| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,¶,¶
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
70% of women with arthritis, multiple sclerosis, and uveitis go into remission during pregnancy (e.g., Refs.13, 14, 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) |
 | (2) |
 |
 |
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, 18, 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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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).
Cell preparation
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).
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 anti-G-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 (Sigma-Aldrich), and 2.5 mM Nitro BT (VWR). G-6-PDase and LDH were stained 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 µg 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 high-sensitivity 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 nitrogen-cooled 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 long-pass dichroic mirror, and a 520-nm long-pass emission filter. Winspec software (Princeton Instruments) was used to analyze spectrophotometric data.
Detection of ROM and NO production
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).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 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.
|
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 pregnant 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).
|
|
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 µg/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 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 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-6-PGDase 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.
|
|
-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 µg/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). 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-anti-6-PGDase as described above. Cells from nonpregnant women exhibited 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 (Fig. 7f). This was confirmed with emission spectroscopy experiments (Fig. 8g). However, RET was not observed between FITC-anti-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 exhibit molecular proximity in cells from both pregnant and nonpregnant women.
|
|
|
|
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 µM 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 µg/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 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 µg/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. 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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, 41, 42, 43, 44, 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 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 enzyme-enzyme 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-6-phosphate 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/6-PGDase complex transport in pregnancy neutrophils. If this normal 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.
|
Footnotes |
|---|
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: hpetty{at}umich.edu
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.
Received for publication December 15, 2003. Accepted for publication March 12, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RIIIB to CR3 proximity on neutrophils. J. Immunol. 164:953. and interleukin-6-mediated cell activation. J. Immunol. 163:4367.-Tubulin complexes: size does matter. Trends Cell Biol. 9:339.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. D. Lippolis Immunological signaling networks: Integrating the body's immune response J Anim Sci, April 1, 2008; 86(14_suppl): E53 - E63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Burvenich, D. D. Bannerman, J. D. Lippolis, L. Peelman, B. J. Nonnecke, M. E. Kehrli Jr., and M. J. Paape Cumulative Physiological Events Influence the Inflammatory Response of the Bovine Udder to Escherichia coli Infections During the Transition Period J Dairy Sci, June 1, 2007; 90(13_suppl): E39 - E54. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Adachi, A. L. Kindzelskii, A. R. Petty, J.-B. Huang, N. Maeda, S. Yotsumoto, Y. Aratani, N. Ohno, and H. R. Petty IFN-{gamma} Primes RAW264 Macrophages and Human Monocytes for Enhanced Oxidant Production in Response to CpG DNA via Metabolic Signaling: Roles of TLR9 and Myeloperoxidase Trafficking. J. Immunol., April 15, 2006; 176(8): 5033 - 5040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. R. Petty, A. L. Kindzelskii, J. Espinoza, and R. Romero Trophoblast contact deactivates human neutrophils. J. Immunol., March 1, 2006; 176(5): 3205 - 3214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Abrahams, I. Visintin, P. B. Aldo, S. Guller, R. Romero, and G. Mor A Role for TLRs in the Regulation of Immune Cell Migration by First Trimester Trophoblast Cells J. Immunol., December 15, 2005; 175(12): 8096 - 8104. [Abstract] [Full Text] [PDF]
|
|
