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Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin
Cellular Immunology 218 (2002) 95–106 www.academicpress.com
Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin Ji-Biao Huang, Andrei L. Kindzelskii, and Howard R. Petty* Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA Received 18 July 2002; accepted 8 October 2002
Abstract Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophilÕs cytosol to its periphery in response to Nformyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 106 to 109 M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chemotactic factor; Metabolism; Glycolytic enzymes
1. Introduction The accumulation of activated neutrophils at an inflammatory site is a multifaceted process. Neutrophils must first bind to then traverse the vascular endothelium into the subjacent extracellular matrix. After chemotaxis through interstitial tissues, neutrophils arrive at inflammatory sites where they participate in host defense. A variety of cellular events contribute to this process including adherence, actin reorganization, chemotaxis, degranulation, phagocytosis, reactive oxygen and nitrogen metabolite production, a complex array of signaling events, and others [1–3]. One feature common to all of these events is that they require large amounts of energy. Since neutrophils rely primarily upon glycolysis for energy production [4], glucose transport is an
*
Corresponding author. Fax: 1-313-577-9008. E-mail address: [email protected] (H.R. Petty).
important contributor to host defense mechanisms. Glucose is required for activation of the hexose monophosphate shunt and superoxide production [5–7]. It also appears to be required for the phagocytosis of certain targets and enhances the phagocytosis of others (e.g., [8]). The rate of facilitated glucose transport is dramatically increased by cell stimulation with fMLP, phorbol 12-myristate 13-acetate (PMA), cytokines, and other activating agents (e.g., [9–13]). Glucose transport is the rate-limiting step in cell metabolism [14]. Studies have suggested that the increase in glucose transport rate is due to the coupling of glucose transport with hexokinase activity [9]. Hexokinase (ATP:D -hexose 6phosphotransferase, EC 2.7.1.1) is found throughout the eukaryotic kingdom and is common among prokaryotes as well [15]. It catalyzes the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, which is required for its entry into glycolysis or the hexose monophosphate shunt via glucose-6-phosphate dehydrogenase. Due to its association with the glucose
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transporter, net glucose uptake is increased; for example, hexokinase association with the glucose transporter eliminates futile cycles (wherein glucose is transported out of the cell via the glucose transporter). Recent studies have shown that hexokinase is translocated into the plasma membraneÕs vicinity during macrophage activation with PMA [16]. Thus, hexokinase translocation may be mechanistically responsible for the increase in glucose influx [10]. Hexokinase translocation is dependent upon an intact cytoskeleton [16]. Indeed, a variety of metabolic enzymes including hexokinase, aldolase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase interact with microfilaments and/or microtubules (e.g., [17–20]). These enzymes are particularly important in cell membrane functions since glycolysis is the preferential energy source for membrane activities (e.g., [21,22]). Moreover, the ability of glycolytic enzymes to be shuttled about within a cell has led to the concept of microcompartmentalization of cell metabolism [19,23]. Microcompartmentalization, in turn, apparently leads to the localized glycolytic production of ATP beneath the plasma membrane [24,25]. Furthermore, we have recently shown that NAD(P)H production is not uniform throughout the neutrophilÕs cytoplasm; NAD(P)H flows throughout a cell as traveling longitudinal or spherical waves that respond to receptor ligation and mediate periodic, localized superoxide release [26–28]. The asymmetry in NAD(P)H flux in response to asymmetric fMLP stimulation (nominally a ‘‘gradient’’) suggested that a mechanism must exist to preferentially activate metabolism at specific sites within a neutrophil. Thus, the changes in metabolic waves observed during neutrophil activation [27] might be explained by metabolic microcompartmentalization, especially hexokinase trafficking to specific membrane sites. In the present study we test the hypothesis that hexokinase translocation accompanies neutrophil activation. Here we report that hexokinase translocation is triggered by a broad variety of activating stimuli under physiologically relevant conditions and that focal accumulation of hexokinase is observed during exposure to an fMLP gradient or latex beads. Complementary support for this hypothesis is also provided by the observation that anti-inflammatory drugs block hexokinase translocation, with parallel changes in ROM production.
2. Methods 2.1. Reagents and antibodies Cytochalasin D, indomethacin, dexamethasone, LY83583, Boc-PLPLP, fMLP, leukotriene B4 (LTB4 ),
lipopolysaccharide (LPS), platelet-activating factor (PAF), PMA, and Brij 58 were obtained from Sigma Chemical (St. Louis, MO). IL-8 and IFN-c were purchased from R & D Systems (Minneapolis, MN). Rabbit anti-BSA and BSA were purchased from Sigma for preparation of immune complexes. Individually packaged aliquots of fluorescein isothiocyanate (FITC) and rhodamine–phalloidin were obtained from Molecular Probes (Eugene, OR). Two lm diameter carboxylate microspheres were obtained from Polysciences (Warrington, PA). Dithiobis(sulfosuccinimidylpropionate) (DSP) was obtained from Pierce Biotechnology (Rockford, IL). Rabbit anti-hexokinase, anti-glucose-6-phosphate dehydrogenase (G-6-PDase), and anti-pyruvate kinase polyclonal antibodies and a goat anti-lactate dehydrogenase polyclonal antibody were obtained from Chemicon International (Temecula, CA). The antiphosphofructokinase antibody was the generous gift of Dr. R. Kemp. FITC-conjugated antibodies were prepared as previously described [29]. 2.2. Cell preparation Neutrophils were isolated from peripheral blood using Ficoll–Hypaque (Sigma) density gradient centrifugation at 300g for 30 min at room temperature [29]. The viability of isolated neutrophils was always >95% as assessed by trypan blue exclusion. Cells were suspended in HBSS (Life Technologies, Grand Island, NY). 2.3. Immunofluorescence staining Neutrophils were seeded onto glass coverslips and then incubated at 37 °C for 1 h with or without various agents or beads. Cells were fixed with 2% paraformaldehyde, permeabilized with 1% Brij, and then fixed with 2% paraformaldehyde at room temperature for 20 min. After washing with HBSS, cells were labeled with appropriately titered FITC-conjugated antibodies at 4 °C for 30 min and then washed five times with HBSS at room temperature with constant shaking for 20 min. Experiments were also performed to ascertain the co-localization of hexokinase and F-actin. However, due to the labile nature of microfilaments, a modification of the fixation protocol of Safiejko-Mroczka and Bell [30] was performed. Cells were first fixed with 1 mM DSP in HBSS at 37 °C for 10 min. Cells were permeabilized with 1% Brij containing 1 mM DSP at room temperature for 20 min and then fixed with 4% paraformaldehyde at 37 °C for 15 min. After washing with PBS, cells were labeled simultaneously with FITC-conjugated anti-hexokinase antibodies and rhodamine–phalloidin (2 lM) at room temperature for 30 min [30].
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2.4. Microscopic assay for ROM release from adherent cells Pericellular release of ROMs from single cells was monitored as described [31]. Briefly, a 2% gelatin mixture containing 100 ng/ml dihydrotetramethylrosamine (H2 TMRos) (Molecular Probes) was warmed to 45 °C. The fluid-phase gelatin was placed on a coverslip (at room temperature) with adherent neutrophils. The slide/ gelatin sandwich was then maintained at 37 °C on a temperature controlled microscope stage (Carl Zeiss, New York). ROMs released by the cells diffused into and were trapped within the gelatin matrix, where they oxidized the H2 TMRos to tetramethylrosamine (TMRos). TMRos fluorescence was excited at 540 nm and its emission was detected using a 590DF30 filter with a 560 long-pass dichroic mirror. The fluorescence intensity was measured and analyzed using a photomultiplier tube (Hamamastu; Bridgewater, NJ), housed in a model D104 fluorescence microscope detection system interfaced with a pentium III computer running FeliX software (Photon Technology International). 2.5. Fluorescence microscopy An axiovert-inverted fluorescence microscope with HBO-100 mercury illumination (Carl Zeiss, New York, NY) interfaced to a Dell 410 workstation via Scion SG-7 video card (Vay Tek, Fairfield, IA) was used. The fluorescence images were collected by an intensified chargecoupled device camera, model XC-77 (Hamamatsu, Hamamatsu City, Japan) and processed with ScionImage Software. A narrow bandpass-discriminating filter set with excitation at 485DF20 nm, emission at 530DF30 nm and a long-pass dichroic mirror at 510 nm was used [29].
