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Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release Mauro Maccarrone,* Natalia Battista,† Mariantonia Meloni,‡ Monica Bari,† Grazia Galleri,‡ Proto Pippia,‡ Augusto Cogoli,§ and Alessandro Finazzi-Agro`† *Department of Biomedical Sciences, University of Teramo, Italy; †Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy; ‡Department of Physiological, Biochemical and Cellular Sciences, University of Sassari, Italy; and §Space Biology, ETH, Zu¨rich, Switzerland

Abstract: Creating conditions similar to those that occur during exposure of cells to microgravity induced a sixfold increase of apoptotic bodies and DNA fragments in human lymphocytes, paralleled by an early (within 2 h) fourfold increase in 5-lipoxygenase (5-LOX) activity and a fivefold decrease in mitochondrial membrane potential and increase in cytochrome c release (within 4 and 8 h, respectively). Similar membrane potential and cytochrome c release were observed in isolated mitochondria treated with physiological amounts of 5-LOX and were enhanced by creating conditions similar to those that occur during exposure of cells to microgravity. 5-LOX inhibitors, 5,8,11,14-eicosatetraynoic acid and caffeic acid, completely prevented apoptosis, whereas the phospholipase A2 inhibitor methyl-arachidonoyl fluorophosphonate and the 5-LOX activating protein inhibitor MK886 reduced it to 65–70%. The intracellular calcium chelator EGTA-acetoxymethylester reduced 5-LOX activity and apoptosis to 30 – 40% of controls, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 was ineffective. The caspase-3 and caspase-9 inhibitors Z-Asp(OCH3)-Glu(OCH3)Val-Asp(OCH3)-fluoromethylketone (FMK) and Z-Leu-Glu(OCH3)-His-Asp(OCH3)-FMK reduced apoptotic bodies to 25–30% of the control cells. Finally, creating conditions similar to those that occur during exposure of cells to microgravity did not induce apoptosis in human lymphoma U937 cells, which did not express an active 5-LOX. J. Leukoc. Biol. 73: 472– 481; 2003. Key Words: arachidonate cascade 䡠 calcium 䡠 caspase 䡠 cyclooxygenase 䡠 mitochondrial membrane potential

INTRODUCTION Experiments performed during spaceflight clearly show that several cellular processes, such as oxidative metabolism, 472

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growth rate, signal transduction, and gene expression, are modified under conditions of weightlessness [1– 4]. For example, these alterations are associated with atrophy in heart, muscle, and bone [5, 6]. In particular, dramatic effects of conditions similar to those that occur during exposure of cells to microgravity have been shown on the activation of human lymphocytes in vitro [7] and have been associated with altered distribution and down-regulation of protein kinase C [8 –10], reduced expression of interleukin-2 (IL-2) and its receptor [11], microtubule anomalies and growth retardation [12], and altered cytoskeletal gene expression [13]. It has been suggested that reduced growth response in lymphocytes during spaceflight might be linked to apoptosis, based on morphological anomalies [12] and cDNA microarray analysis [13] of space-flown human lymphoblastoid (Jurkat) cells. Although there is no coherent explanation for these observations, and it is not known which biomolecules might act as “gravity responders,” recent evidence seems to suggest that inhibition of lymphocyte proliferation depends on alterations occurring within the first few hours of microgravity [14]. 5-Lipoxygenase (5-LOX) plays a central role in IL-2 expression [15] and activation of human lymphocytes [16] and is involved in the initiation of programmed death (apoptosis) triggered by several stimuli in different human cells (reviewed in ref. [17]). Remarkably, recent in vitro studies performed in the course of the 28th parabolic flight campaign of the European Space Agency have demonstrated that low gravity (⬃10⫺2 g) directly enhances the catalytic efficiency of pure LOX up to ⬃fourfold over the ground (1 g) controls [18]. Here, we created conditions similar to those that occur during exposure of cells to microgravity with the random positioning machine (RPM) [11] and with the rotary cell culture system (RCCS) [9] to ascertain whether these conditions might induce apoptosis in human lymphocytes. The possible involvement of the arachidonate

Correspondence: Mauro Maccarrone, Ph.D., Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. E-mail: [email protected] Received June 13, 2002; revised November 15, 2002; accepted December 24, 2002; doi: 10.1189/jlb.0602295.

