolecular complexes of 6-PGDase and G-6-PDase. Furthermore, this finding is consistent with the fact that colchicine does not decrease the amount of superoxide produced (23), which would have been expected if microtubules were required for substrate channeling. Our work suggests that 6-PGDase and G-6-PDase form a supramolecular complex in cells, which facilitates the production of NADPH by the HMS. In nonpregnant women, the complex is found in an anterograde distribution at the cell periphery. The peripheral distribution of the 6-PGDase/G-6-PDase complex facilitates its coupling with hexokinase, thereby promoting NADPH production. However, neutrophils from pregnant women are characterized by retrograde motion in a colchicine-sensitive (microtubule-dependent) fashion to the MTOC. This configuration of enzymes does not make G-6-P, which is produced at the plasma membrane, readily available to G-6-PDase/6-PGDase complex, which is located at the MTOC. This allows G-6-P to undergo glycolytic conversion by phosphoglucose isomerase to fructose-6phosphate and irreversible metabolism by PFK. Thus, NADPH production is blunted in cells from pregnant women. The reduction in activated levels of superoxide production may explain the increased susceptibility to certain infectious diseases and the decreased symptoms observed during certain chronic inflammatory diseases during pregnancy. The HMS plays a key role in the synthesis of ribose 5-phosphate, which is required for cell proliferation, and in NADPH production, which participates in biosynthetic pathways and in superoxide and NO production. The physical uncoupling of the 6-PGDase/G-6-PDase complex from peripheral cellular metabolism reduces the efficiency of NADPH production, at least under conditions of normal glucose concentrations. Consequently, reduced NADPH availability decreases superoxide production by the NADPH oxidase and NO synthesis by the NO synthase. This is quite reasonable given the observation that superoxide and NO production oscillate in both time and space with the intracellular NAD(P)H concentration (34, 35, 39). Furthermore, we experimentally showed that ROM and NO production in activated neutrophils was reduced in cells from pregnant women in comparison with activated cells from nonpregnant individuals. To our knowledge, this is the first time that pregnancy-associated changes in NO production have been reported. The reduction in oxidant production by cells from pregnant women may help to minimize oxidative damage to the conceptus (47). Several additional implications of regulatory enzyme translocation might also be considered. Inasmuch as immunoregulation is an important aspect of pregnancy and because oxidant stress has been associated with several pregnancy-related clinical conditions (38, 47), the evaluation of G-6-PDase or 6-PGDase translocation may provide a novel means of monitoring pregnancy. We are presently studying the mechanism promoting retrograde G-6-PDase/6PGDase complex transport in pregnancy neutrophils. If this normal METABOLIC REGULATION IN PREGNANCY The Journal of Immunology 30. Kindzelskii, A. L., Z. Yang, G. J. Nabel, R. F. Todd III, and H. R. Petty. 2000. Ebola virus secretory glycoprotein (sGP) disrupts Fc␥RIIIB to CR3 proximity on neutrophils. J. Immunol. 164:953. 31. Kindzelskii, A. L., M.-J. Zhou, R. P. Haugland, and H. R. Petty. 1998. Oscillatory pericellular proteolysis and oxidant deposition during neutrophil migration. Biophys. J. 74:90. 32. Adachi, Y., A. L. Kindzelskii, N. Ohno, T. 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Knull. 1997. A glycolytic enzyme binding domain on tubulin. Arch. Biochem. Biophys. 338:237. 46. Debnam, P. M., G. Shearer, L. Blackwood, and D. H. Kohl. 1997. Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes. Eur. J. Biochem. 246:283. 47. Ornoy, A., D. Kimyagarov, P. Yaffee, R. Abir, I. Raz, and R. Kohen. 1996. Role of reactive oxygen species in diabetes-induced embryotoxicity: studies on preimplantation mouse embryos cultured in serum from diabetic pregnant women. Isr. J. Med. Sci. 32:1066. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017