3. Results 3.1. Hexokinase translocates to the cell periphery during fMLP stimulation In the present study we test the hypothesis that hexokinase is recruited into the vicinity of the neutrophilÕs plasma membrane in response to activating stimuli and phagocytosis. In the first series of experiments we examined the effect of the chemotactic factor fMLP on the intracellular distribution of hexokinase. Adherent neutrophils were incubated with or without 1 lM fMLP, a formyl peptide receptor agonist, at 37 °C for 1 h. In a parallel series of experiments cells were incubated in the presence or absence of 1 lM Boc-PLPLP, a formyl peptide receptor antagonist, at 37 °C for 1 h. Cells were fixed as described above then labeled with FITC-conjugated anti-hexokinase. Since neutrophils are small
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cells, the hexokinase distribution was characterized as either random or peripheral for these experiments with small activating stimuli. As shown in Fig. 1A, hexokinase was randomly distributed in unstimulated neutrophils. Similarly, the intracellular distributions of hexokinase did not change during exposure to BocPLPLP (Fig. 1B). However, incubation with fMLP dramatically effects the intracellular distribution of hexokinase (Fig. 1C). For fMPL-mediated activation, 100% of the cells displayed a peripheral distribution of hexokinase as shown in Fig. 1C. Thus, hexokinase is translocated from throughout the cytoplasm to the periphery of the cell in response to formyl peptide receptor agonists, but not antagonists. Recent studies have identified the presence of both high (Kd nM) and low (Kd lM) affinity receptors for fMLP [32]. Therefore, we examined the effect of other concentrations of fMLP on hexokinase translocation. Experiments were performed as described in the preceding paragraph. Fig. 2 shows that 109 M fMLP effectively promotes hexokinase translocation to the cell periphery, although lower concentrations such as 1011 M do not. Hence, hexokinase translocation is observed in response to a broad range of fMLP concentrations (106 –109 M). 3.2. Specificity of hexokinase translocation during neutrophil activation To determine if additional enzymes associated with glucose metabolism undergo translocation during neutrophil activation, we performed a series of immunofluorescence microscopy studies. Neutrophils were untreated or treated with Boc-PLPLP or fMLP as described above. Cells were fixed then stained with FITC conjugates of anti-G-6-PDase (Figs. 1D–F), anti-phosphofructokinase (Figs. 1G–I), anti-pyruvate kinase (Figs. 1J–L), and anti-lactate dehydrogenase (Figs. 1M– O). During all experimental conditions G-6-PDase was located in the vicinity of the plasma membrane. Inasmuch as G-6-PDase is the rate-controlling step of the hexose monophosphate shunt and provides NADPH for the synthesis of superoxide anions, it localization near the plasma membrane is not surprising. Although BocPLPLP had no effect upon the distribution of phosphofructokinase and pyruvate kinase, some punctate accumulation of phosphofructokinase was noted in the presence of fMLP on polarized, but not spherical cells (Figs. 1I and L). Thus, cell activation results in dramatic changes in hexokinase distribution, which are not found for several other enzymes participating in glucose metabolism. 3.3. Kinetics of hexokinase translocation We next examined the time-dependence of hexokinase translocation in response to fMLP. Neutrophils
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Fig. 1. Fluorescence microscopic localization of hexokinase in human neutrophils. Cells were incubated with buffer alone (unstimulated) (A, D, G, J, and M), Boc-PLPLP (1 lM) (B, E, H, K, and N), or fMLP (1 lM) (C, F, I, L, and O) at 37 °C for 1 h then fixed. Samples were labeled with FITC-conjugated anti-hexokinase (A–C), glucose-6-phosphate dehydrogenase (D–F), anti-phosphofructokinase (G–I), pyruvate kinase (J–L), and anti-lactate dehydrogenase (M–O) antibodies as described in Section 2. Three to five individual experiments were performed and representative fluorescence images are shown. Hexokinase translocates to the cell periphery during fMLP stimulation (627).
were treated with various doses of fMLP for 1, 5, 10, and 20 min. Cells were then fixed and stained with FITC-conjugated anti-hexokinase. Fig. 3 shows representative examples of neutrophils treated with 109 M fMLP. Extensive clumping and translocation of hexokinase is evident after 5 min of incubation. Similar ob-
servations were made at 106 M fMLP (data not shown). Thus, a finite amount of time is required for hexokinase redistribution during neutrophil activation. Furthermore, this time frame is not surprising since, in the absence of extensive mixing, about 3 min is required for diffusion of an activating substance to a cell.
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Fig. 2. Hexokinase translocation at various doses of fMLP. Neutrophils were incubated with or without fMLP (106 –1012 M) for 1 h at 37 °C followed by fixation and labeling with anti-hexokinase antibody. As panels A–D show, 1 lM–nM fMLP was sufficient to stimulate extensive translocation and clumping of hexokinase. Significant translocation was not observed at 1010 , 1011 , and 1012 M (panels E–G, respectively) and in the absence of fMLP (panel H), although some clumping may be detected in the micrographs (n ¼ 3) (867).
Fig. 3. Kinetics of hexokinase translocation. Neutrophils were incubated with 1 nM fMLP for various periods of time at 37 °C followed by fixation and labeling with anti-hexokinase antibody. Although translocation was not evident at 1 min (A), extensive translocation and clumping in the vicinity of the plasma membrane was found at 5 and 10 min of incubation (panels B and C, respectively), which persisted for long periods of time. Hexokinase translocation was not observed in the absence of fMLP (panels D–F, respectively) after corresponding incubation times (n ¼ 4) (956).
3.4. Effect of a chemotactic gradient on hexokinase localization
3.5. Hexokinase translocates to the neutrophil periphery in response to a variety of activating stimuli
Inasmuch as fMLP is a chemotactic factor, we examined the distribution of hexokinase when cells were exposed to a gradient of fMLP. An fMLP gradient was formed by placing 15 ll of a 5 lM solution of fMLP at one end of a coverslip. After 10 min at 37 °C to allow time for cell polarization, neutrophils were fixed and stained. As shown in Fig. 4, hexokinase accumulates at the leading edge of neutrophils exposed to an fMLP gradient. Thus, hexokinase is translocated to the periphery of the cell during exposure to uniformly applied fMLP and specifically to the lamellipodium when the stimulant is applied in the form of a gradient.
We next sought to determine if hexokinase translocation was a general property of neutrophil activation. Neutrophils were treated at 37 °C with the activating/ priming stimuli: IFN-c (50 U/ml for 2 h), IL-8 (50 ng/ml for 45 min), LTB4 (1 lg=ml for 1 h), PMA (5 nM, 1 h), immune complexes (10 lg=ml for 1 h), LPS (50 ng/ml for 20 min), and PAF (100 nM for 30 min). The conditions and concentrations of these reagents were chosen based upon prior findings [27]. Representative photomicrographs are shown in Fig. 5. In all cases substantial translocation into the vicinity of the plasma membrane was noted for hexokinase, although this was not ob-
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Fig. 4. Hexokinase accumulates at the lamellipodium of neutrophils exposed to a gradient of fMLP. Panels A and C show bright-field micrographs to illustrate the orientation of the cells whereas panel B and D show immunofluorescence microscopy of hexokinase. In panels A and B, cells were exposed to an fMLP gradient by placing 15 ll of an fMLP solution (5 lM) at one side of a cover glass on a microscope slide. This was incubated at 37 °C for 10 min followed by fixation and labeling with FITC-conjugated anti-hexokinase antibody. The white arrow points in the direction of decreasing fMLP concentration. In panels C and D, cells were incubated without fMLP to serve as a negative control. Four individual experiments were performed and the typical DIC and fluorescence images are shown (815).