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cascade and mitochondrial alterations in the apoptotic program were also investigated. Taken together, the data indicate that creating conditions similar to those that occur during exposure of cells to microgravity induces lymphocyte apoptosis and that 5-LOX is a “gravity responder” that executes this process. The ability of 5-LOX to uncouple mitochondria, leading to cytochrome c release and caspase activation, seems to be the critical event along the apoptotic pathway, suggesting that creating conditions similar to those that occur during exposure of cells to microgravity triggers a signaling cascade common to other unrelated proapoptotic stimuli.

MATERIALS AND METHODS Materials Chemicals were of the purest analytical grade. Actinomycin D, cycloheximide, 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid (CA), indomethacin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), ionomycin, cytochrome c, and cyclosporin A were purchased from Sigma Chemical Co. (St. Louis, MO). EGTA-acetoxymethylester (AM), caspase-3 inhibitor II [Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethylketone (Z-DEVD-FMK)], caspase-9 inhibitor I [Z-Leu-Glu(OCH3)-His-Asp(OCH3)-FMK (Z-LEHD-FMK)], and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 were from Calbiochem (La Jolla, CA). Methyl-arachidonoyl fluorophosphonate (MAFP), authentic prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 9-hydroperoxyoctadecadienoic acid (9-HPOD) were from Cayman Chemicals (Ann Arbor, MI). 5,5⬘,6,6⬘-Tetrachloro1,1⬘,3,3⬘-tetra-ethylbenzimidazol-carbocyanine iodide (JC-1) and 1-[2-amino5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-[2-amino-5-methylphenoxy]ethane-N,N,N⬘,N⬘-tetra-AM (Fluo-3 AM) were from Molecular Probes (Eugene, OR). Anti-human 5-LOX rabbit polyclonal antibodies were a kind gift of Dr. Anthony W. Ford-Hutchinson (Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Canada), and anti-LOX monoclonal antibodies (mAb) were produced at CIVO-TNO (Zeist, The Netherlands) using soybean (Glycine max) 15-LOX and a single hybridoma clone as described [19]. These anti-LOX mAb have been shown to inhibit the activity of soybean and lentil (Lens culinaris) 15-LOX [19] and barley (Hordeum vulgare) 5-LOX (unpublished), as well as the cellular 15-LOX activity upon electrotransfer into intact lentil cells [19]. Anticytochrome c mAb (clone 7H8.2C12) were purchased from PharMingen (San Diego, CA), and goat anti-mouse or anti-rabbit alkaline phosphatase conjugates (GAM-AP or GAR-AP, respectively) were from BioRad (Hercules, CA). Nonimmune mouse serum was from Nordic Immunology (Tilburg, The Netherlands). Pure 5-LOX (1 unit⫽amount of enzyme oxygenating 1 nmol arachidonate per min) was prepared from ungerminated barley as reported [20].

Creating conditions similar to those that occur during exposure of cells to microgravity Conditions similar to those that occur during exposure of cells to microgravity were created by the three-dimensional clinostat developed by Fokker Space (Leiden, The Netherlands), also called RPM. The RPM consists of a frame that rotates within a second rotating frame, and each frame is driven by a separate motor. Rotation of each frame is random, autonomous, and regulated by computer software. Each frame rotates at a speed of 60° 䡠 s–1, which corresponds to a force of gravity of ⬃2 x 10⫺3 g at the center of the frame. The RPM was located in a room at 37°C, and a box containing the cell cultures, sealed in 2 ml Eppendorf tubes, was placed at the center of the inner frame. Control (static) cultures were placed at the basement of the RPM. To avoid the presence of air bubbles, which could lead to shear force damage of the cells on the RPM, the Eppendorf tubes were completely filled [11]. In the experiments on DNA fragmentation in human lymphocytes and in those with isolated mitochondria, the same level of force of gravity (⬃2x10⫺3 g) was obtained by means of the RCCS, developed by the National Aeronautics and Space Administration (Washington, DC) and manufactured by Synthecon (Houston, TX) [9]. In this case, cell cultures and mitochondria suspensions were placed in 50

ml vessels and rotated at a speed of 7.2 rpm, according to the manufacturer’s instructions.