served for lactate dehydrogenase. Therefore, hexokinase translocation is not a specific property of fMLPtreated neutrophils, but rather a more general feature of molecules promoting the priming or activation of these cells. 3.6. Hexokinase translocates into the vicinity of captured beads in neutrophils Since phagocytosis is accompanied by significant changes in cell metabolism and organization, we investigated the intracellular distribution of hexokinase following incubation with target particles. Latex beads were chosen for these microscopic studies because they have a very distinctive appearance and therefore could be unambiguously correlated with the site of hexokinase accumulation. Cells were incubated with 2 lm diameter unopsonized latex beads (0.5 ll of the stock bead solution added to 100 ll of cell suspension) at 37 °C for 1 h followed by fixation and labeling with FITC-conjugated anti-hexokinase or FITC-conjugated anti-lactate dehydrogenase antibody. As shown in Figs. 6A–C, hexokinase accumulated in the vicinity of captured beads. The redistribution of hexokinase is noticeable by comparing the DIC and the fluorescence micrographs in panels A and B. In contrast to the peripheral distribution of hexokinase found for fMLP treatment, after incubation with latex beads hexokinase was found in (1) the cyto-
plasm, (2) near the plasma membrane, and (3) in the vicinity of the target. In addition, quantitative line profile analyses also support this conclusion (Fig. 6C). However, no significant changes in lactate dehydrogenase distribution were noted (Fig. 6D–F). To provide further quantitative data to sort out these labeling differences, digital images were analyzed using software that calculates the mean fluorescence intensity of an adjustable pre-defined area in the micrographs. In this way the fluorescence intensity surrounding the target(s) and that of the entire cell were determined. When latex beads and neutrophils were incubated together for 10 min, very little hexokinase accumulated at the bead(s) (Fig. 7). However, roughly 25% of the total hexokinase accumulated at the bead(s) at 1 h (Fig. 7). Thus, hexokinase traffics to the site of target binding and uptake during phagocytosis. Since exposure to beads and to fMLP led to hexokinase redistribution in neutrophils, we examined the effect of simultaneous exposure to both of these stimuli. Adherent neutrophils were incubated simultaneously with 1 lM fMLP and latex beads at 37 °C as described above. Cells were fixed and stained with FITC-conjugated anti-hexokinase. At short co-incubation times (10 min), fMLP promoted hexokinase accumulation near the beads (Fig. 7). Furthermore, double labeling experiments indicated that hexokinase and F-actin accumulated at the site of phagocytosis (Fig. 8), which is consistent with the potential role of microfilaments. When cells were incubated in the presence of fMLP and beads for 1 h a peripheral, not a target-associated, hexokinase distribution was observed. Similarly, F-actin is lost from the phagosome after 1 h as well (data not shown and [33]). Thus, hexokinase trafficking to latex beads is faster in the presence of fMLP which is followed by loss of hexokinase and F-actin from the vicinity of targets. No changes in lactate dehydrogenase distribution were noted (data not shown). 3.7. Inhibitory effects of cytochalasin D and anti-inflammatory agents on hexokinase translocation We next sought to explore potential mechanisms contributing to hexokinase translocation in neutrophils. Since hexokinase translocation accompanies cytoskeletal remodeling, we investigated the effects of colchicine and cytochalasin D, inhibitors of microtubules and microfilaments, respectively, on hexokinase distribution. Neutrophils were pre-treated with 0.3 lg=ml colchicine for 30 min at 37 °C then incubated with 1 lM fMLP for another 1 h. As Fig. 9B shows, hexokinase was translocated to the periphery of neutrophils after treatment with both colchicine and fMLP. This suggests that microtubules are not responsible for hexokinase trafficking in neutrophils. As shown in Fig. 9C, cytochalasin D (10 lg=ml) prevented the translocation of hexokinase to
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Fig. 5. Representative fluorescence studies showing that hexokinase translocates to the neutrophil periphery in response to a variety of activating stimuli. Neutrophils were incubated at 37 °C for 1 h in the absence of stimuli (A and B) or treated with IFN-c (50 U/ml) at 37 °C for 2 h (C and D), IL8 (50 ng/ml) at 37 °C for 45 min (E and F), LTB4 (1 lg=ml) at 37 °C for 1 h (G and H), PMA (5 nM) at 37 °C for 1 h (I and J), LPS (50 ng/ml) at 37 °C for 20 min (K and L), immune complexes (10 lg=ml) at 37 °C for 1 h (M and N) as well as PAF (100 nM) at 37 °C for 30 min (O and P). After treatment, cells were labeled with FITC-conjugated anti-hexokinase antibody (A, C, E, G, I, K, M, and O) or FITC-conjugated anti-lactate dehydrogenase antibody (B, D, F, H, J, L, N, and P). At least three separate repetitions for each of the experiments shown were performed (587).
the cell periphery in the presence of 1 lM fMLP. Thus, hexokinase trafficking appears to depend upon microfilaments, but not microtubules. Inasmuch as activating stimuli promoted hexokinase redistribution, we next examined the effect of anti-inflammatory agents. In the first series of experiments we tested the effect of dexamethasone on hexokinase translocation in neutrophils. Cells were incubated with 2 lM dexamethasone for 45 min at 37 °C followed by the addition of 1 lM fMLP for 1 h. Dexamethasone inhibited the accumulation of hexokinase at the cell periphery (Fig. 9E). This result is not surprising since previous workers have shown that dexamethasone blocks the coupling of hexokinase with the glucose transporter [34]. We next examined the effect of indomethacin, a cyclooxygenase inhibitor that blocks PGH synthesis in
response to inflammatory stimuli. It has been reported to diminish neutrophil oxidative metabolism (e.g., [35]). Cells were incubated with 5 lM indomethacin and 1 lM fMLP for 1 h at 37 °C. Our observations show that indomethacin effectively inhibited hexokinase translocation in response to fMLP in neutrophils (Fig. 9). An important difference between the effects of cytochalasin D and indomethacin is that neutrophils could polarize in the presence of indomethacin (Fig. 9D), but not in the presence of the cytoskeletal-disrupting drug cytochalasin D where the cells are spherical (Fig. 9C). Thus, two different but widely prescribed drugs, dexamethasone and indomethacin, share a downstream mechanistic step—the inhibition of hexokinase translocation to the cell periphery and its accompanying up-regulation of cell metabolism.
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Fig. 6. Hexokinase is recruited to the vicinity of captured beads within neutrophils. Cells were incubated with 2 lm diameter nonopsonized latex beads at 37 °C for 1 h and labeled with FITC-conjugated anti-hexokinase (A, B, and C) or FITC-conjugated anti-lactate dehydrogenase (D, E, and F) antibodies. DIC micrographs (A and D), fluorescence images (B and E), and line profile analyses of fluorescence images (C and F) are shown. The locations of the spherical latex beads are shown in panels A and D with white arrows. Panel B shows the accumulation of hexokinase in the region of the latex beads in these two cells, although no such changes are noted for lactate dehydrogenase (panel E). The line profile analyses (fluorescence intensity vs. pixel number) of panels C and F correspond to the white lines drawn through the micrographs of panels B and E, respectively. Both the enrichment in hexokinase and the location of the beads (see black arrows) are apparent in the line profile analyses in panel C. These features were not observed for lactate dehydrogenase (F). At least three individual experiments were performed for each of these studies (1680).
3.8. Superoxide production by adherent neutrophils We next sought to confirm the physiological relevance and extend our findings by measuring the production of ROMs. ROM release into the extracellular environment was detected using H2 -TMRos, a nonfluorescent molecule that becomes fluorescent upon exposure to ROMs. The time-dependence of ROM release was assessed on a single cell basis at 37 °C as previously described [31]. Low levels of ROMs were detected for adherent neutrophils (Fig. 10, trace 3), as previously described [31,36]. As anticipated, treatment with 50 nM fMLP substantially increased the rate of ROM production (Fig. 10, trace 1). Furthermore, pre-treatment with indomethacin or dexamethasone, as described above, reduced ROM release to baseline levels associated with adherent, polarized neutrophils (Fig. 10, traces 4 and 5). Thus, ROM production parallels the translocation of hexokinase to the cell periphery. We next evaluated the possibility that hexokinase translocation was required for enhanced ROM production. The reagent LY83583 was employed. It has been found to dramatically increase glucose transport into cells [37]. Cells were incubated with LY83583 (1 lg=ml) for 15 min at room temperature followed by
microscopy at 37 °C. No change in the intracellular location of hexokinase was observed using the protocols given above (data not shown). However, the rate of ROM release was increased to levels comparable with that of fMLP treatment (Fig. 10, trace 2). Indomethacin was unable to reverse the effect of LY83583 on ROM production (data not shown), in contrast to its effect on fMLP-mediated stimulation (see above). Although LY83583 has multiple effects on cells as do many other drugs, this reagent: (1) provides evidence consistent with a role of glucose transport in cell activation and (2) suggests that hexokinase translocation is not always required for high levels of ROM production, but accompanies all of the tested physiological stimuli (Fig. 5).
4. Discussion The translocation of kinases, such as protein kinase C, to the plasma membrane is well known in leukocytes. In the present study we have characterized the translocation of another kinase, hexokinase—a key enzyme in the control of cell metabolism, from the cytoplasm to the peri-membrane region during neutrophil activation. This effect is specific for productive receptor occupancy
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Fig. 7. Quantitative analyses of hexokinase translocation. The percentage of hexokinase translocated to the vicinity of cell-associated latex beads are shown. The effects of incubation time and fMLP addition on hexokinase accumulation are illustrated. Cells were incubated with 2 lm diameter nonopsonized latex beads at 37 °C for 10 min or 1 h in the presence or absence of fMLP then labeled with FITCconjugated anti-hexokinase. Neutrophil activation with fMLP accelerates the delivery of hexokinase to the vicinity of latex beads. At least three individual experiments were performed. *P < 0:001.
Fig. 9. Hexokinase trafficking is affected by the cytoskeleton and antiinflammatory compounds. Cells incubated with fMLP at 37 °C for 1 h (1 lM) (A) showed hexokinase clumps in the vicinity of the plasma membrane. Peripheral labeling was also observed after pretreatment with colchicine (300 ng/ml) at 37 °C for 30 min followed by addition of fMLP (1 lM) for 1 h at 37 °C (B). However, exposure to cytochalasin D (5 lg=ml) (panel C) or dexamethasone (2 lM) (panel E) at 37 °C for 1 h followed by incubation with fMLP (1 lM) at 37 °C for 1 h led to essentially random labeling with anti-hexokinase antibody. Similarly, indomethacin (5 lM) treatment (panel D) at 37 °C for 1 h with fMLP led to no significant changes in hexokinase distribution. At least three individual experiments were performed for each of the studies shown (627).
Fig. 8. Colocalization of F-actin and hexokinase in the vicinity of neutrophil-associated beads. Cells were incubated with 2 mm diameter unopsonized latex beads and fMLP (1 mM) at 37 °C for 10 min then labeled with FITC-conjugated anti-hexokinase (C and F) and rhodamine-conjugated phalloidin (B and E). DIC micrographs (A and D) and fluorescence images (B, C, E, and F) are shown. The locations of the spherical latex beads are shown in panels A and D with white arrows. Panels B and E show the accumulation of F-actin in the region of the latex beads in these two cells. The location of hexokinase labeling is illustrated in panels C and F. Note the co-localization of these two labels. At least three individual experiments were performed for each of these studies. (600 for panels A–C, 1680 for panels D–F.)
since the formyl peptide receptor antagonist Boc-PLPLP did not have an effect on cells. This was not a generalized redistribution of metabolic enzymes because cell activation had no effect on other enzymes such as lactate dehydrogenase. Naftalin and co-workers [16] first re-
ported hexokinase translocation in rat macrophages in response to PMA. This observation seems to have been largely overlooked, presumably in the mistaken belief that biochemistry is homogeneously distributed within cells. Not only can metabolic reactions be sequestered in specific organelles, but they can be localized to specific regions of the cytoplasm or membranes. Theoretical physics has predicted that metabolism is not uniformly distributed in cells and the cytoskeleton could be a factor contributing to chemical instabilities within cells (e.g., [38,39]). Recent studies from this laboratory have shown that metabolism is not spatially or temporally uniform within living adherent or polarized neutrophils; NAD(P)H production is observed as spherical or nominally longitudinal waves traveling with specific directions, velocities, and shapes [26–28]. We therefore hypothesized that: (1) hexokinase translocation to the plasma membrane or regions thereof constituted a
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Fig. 10. Rates of ROM release by neutrophils. ROM release was measured during cell migration through a gelatin matrix containing H2 -TMRos, as described [39]. High rates of ROM production are observed during incubation with 50 nM fMLP (trace 1) and 1 lg=ml LY83583 (trace 2). However, adherent cells produce lower levels of ROMs in the absence of these reagents (trace 3). Enhanced ROM production is blocked by incubation with 2 lM dexamethasone (trace 4) and 5 lM indomethacin (trace 5).
spatial chemical change leading to alterations in metabolic wave properties and (2) hexokinase translocation is an important aspect of neutrophil activation. We have shown that hexokinase is translocated to the periphery of normal adherent human neutrophils in response to fMLP. Hexokinase translocation was observed over a range of fMLP concentrations from 106 to 109 M, which is consistent with the physiologically relevant concentrations associated with receptor activation. Hexokinase underwent extensive translocation in less than 5 min. This observation is consistent with the facts that about 3 min is required for fMLP diffusion to the cell in this unstirred system and another study has shown that the maximal rate of superoxide production is only achieved at about 1.5 min [40]. Although fMLP stimulation of stirred cells in suspension begins with seconds, this is not directly comparable with our work because: (1) our samples were unstirred and (2) our cells were adherent, not in suspension. As Nathan [41] has shown, adherent neutrophils display properties distinct from suspended cells; for example, the kinetics of activation differs and oxidant release is enhanced in adherent neutrophils. The potential physiological significance of these findings in regard to ROM release is also supported by the fact that several pro-inflammatory stimuli share this effect on cells whereas anti-inflammatory drugs have just the opposite effect. We hypothesized that hexokinase translocation may broadly participate in neutrophil activation. Therefore, we tested a variety of stimulatory agents for their effect of hexokinase trafficking. We found that diverse neutrophil-activating reagents, including fMLP, LPS, latex beads, IL-8, PMA, PAF, LTB4 , immune complexes, and IFN-c, all promoted hexokinase translocation at phys-
iologically appropriate doses. Thus, hexokinase translocation appears to be a general property of neutrophil activation, not a specific response to one agent such as fMLP. Hexokinase accumulation was, essentially, in the form of clumps about the cell periphery. In contrast, the asymmetric addition of fMLP to neutrophils results in asymmetric accumulation of hexokinase at a cellÕs lamellipodium. Moreover, phagocytic stimuli lead to hexokinase translocation about the periphery of the target. In both of these cases, hexokinase traffics to the region of membrane perturbation. We previously found that metabolic (NAD(P)H) waves in neutrophils correspond to sites of fMLP binding [27]. Hexokinase accumulation and the consequent increase in glucose transport at these local sites, which are undergoing substantial cytoskeletal re-organization, may account for these earlier findings. Although a variety of molecules may accumulate at sites of phagocytosis, our finding that hexokinase is translocated to the vicinity of captured beads appears to be the first metabolic enzyme noted to behave in this way. As mentioned below, microfilaments appear to be important in hexokinase translocation. Although microfilaments may promote hexokinase trafficking to phagosomes, they may not be entirely responsible for retention since microfilaments surrounding phagosomes disassemble [33] whereas hexokinase translocation is stable for at least an hour. In the presence of both fMLP and beads, hexokinase was observed in the vicinity of beads at 10 min, but not after 1 h. Thus, hexokinase translocation to the vicinity of the target appears to be faster in the presence of fMLP. As might be expected, the trafficking properties of hexokinase depend upon the physiological stimuli. The accumulation of hexokinase near phagosomes raises the interesting question of what biological function could hexokinase serve at a phagosome? One speculative possibility is that it is present to starve the target. The presence of hexokinase would accelerate the removal of glucose from the phagolysosome. Thus, glucose captured from the medium during phagocytosis, released from a target due to the formation of membrane lesions, or glucose formed by the action of phosphatases within the phagolysosome, glycosidases, or other enzymes of the phagolysosome would be removed by the neutrophil. Transport across the targetÕs membrane would be futile in the presence of membrane lesions. Thus, hexokinase trafficking could enhance target killing. Several metabolic enzymes may be associated with cytoskeletal elements. For example, aldolase and phosphofructokinase are associated with microfilaments (e.g., [42,43]). Moreover, hexokinase translocation to the cell periphery is sensitive to cytochalasin D [16], which has been confirmed in this study. Cytoskeletal
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associations may promote the delivery of metabolic machinery to specific sites to meet local metabolic demands [16–24]. Our findings are also consistent with prior experimental studies of oxidant production. Although studies of neutrophils in suspension have indicated that cytochalasin B promotes fMLP-associated superoxide production [44], studies of adherent neutrophils have indicated that cytochalasin D blocks superoxide production [36] during spreading. Recent studies have shown that cytochalasin inhibits postphagocytosis oxidant production in phagosomes [45]. Thus, the microfilament-mediated redistribution of hexokinase may contribute to certain effector activities. To further examine the participation of hexokinase translocation in neutrophil activation, we tested the effect of anti-inflammatory agents on fMLP-mediated stimulation. Our findings appear to be the first to show that indomethacin dramatically inhibits hexokinase translocation to the peri-membrane region of the cell. Thus, inflammatory signals promote hexokinase translocation to the cell periphery whereas the anti-inflammatory drug indomethacin inhibits this translocation. This finding may contribute to a better understanding of the mechanisms of actions of anti-inflammatory drugs. For example, it is well known that indomethacin reduces superoxide production, as well as other effector responses, of stimulated neutrophils (e.g., [35]). It is also well known that indomethacin and other NSAIDs (nonsteroidal anti-inflammatory drugs) inhibit the activity of cyclooxygenases [46], although the links between cyclooxygenase inhibition and diminished effector responses have not been fully characterized. Moreover, cyclooxygenase-independent pathways of NSAIDs have been increasingly recognized in recent years [46]. We speculate that inhibition of hexokinase translocation may contribute to the anti-inflammatory effects of certain drugs by either cyclooxygenase-dependent and/or cyclooxygenase-independent pathways. For example, maximal production of superoxide anions requires a substantial carbon flux through the hexose monophosphate shunt, which would be limited in the absence of up-regulated glucose transport and the availability of glucose-6-phosphate at the level of the plasma membrane. Furthermore, this concept may apply to both indomethacin and dexamethasone. Although the present study has focused on hexokinase trafficking during neutrophil activation, the broad importance of metabolic changes in immunologic and inflammatory processes [47] suggests that the concepts outlined above may be applicable in additional physiological settings.
Acknowledgments This work has been supported by grants from the NIH (AI51789) and the National Multiple Sclerosis
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Society to H.R.P. We thank Dr. T. Mair for a helpful discussion.