Lymphocyte isolation, culture, and treatment Blood samples (20 ml per donor) were drawn from the antecubital vein of healthy donors (age range 28 –35 years), who gave informed consent to the study, and were collected into heparinized sterile tubes. Peripheral lymphocytes were purified by gradient centrifugation using the density separation medium Lymphoprep (Nycomed Pharma, Oslo, Norway) and consisted of T lymphocytes (⬃85%), B cells (⬃10%), and monocytes (⬃5%) [21]. Isolated lymphocytes were resuspended in RPMI-1640 medium (Gibco, Paisley, UK), supplemented with 25 mM Hepes, 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) at a density of 1.5 x 106 cells/ml [21] and were stored at 37°C for at least 4 h before exposure to conditions similar to those that occur during exposure of cells to microgravity to allow them to recover from the potential stress of the centrifugation steps during isolation [11]. Incubation of lymphocytes with different inhibitors was performed at 37°C at the indicated concentrations and for the indicated periods of time. Controls were incubated with vehicles alone. Human lymphoma U937 cells were cultured and treated as the lymphocytes [21]. The U937 cell line was obtained from an individual with histiocytic lymphoma and has monocyte-like characteristics; therefore, it cannot be considered a pure model for lymphocytes. However, it is widely used as a model for immune cells [17, 22, 23]. Electrotransfer of anti-LOX mAb (200 ␮g/test) into lymphocytes (106 cells/ test) was performed with a Gene Pulser II Plus apparatus (Bio-Rad). Exponentially decaying pulses were generated and delivered to cells suspended in phosphate-buffered saline (PBS; 0.7 ml/test) in sterile, disposable electroporation cuvettes (Bio-Rad) of 0.4 cm path length [22]. Lymphocytes were electroporated at a capacitance of 125 ␮F and a field strength of 800 V/cm with a time constant of 1.5 ⫾ 0.2 ms. Control cells were electroporated under the same experimental conditions in the presence of nonimmune mouse serum (200 ␮g/test) as reported [22]. After electroporation, cells were kept for 5 min at 4°C, and then they were washed twice in PBS and were subjected to conditions similar to those that occur during exposure of cells to microgravity, as described for the nonelectroporated cells. Under these experimental conditions, ⬃1.0 pg/cell (2.5 ␮g/mg protein) mAb was transferred inside the cells, where they could inhibit their intracellular target [22].

Evaluation of cell death After incubation for the indicated periods of time in culture medium, apoptosis was estimated in all experiments by cytofluorimetric analysis in a FACScalibur flow cytometer (Becton Dickinson, Bedford, MA), which quantified apoptotic body formation in dead cells by staining with propidium iodide (50 ␮g/ml, also pretreated with 15,000 U/ml RNase to reduce noise), as reported [24]. Cells were fixed with cold methanol:acetone (4:1 v/v) diluted 1:1 in PBS and were stored at 4°C. Cells were excited at 488 nm using a 15 mW Argon laser, and the fluorescence was monitored at 570 nm. Events were triggered by the forward scattering signal and gated for FL2-A/FL2-W to skip aggregates. Ten-thousand events were evaluated using the Cell Quest Program. Controls of different batches of cells contained less than 4.0 ⫾ 1.0 apoptotic bodies every 100 cells analyzed. Apoptosis was also quantified by the cell-death detection enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Mannheim, Mannheim, Germany), based on the evaluation of DNA fragmentation by an immunoassay for histone-associated DNA fragments in the cell cytoplasm [22].