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Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin Ji-Biao Huang, Andrei L. Kindzelskii, and Howard R. Petty* Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA Received 18 July 2002; accepted 8 October 2002
Abstract Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophilÕs cytosol to its periphery in response to Nformyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 106 to 109 M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Chemotactic factor; Metabolism; Glycolytic enzymes
1. Introduction The accumulation of activated neutrophils at an inflammatory site is a multifaceted process. Neutrophils must first bind to then traverse the vascular endothelium into the subjacent extracellular matrix. After chemotaxis through interstitial tissues, neutrophils arrive at inflammatory sites where they participate in host defense. A variety of cellular events contribute to this process including adherence, actin reorganization, chemotaxis, degranulation, phagocytosis, reactive oxygen and nitrogen metabolite production, a complex array of signaling events, and others [1–3]. One feature common to all of these events is that they require large amounts of energy. Since neutrophils rely primarily upon glycolysis for energy production [4], glucose transport is an
*
Corresponding author. Fax: 1-313-577-9008. E-mail address: [email protected] (H.R. Petty).
important contributor to host defense mechanisms. Glucose is required for activation of the hexose monophosphate shunt and superoxide production [5–7]. It also appears to be required for the phagocytosis of certain targets and enhances the phagocytosis of others (e.g., [8]). The rate of facilitated glucose transport is dramatically increased by cell stimulation with fMLP, phorbol 12-myristate 13-acetate (PMA), cytokines, and other activating agents (e.g., [9–13]). Glucose transport is the rate-limiting step in cell metabolism [14]. Studies have suggested that the increase in glucose transport rate is due to the coupling of glucose transport with hexokinase activity [9]. Hexokinase (ATP:D -hexose 6phosphotransferase, EC 2.7.1.1) is found throughout the eukaryotic kingdom and is common among prokaryotes as well [15]. It catalyzes the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, which is required for its entry into glycolysis or the hexose monophosphate shunt via glucose-6-phosphate dehydrogenase. Due to its association with the glucose
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transporter, net glucose uptake is increased; for example, hexokinase association with the glucose transporter eliminates futile cycles (wherein glucose is transported out of the cell via the glucose transporter). Recent studies have shown that hexokinase is translocated into the plasma membraneÕs vicinity during macrophage activation with PMA [16]. Thus, hexokinase translocation may be mechanistically responsible for the increase in glucose influx [10]. Hexokinase translocation is dependent upon an intact cytoskeleton [16]. Indeed, a variety of metabolic enzymes including hexokinase, aldolase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase interact with microfilaments and/or microtubules (e.g., [17–20]). These enzymes are particularly important in cell membrane functions since glycolysis is the preferential energy source for membrane activities (e.g., [21,22]). Moreover, the ability of glycolytic enzymes to be shuttled about within a cell has led to the concept of microcompartmentalization of cell metabolism [19,23]. Microcompartmentalization, in turn, apparently leads to the localized glycolytic production of ATP beneath the plasma membrane [24,25]. Furthermore, we have recently shown that NAD(P)H production is not uniform throughout the neutrophilÕs cytoplasm; NAD(P)H flows throughout a cell as traveling longitudinal or spherical waves that respond to receptor ligation and mediate periodic, localized superoxide release [26–28]. The asymmetry in NAD(P)H flux in response to asymmetric fMLP stimulation (nominally a ‘‘gradient’’) suggested that a mechanism must exist to preferentially activate metabolism at specific sites within a neutrophil. Thus, the changes in metabolic waves observed during neutrophil activation [27] might be explained by metabolic microcompartmentalization, especially hexokinase trafficking to specific membrane sites. In the present study we test the hypothesis that hexokinase translocation accompanies neutrophil activation. Here we report that hexokinase translocation is triggered by a broad variety of activating stimuli under physiologically relevant conditions and that focal accumulation of hexokinase is observed during exposure to an fMLP gradient or latex beads. Complementary support for this hypothesis is also provided by the observation that anti-inflammatory drugs block hexokinase translocation, with parallel changes in ROM production.
2. Methods 2.1. Reagents and antibodies Cytochalasin D, indomethacin, dexamethasone, LY83583, Boc-PLPLP, fMLP, leukotriene B4 (LTB4 ),
lipopolysaccharide (LPS), platelet-activating factor (PAF), PMA, and Brij 58 were obtained from Sigma Chemical (St. Louis, MO). IL-8 and IFN-c were purchased from R & D Systems (Minneapolis, MN). Rabbit anti-BSA and BSA were purchased from Sigma for preparation of immune complexes. Individually packaged aliquots of fluorescein isothiocyanate (FITC) and rhodamine–phalloidin were obtained from Molecular Probes (Eugene, OR). Two lm diameter carboxylate microspheres were obtained from Polysciences (Warrington, PA). Dithiobis(sulfosuccinimidylpropionate) (DSP) was obtained from Pierce Biotechnology (Rockford, IL). Rabbit anti-hexokinase, anti-glucose-6-phosphate dehydrogenase (G-6-PDase), and anti-pyruvate kinase polyclonal antibodies and a goat anti-lactate dehydrogenase polyclonal antibody were obtained from Chemicon International (Temecula, CA). The antiphosphofructokinase antibody was the generous gift of Dr. R. Kemp. FITC-conjugated antibodies were prepared as previously described [29]. 2.2. Cell preparation Neutrophils were isolated from peripheral blood using Ficoll–Hypaque (Sigma) density gradient centrifugation at 300g for 30 min at room temperature [29]. The viability of isolated neutrophils was always >95% as assessed by trypan blue exclusion. Cells were suspended in HBSS (Life Technologies, Grand Island, NY). 2.3. Immunofluorescence staining Neutrophils were seeded onto glass coverslips and then incubated at 37 °C for 1 h with or without various agents or beads. Cells were fixed with 2% paraformaldehyde, permeabilized with 1% Brij, and then fixed with 2% paraformaldehyde at room temperature for 20 min. After washing with HBSS, cells were labeled with appropriately titered FITC-conjugated antibodies at 4 °C for 30 min and then washed five times with HBSS at room temperature with constant shaking for 20 min. Experiments were also performed to ascertain the co-localization of hexokinase and F-actin. However, due to the labile nature of microfilaments, a modification of the fixation protocol of Safiejko-Mroczka and Bell [30] was performed. Cells were first fixed with 1 mM DSP in HBSS at 37 °C for 10 min. Cells were permeabilized with 1% Brij containing 1 mM DSP at room temperature for 20 min and then fixed with 4% paraformaldehyde at 37 °C for 15 min. After washing with PBS, cells were labeled simultaneously with FITC-conjugated anti-hexokinase antibodies and rhodamine–phalloidin (2 lM) at room temperature for 30 min [30].
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2.4. Microscopic assay for ROM release from adherent cells Pericellular release of ROMs from single cells was monitored as described [31]. Briefly, a 2% gelatin mixture containing 100 ng/ml dihydrotetramethylrosamine (H2 TMRos) (Molecular Probes) was warmed to 45 °C. The fluid-phase gelatin was placed on a coverslip (at room temperature) with adherent neutrophils. The slide/ gelatin sandwich was then maintained at 37 °C on a temperature controlled microscope stage (Carl Zeiss, New York). ROMs released by the cells diffused into and were trapped within the gelatin matrix, where they oxidized the H2 TMRos to tetramethylrosamine (TMRos). TMRos fluorescence was excited at 540 nm and its emission was detected using a 590DF30 filter with a 560 long-pass dichroic mirror. The fluorescence intensity was measured and analyzed using a photomultiplier tube (Hamamastu; Bridgewater, NJ), housed in a model D104 fluorescence microscope detection system interfaced with a pentium III computer running FeliX software (Photon Technology International). 2.5. Fluorescence microscopy An axiovert-inverted fluorescence microscope with HBO-100 mercury illumination (Carl Zeiss, New York, NY) interfaced to a Dell 410 workstation via Scion SG-7 video card (Vay Tek, Fairfield, IA) was used. The fluorescence images were collected by an intensified chargecoupled device camera, model XC-77 (Hamamatsu, Hamamatsu City, Japan) and processed with ScionImage Software. A narrow bandpass-discriminating filter set with excitation at 485DF20 nm, emission at 530DF30 nm and a long-pass dichroic mirror at 510 nm was used [29].
3. Results 3.1. Hexokinase translocates to the cell periphery during fMLP stimulation In the present study we test the hypothesis that hexokinase is recruited into the vicinity of the neutrophilÕs plasma membrane in response to activating stimuli and phagocytosis. In the first series of experiments we examined the effect of the chemotactic factor fMLP on the intracellular distribution of hexokinase. Adherent neutrophils were incubated with or without 1 lM fMLP, a formyl peptide receptor agonist, at 37 °C for 1 h. In a parallel series of experiments cells were incubated in the presence or absence of 1 lM Boc-PLPLP, a formyl peptide receptor antagonist, at 37 °C for 1 h. Cells were fixed as described above then labeled with FITC-conjugated anti-hexokinase. Since neutrophils are small
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cells, the hexokinase distribution was characterized as either random or peripheral for these experiments with small activating stimuli. As shown in Fig. 1A, hexokinase was randomly distributed in unstimulated neutrophils. Similarly, the intracellular distributions of hexokinase did not change during exposure to BocPLPLP (Fig. 1B). However, incubation with fMLP dramatically effects the intracellular distribution of hexokinase (Fig. 1C). For fMPL-mediated activation, 100% of the cells displayed a peripheral distribution of hexokinase as shown in Fig. 1C. Thus, hexokinase is translocated from throughout the cytoplasm to the periphery of the cell in response to formyl peptide receptor agonists, but not antagonists. Recent studies have identified the presence of both high (Kd nM) and low (Kd lM) affinity receptors for fMLP [32]. Therefore, we examined the effect of other concentrations of fMLP on hexokinase translocation. Experiments were performed as described in the preceding paragraph. Fig. 2 shows that 109 M fMLP effectively promotes hexokinase translocation to the cell periphery, although lower concentrations such as 1011 M do not. Hence, hexokinase translocation is observed in response to a broad range of fMLP concentrations (106 –109 M). 3.2. Specificity of hexokinase translocation during neutrophil activation To determine if additional enzymes associated with glucose metabolism undergo translocation during neutrophil activation, we performed a series of immunofluorescence microscopy studies. Neutrophils were untreated or treated with Boc-PLPLP or fMLP as described above. Cells were fixed then stained with FITC conjugates of anti-G-6-PDase (Figs. 1D–F), anti-phosphofructokinase (Figs. 1G–I), anti-pyruvate kinase (Figs. 1J–L), and anti-lactate dehydrogenase (Figs. 1M– O). During all experimental conditions G-6-PDase was located in the vicinity of the plasma membrane. Inasmuch as G-6-PDase is the rate-controlling step of the hexose monophosphate shunt and provides NADPH for the synthesis of superoxide anions, it localization near the plasma membrane is not surprising. Although BocPLPLP had no effect upon the distribution of phosphofructokinase and pyruvate kinase, some punctate accumulation of phosphofructokinase was noted in the presence of fMLP on polarized, but not spherical cells (Figs. 1I and L). Thus, cell activation results in dramatic changes in hexokinase distribution, which are not found for several other enzymes participating in glucose metabolism. 3.3. Kinetics of hexokinase translocation We next examined the time-dependence of hexokinase translocation in response to fMLP. Neutrophils
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Fig. 1. Fluorescence microscopic localization of hexokinase in human neutrophils. Cells were incubated with buffer alone (unstimulated) (A, D, G, J, and M), Boc-PLPLP (1 lM) (B, E, H, K, and N), or fMLP (1 lM) (C, F, I, L, and O) at 37 °C for 1 h then fixed. Samples were labeled with FITC-conjugated anti-hexokinase (A–C), glucose-6-phosphate dehydrogenase (D–F), anti-phosphofructokinase (G–I), pyruvate kinase (J–L), and anti-lactate dehydrogenase (M–O) antibodies as described in Section 2. Three to five individual experiments were performed and representative fluorescence images are shown. Hexokinase translocates to the cell periphery during fMLP stimulation (627).
were treated with various doses of fMLP for 1, 5, 10, and 20 min. Cells were then fixed and stained with FITC-conjugated anti-hexokinase. Fig. 3 shows representative examples of neutrophils treated with 109 M fMLP. Extensive clumping and translocation of hexokinase is evident after 5 min of incubation. Similar ob-
servations were made at 106 M fMLP (data not shown). Thus, a finite amount of time is required for hexokinase redistribution during neutrophil activation. Furthermore, this time frame is not surprising since, in the absence of extensive mixing, about 3 min is required for diffusion of an activating substance to a cell.
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Fig. 2. Hexokinase translocation at various doses of fMLP. Neutrophils were incubated with or without fMLP (106 –1012 M) for 1 h at 37 °C followed by fixation and labeling with anti-hexokinase antibody. As panels A–D show, 1 lM–nM fMLP was sufficient to stimulate extensive translocation and clumping of hexokinase. Significant translocation was not observed at 1010 , 1011 , and 1012 M (panels E–G, respectively) and in the absence of fMLP (panel H), although some clumping may be detected in the micrographs (n ¼ 3) (867).
Fig. 3. Kinetics of hexokinase translocation. Neutrophils were incubated with 1 nM fMLP for various periods of time at 37 °C followed by fixation and labeling with anti-hexokinase antibody. Although translocation was not evident at 1 min (A), extensive translocation and clumping in the vicinity of the plasma membrane was found at 5 and 10 min of incubation (panels B and C, respectively), which persisted for long periods of time. Hexokinase translocation was not observed in the absence of fMLP (panels D–F, respectively) after corresponding incubation times (n ¼ 4) (956).
3.4. Effect of a chemotactic gradient on hexokinase localization
3.5. Hexokinase translocates to the neutrophil periphery in response to a variety of activating stimuli
Inasmuch as fMLP is a chemotactic factor, we examined the distribution of hexokinase when cells were exposed to a gradient of fMLP. An fMLP gradient was formed by placing 15 ll of a 5 lM solution of fMLP at one end of a coverslip. After 10 min at 37 °C to allow time for cell polarization, neutrophils were fixed and stained. As shown in Fig. 4, hexokinase accumulates at the leading edge of neutrophils exposed to an fMLP gradient. Thus, hexokinase is translocated to the periphery of the cell during exposure to uniformly applied fMLP and specifically to the lamellipodium when the stimulant is applied in the form of a gradient.
We next sought to determine if hexokinase translocation was a general property of neutrophil activation. Neutrophils were treated at 37 °C with the activating/ priming stimuli: IFN-c (50 U/ml for 2 h), IL-8 (50 ng/ml for 45 min), LTB4 (1 lg=ml for 1 h), PMA (5 nM, 1 h), immune complexes (10 lg=ml for 1 h), LPS (50 ng/ml for 20 min), and PAF (100 nM for 30 min). The conditions and concentrations of these reagents were chosen based upon prior findings [27]. Representative photomicrographs are shown in Fig. 5. In all cases substantial translocation into the vicinity of the plasma membrane was noted for hexokinase, although this was not ob-
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Fig. 4. Hexokinase accumulates at the lamellipodium of neutrophils exposed to a gradient of fMLP. Panels A and C show bright-field micrographs to illustrate the orientation of the cells whereas panel B and D show immunofluorescence microscopy of hexokinase. In panels A and B, cells were exposed to an fMLP gradient by placing 15 ll of an fMLP solution (5 lM) at one side of a cover glass on a microscope slide. This was incubated at 37 °C for 10 min followed by fixation and labeling with FITC-conjugated anti-hexokinase antibody. The white arrow points in the direction of decreasing fMLP concentration. In panels C and D, cells were incubated without fMLP to serve as a negative control. Four individual experiments were performed and the typical DIC and fluorescence images are shown (815).
served for lactate dehydrogenase. Therefore, hexokinase translocation is not a specific property of fMLPtreated neutrophils, but rather a more general feature of molecules promoting the priming or activation of these cells. 3.6. Hexokinase translocates into the vicinity of captured beads in neutrophils Since phagocytosis is accompanied by significant changes in cell metabolism and organization, we investigated the intracellular distribution of hexokinase following incubation with target particles. Latex beads were chosen for these microscopic studies because they have a very distinctive appearance and therefore could be unambiguously correlated with the site of hexokinase accumulation. Cells were incubated with 2 lm diameter unopsonized latex beads (0.5 ll of the stock bead solution added to 100 ll of cell suspension) at 37 °C for 1 h followed by fixation and labeling with FITC-conjugated anti-hexokinase or FITC-conjugated anti-lactate dehydrogenase antibody. As shown in Figs. 6A–C, hexokinase accumulated in the vicinity of captured beads. The redistribution of hexokinase is noticeable by comparing the DIC and the fluorescence micrographs in panels A and B. In contrast to the peripheral distribution of hexokinase found for fMLP treatment, after incubation with latex beads hexokinase was found in (1) the cyto-
plasm, (2) near the plasma membrane, and (3) in the vicinity of the target. In addition, quantitative line profile analyses also support this conclusion (Fig. 6C). However, no significant changes in lactate dehydrogenase distribution were noted (Fig. 6D–F). To provide further quantitative data to sort out these labeling differences, digital images were analyzed using software that calculates the mean fluorescence intensity of an adjustable pre-defined area in the micrographs. In this way the fluorescence intensity surrounding the target(s) and that of the entire cell were determined. When latex beads and neutrophils were incubated together for 10 min, very little hexokinase accumulated at the bead(s) (Fig. 7). However, roughly 25% of the total hexokinase accumulated at the bead(s) at 1 h (Fig. 7). Thus, hexokinase traffics to the site of target binding and uptake during phagocytosis. Since exposure to beads and to fMLP led to hexokinase redistribution in neutrophils, we examined the effect of simultaneous exposure to both of these stimuli. Adherent neutrophils were incubated simultaneously with 1 lM fMLP and latex beads at 37 °C as described above. Cells were fixed and stained with FITC-conjugated anti-hexokinase. At short co-incubation times (10 min), fMLP promoted hexokinase accumulation near the beads (Fig. 7). Furthermore, double labeling experiments indicated that hexokinase and F-actin accumulated at the site of phagocytosis (Fig. 8), which is consistent with the potential role of microfilaments. When cells were incubated in the presence of fMLP and beads for 1 h a peripheral, not a target-associated, hexokinase distribution was observed. Similarly, F-actin is lost from the phagosome after 1 h as well (data not shown and [33]). Thus, hexokinase trafficking to latex beads is faster in the presence of fMLP which is followed by loss of hexokinase and F-actin from the vicinity of targets. No changes in lactate dehydrogenase distribution were noted (data not shown). 3.7. Inhibitory effects of cytochalasin D and anti-inflammatory agents on hexokinase translocation We next sought to explore potential mechanisms contributing to hexokinase translocation in neutrophils. Since hexokinase translocation accompanies cytoskeletal remodeling, we investigated the effects of colchicine and cytochalasin D, inhibitors of microtubules and microfilaments, respectively, on hexokinase distribution. Neutrophils were pre-treated with 0.3 lg=ml colchicine for 30 min at 37 °C then incubated with 1 lM fMLP for another 1 h. As Fig. 9B shows, hexokinase was translocated to the periphery of neutrophils after treatment with both colchicine and fMLP. This suggests that microtubules are not responsible for hexokinase trafficking in neutrophils. As shown in Fig. 9C, cytochalasin D (10 lg=ml) prevented the translocation of hexokinase to
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Fig. 5. Representative fluorescence studies showing that hexokinase translocates to the neutrophil periphery in response to a variety of activating stimuli. Neutrophils were incubated at 37 °C for 1 h in the absence of stimuli (A and B) or treated with IFN-c (50 U/ml) at 37 °C for 2 h (C and D), IL8 (50 ng/ml) at 37 °C for 45 min (E and F), LTB4 (1 lg=ml) at 37 °C for 1 h (G and H), PMA (5 nM) at 37 °C for 1 h (I and J), LPS (50 ng/ml) at 37 °C for 20 min (K and L), immune complexes (10 lg=ml) at 37 °C for 1 h (M and N) as well as PAF (100 nM) at 37 °C for 30 min (O and P). After treatment, cells were labeled with FITC-conjugated anti-hexokinase antibody (A, C, E, G, I, K, M, and O) or FITC-conjugated anti-lactate dehydrogenase antibody (B, D, F, H, J, L, N, and P). At least three separate repetitions for each of the experiments shown were performed (587).
the cell periphery in the presence of 1 lM fMLP. Thus, hexokinase trafficking appears to depend upon microfilaments, but not microtubules. Inasmuch as activating stimuli promoted hexokinase redistribution, we next examined the effect of anti-inflammatory agents. In the first series of experiments we tested the effect of dexamethasone on hexokinase translocation in neutrophils. Cells were incubated with 2 lM dexamethasone for 45 min at 37 °C followed by the addition of 1 lM fMLP for 1 h. Dexamethasone inhibited the accumulation of hexokinase at the cell periphery (Fig. 9E). This result is not surprising since previous workers have shown that dexamethasone blocks the coupling of hexokinase with the glucose transporter [34]. We next examined the effect of indomethacin, a cyclooxygenase inhibitor that blocks PGH synthesis in
response to inflammatory stimuli. It has been reported to diminish neutrophil oxidative metabolism (e.g., [35]). Cells were incubated with 5 lM indomethacin and 1 lM fMLP for 1 h at 37 °C. Our observations show that indomethacin effectively inhibited hexokinase translocation in response to fMLP in neutrophils (Fig. 9). An important difference between the effects of cytochalasin D and indomethacin is that neutrophils could polarize in the presence of indomethacin (Fig. 9D), but not in the presence of the cytoskeletal-disrupting drug cytochalasin D where the cells are spherical (Fig. 9C). Thus, two different but widely prescribed drugs, dexamethasone and indomethacin, share a downstream mechanistic step—the inhibition of hexokinase translocation to the cell periphery and its accompanying up-regulation of cell metabolism.
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Fig. 6. Hexokinase is recruited to the vicinity of captured beads within neutrophils. Cells were incubated with 2 lm diameter nonopsonized latex beads at 37 °C for 1 h and labeled with FITC-conjugated anti-hexokinase (A, B, and C) or FITC-conjugated anti-lactate dehydrogenase (D, E, and F) antibodies. DIC micrographs (A and D), fluorescence images (B and E), and line profile analyses of fluorescence images (C and F) are shown. The locations of the spherical latex beads are shown in panels A and D with white arrows. Panel B shows the accumulation of hexokinase in the region of the latex beads in these two cells, although no such changes are noted for lactate dehydrogenase (panel E). The line profile analyses (fluorescence intensity vs. pixel number) of panels C and F correspond to the white lines drawn through the micrographs of panels B and E, respectively. Both the enrichment in hexokinase and the location of the beads (see black arrows) are apparent in the line profile analyses in panel C. These features were not observed for lactate dehydrogenase (F). At least three individual experiments were performed for each of these studies (1680).
3.8. Superoxide production by adherent neutrophils We next sought to confirm the physiological relevance and extend our findings by measuring the production of ROMs. ROM release into the extracellular environment was detected using H2 -TMRos, a nonfluorescent molecule that becomes fluorescent upon exposure to ROMs. The time-dependence of ROM release was assessed on a single cell basis at 37 °C as previously described [31]. Low levels of ROMs were detected for adherent neutrophils (Fig. 10, trace 3), as previously described [31,36]. As anticipated, treatment with 50 nM fMLP substantially increased the rate of ROM production (Fig. 10, trace 1). Furthermore, pre-treatment with indomethacin or dexamethasone, as described above, reduced ROM release to baseline levels associated with adherent, polarized neutrophils (Fig. 10, traces 4 and 5). Thus, ROM production parallels the translocation of hexokinase to the cell periphery. We next evaluated the possibility that hexokinase translocation was required for enhanced ROM production. The reagent LY83583 was employed. It has been found to dramatically increase glucose transport into cells [37]. Cells were incubated with LY83583 (1 lg=ml) for 15 min at room temperature followed by
microscopy at 37 °C. No change in the intracellular location of hexokinase was observed using the protocols given above (data not shown). However, the rate of ROM release was increased to levels comparable with that of fMLP treatment (Fig. 10, trace 2). Indomethacin was unable to reverse the effect of LY83583 on ROM production (data not shown), in contrast to its effect on fMLP-mediated stimulation (see above). Although LY83583 has multiple effects on cells as do many other drugs, this reagent: (1) provides evidence consistent with a role of glucose transport in cell activation and (2) suggests that hexokinase translocation is not always required for high levels of ROM production, but accompanies all of the tested physiological stimuli (Fig. 5).
4. Discussion The translocation of kinases, such as protein kinase C, to the plasma membrane is well known in leukocytes. In the present study we have characterized the translocation of another kinase, hexokinase—a key enzyme in the control of cell metabolism, from the cytoplasm to the peri-membrane region during neutrophil activation. This effect is specific for productive receptor occupancy
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Fig. 7. Quantitative analyses of hexokinase translocation. The percentage of hexokinase translocated to the vicinity of cell-associated latex beads are shown. The effects of incubation time and fMLP addition on hexokinase accumulation are illustrated. Cells were incubated with 2 lm diameter nonopsonized latex beads at 37 °C for 10 min or 1 h in the presence or absence of fMLP then labeled with FITCconjugated anti-hexokinase. Neutrophil activation with fMLP accelerates the delivery of hexokinase to the vicinity of latex beads. At least three individual experiments were performed. *P < 0:001.
Fig. 9. Hexokinase trafficking is affected by the cytoskeleton and antiinflammatory compounds. Cells incubated with fMLP at 37 °C for 1 h (1 lM) (A) showed hexokinase clumps in the vicinity of the plasma membrane. Peripheral labeling was also observed after pretreatment with colchicine (300 ng/ml) at 37 °C for 30 min followed by addition of fMLP (1 lM) for 1 h at 37 °C (B). However, exposure to cytochalasin D (5 lg=ml) (panel C) or dexamethasone (2 lM) (panel E) at 37 °C for 1 h followed by incubation with fMLP (1 lM) at 37 °C for 1 h led to essentially random labeling with anti-hexokinase antibody. Similarly, indomethacin (5 lM) treatment (panel D) at 37 °C for 1 h with fMLP led to no significant changes in hexokinase distribution. At least three individual experiments were performed for each of the studies shown (627).
Fig. 8. Colocalization of F-actin and hexokinase in the vicinity of neutrophil-associated beads. Cells were incubated with 2 mm diameter unopsonized latex beads and fMLP (1 mM) at 37 °C for 10 min then labeled with FITC-conjugated anti-hexokinase (C and F) and rhodamine-conjugated phalloidin (B and E). DIC micrographs (A and D) and fluorescence images (B, C, E, and F) are shown. The locations of the spherical latex beads are shown in panels A and D with white arrows. Panels B and E show the accumulation of F-actin in the region of the latex beads in these two cells. The location of hexokinase labeling is illustrated in panels C and F. Note the co-localization of these two labels. At least three individual experiments were performed for each of these studies. (600 for panels A–C, 1680 for panels D–F.)
since the formyl peptide receptor antagonist Boc-PLPLP did not have an effect on cells. This was not a generalized redistribution of metabolic enzymes because cell activation had no effect on other enzymes such as lactate dehydrogenase. Naftalin and co-workers [16] first re-
ported hexokinase translocation in rat macrophages in response to PMA. This observation seems to have been largely overlooked, presumably in the mistaken belief that biochemistry is homogeneously distributed within cells. Not only can metabolic reactions be sequestered in specific organelles, but they can be localized to specific regions of the cytoplasm or membranes. Theoretical physics has predicted that metabolism is not uniformly distributed in cells and the cytoskeleton could be a factor contributing to chemical instabilities within cells (e.g., [38,39]). Recent studies from this laboratory have shown that metabolism is not spatially or temporally uniform within living adherent or polarized neutrophils; NAD(P)H production is observed as spherical or nominally longitudinal waves traveling with specific directions, velocities, and shapes [26–28]. We therefore hypothesized that: (1) hexokinase translocation to the plasma membrane or regions thereof constituted a
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Fig. 10. Rates of ROM release by neutrophils. ROM release was measured during cell migration through a gelatin matrix containing H2 -TMRos, as described [39]. High rates of ROM production are observed during incubation with 50 nM fMLP (trace 1) and 1 lg=ml LY83583 (trace 2). However, adherent cells produce lower levels of ROMs in the absence of these reagents (trace 3). Enhanced ROM production is blocked by incubation with 2 lM dexamethasone (trace 4) and 5 lM indomethacin (trace 5).
spatial chemical change leading to alterations in metabolic wave properties and (2) hexokinase translocation is an important aspect of neutrophil activation. We have shown that hexokinase is translocated to the periphery of normal adherent human neutrophils in response to fMLP. Hexokinase translocation was observed over a range of fMLP concentrations from 106 to 109 M, which is consistent with the physiologically relevant concentrations associated with receptor activation. Hexokinase underwent extensive translocation in less than 5 min. This observation is consistent with the facts that about 3 min is required for fMLP diffusion to the cell in this unstirred system and another study has shown that the maximal rate of superoxide production is only achieved at about 1.5 min [40]. Although fMLP stimulation of stirred cells in suspension begins with seconds, this is not directly comparable with our work because: (1) our samples were unstirred and (2) our cells were adherent, not in suspension. As Nathan [41] has shown, adherent neutrophils display properties distinct from suspended cells; for example, the kinetics of activation differs and oxidant release is enhanced in adherent neutrophils. The potential physiological significance of these findings in regard to ROM release is also supported by the fact that several pro-inflammatory stimuli share this effect on cells whereas anti-inflammatory drugs have just the opposite effect. We hypothesized that hexokinase translocation may broadly participate in neutrophil activation. Therefore, we tested a variety of stimulatory agents for their effect of hexokinase trafficking. We found that diverse neutrophil-activating reagents, including fMLP, LPS, latex beads, IL-8, PMA, PAF, LTB4 , immune complexes, and IFN-c, all promoted hexokinase translocation at phys-
iologically appropriate doses. Thus, hexokinase translocation appears to be a general property of neutrophil activation, not a specific response to one agent such as fMLP. Hexokinase accumulation was, essentially, in the form of clumps about the cell periphery. In contrast, the asymmetric addition of fMLP to neutrophils results in asymmetric accumulation of hexokinase at a cellÕs lamellipodium. Moreover, phagocytic stimuli lead to hexokinase translocation about the periphery of the target. In both of these cases, hexokinase traffics to the region of membrane perturbation. We previously found that metabolic (NAD(P)H) waves in neutrophils correspond to sites of fMLP binding [27]. Hexokinase accumulation and the consequent increase in glucose transport at these local sites, which are undergoing substantial cytoskeletal re-organization, may account for these earlier findings. Although a variety of molecules may accumulate at sites of phagocytosis, our finding that hexokinase is translocated to the vicinity of captured beads appears to be the first metabolic enzyme noted to behave in this way. As mentioned below, microfilaments appear to be important in hexokinase translocation. Although microfilaments may promote hexokinase trafficking to phagosomes, they may not be entirely responsible for retention since microfilaments surrounding phagosomes disassemble [33] whereas hexokinase translocation is stable for at least an hour. In the presence of both fMLP and beads, hexokinase was observed in the vicinity of beads at 10 min, but not after 1 h. Thus, hexokinase translocation to the vicinity of the target appears to be faster in the presence of fMLP. As might be expected, the trafficking properties of hexokinase depend upon the physiological stimuli. The accumulation of hexokinase near phagosomes raises the interesting question of what biological function could hexokinase serve at a phagosome? One speculative possibility is that it is present to starve the target. The presence of hexokinase would accelerate the removal of glucose from the phagolysosome. Thus, glucose captured from the medium during phagocytosis, released from a target due to the formation of membrane lesions, or glucose formed by the action of phosphatases within the phagolysosome, glycosidases, or other enzymes of the phagolysosome would be removed by the neutrophil. Transport across the targetÕs membrane would be futile in the presence of membrane lesions. Thus, hexokinase trafficking could enhance target killing. Several metabolic enzymes may be associated with cytoskeletal elements. For example, aldolase and phosphofructokinase are associated with microfilaments (e.g., [42,43]). Moreover, hexokinase translocation to the cell periphery is sensitive to cytochalasin D [16], which has been confirmed in this study. Cytoskeletal
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associations may promote the delivery of metabolic machinery to specific sites to meet local metabolic demands [16–24]. Our findings are also consistent with prior experimental studies of oxidant production. Although studies of neutrophils in suspension have indicated that cytochalasin B promotes fMLP-associated superoxide production [44], studies of adherent neutrophils have indicated that cytochalasin D blocks superoxide production [36] during spreading. Recent studies have shown that cytochalasin inhibits postphagocytosis oxidant production in phagosomes [45]. Thus, the microfilament-mediated redistribution of hexokinase may contribute to certain effector activities. To further examine the participation of hexokinase translocation in neutrophil activation, we tested the effect of anti-inflammatory agents on fMLP-mediated stimulation. Our findings appear to be the first to show that indomethacin dramatically inhibits hexokinase translocation to the peri-membrane region of the cell. Thus, inflammatory signals promote hexokinase translocation to the cell periphery whereas the anti-inflammatory drug indomethacin inhibits this translocation. This finding may contribute to a better understanding of the mechanisms of actions of anti-inflammatory drugs. For example, it is well known that indomethacin reduces superoxide production, as well as other effector responses, of stimulated neutrophils (e.g., [35]). It is also well known that indomethacin and other NSAIDs (nonsteroidal anti-inflammatory drugs) inhibit the activity of cyclooxygenases [46], although the links between cyclooxygenase inhibition and diminished effector responses have not been fully characterized. Moreover, cyclooxygenase-independent pathways of NSAIDs have been increasingly recognized in recent years [46]. We speculate that inhibition of hexokinase translocation may contribute to the anti-inflammatory effects of certain drugs by either cyclooxygenase-dependent and/or cyclooxygenase-independent pathways. For example, maximal production of superoxide anions requires a substantial carbon flux through the hexose monophosphate shunt, which would be limited in the absence of up-regulated glucose transport and the availability of glucose-6-phosphate at the level of the plasma membrane. Furthermore, this concept may apply to both indomethacin and dexamethasone. Although the present study has focused on hexokinase trafficking during neutrophil activation, the broad importance of metabolic changes in immunologic and inflammatory processes [47] suggests that the concepts outlined above may be applicable in additional physiological settings.
Acknowledgments This work has been supported by grants from the NIH (AI51789) and the National Multiple Sclerosis
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Society to H.R.P. We thank Dr. T. Mair for a helpful discussion.
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