Assay of arachidonate-degrading enzymes and of their metabolites Arachidonic acid is converted into bioactive compounds such as PGs and LTs through the “cyclooxygenase branch” and the “lipoxygenase branch” of the arachidonate cascade, respectively [25]. The activity of cyclooxygenase (COX; PG-H synthase; E.C. 1.14.99.1) was measured polarographically at 30°C in 0.1 M potassium phosphate, pH 7.2, 1 mM phenol, and 75 ␮M arachidonic acid [26] and was expressed as nmol O2 consumed per min per ␮g protein. The activity of 5-LOX (arachidonate:oxygen 5-oxidoreductase; E.C. 1.13.11.34) was measured by incubating cell extracts for 10 min at 37°C in the presence of 1 mM adenosine 5⬘-triphosphate, 2 mM CaCl2, and 40 ␮M arachidonic acid [27]. 5-LOX activity was expressed as nmol 5-HPETE formed per min per mg

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protein. The activity of 12- and 15-LOX in the cells was measured under the same experimental conditions in the presence of 40 ␮M arachidonic acid only [27, 28]. The activity of 12- and 15-LOX was expressed as nmol 12- or 15-HPETE formed per min per mg protein. The amount of proteins in cell extracts was determined according to Bradford [29]. Arachidonate metabolites PGE2 and LTB4 were extracted from intact lymphocytes or from cell supernatants on octadecyl-solid phase extraction columns (Baker, Deventer, The Netherlands) and were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 3 x 3 CR column (SGE, Austin, TX), as reported [27]. RP-HPLC was performed on a Perkin Elmer 1022 LC Plus liquid chromatograph at a flow rate of 1.2 ml/min, using methanol/water/ trifluoroacetic acid (70:30:0.07 by vol) as mobile phase. Chromatograms were recorded at 270 nm, assessing peak identity by comparison with authentic standards. Quantitative determinations were performed by integrating peak areas of each compound. The effect of various inhibitors on cellular activity of 5-LOX, LTB4 content, cytochrome c release, and apoptotic body formation was evaluated by treating intact lymphocytes with each compound for the indicated periods of time and then performing the biochemical assays as detailed above. Inhibitors were used at concentrations found to be effective on their target enzymes in preliminary experiments and were in keeping with previous investigations [17, 22, 30]. ELISA determined the content of 5-LOX protein [22] by coating each well overnight with cell extracts (25 ␮g/well) and reacting with rabbit anti-5-LOX (diluted 1:200) polyclonal antibodies as first antibody. GAR-AP conjugates were used as second antibody (diluted 1:2000), and color development of the alkaline phosphatase reaction was followed at 405 nm, using p-nitrophenylphosphate as substrate [22].

Isolation of mitochondria Mitochondria were isolated from the liver of Wistar rats (weighing ⬃350 g) by standard differential centrifugation, followed by Percoll purification [31]. Mitochondria isolated by this procedure were ⬃90% intact by cytofluorimetric analysis in a FACScalibur flow cytometer (Becton Dickinson), corroborating previous functional [31] and morphological [32] data on preparations obtained with the same method. Isolated mitochondria were resuspended in 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 0.1% bovine serum albumin, 15 mM Hepes (pH 7.4) buffer [17], at a protein concentration of 10 mg/ml and were used as such for the treatment with pure 5-LOX, 5-HPETE, or 9-HPOD and subsequent biochemical analyses. In some experiments, mitochondria were incubated with 5-LOX (100 units/test) for 2 h in the RCCS to check the effect of conditions similar to those that occur during exposure of cells to microgravity.

Measurement of intracellular calcium, mitochondrial membrane potential, and cytochrome c release Intracellular calcium concentration and mitochondrial membrane potential were evaluated by flow cytometric analysis in a FACScalibur flow cytometer (Becton Dickinson). Cytoplasmic-free calcium was measured using the fluorescent Ca2⫹ indicator Fluo-3 AM, as reported [33]. Immediately before exposure to conditions similar to those that occur during microgravity, lymphocytes were collected by centrifugation and washed twice in Ca2⫹- and Mg2⫹-free PBS. Then, Fluo-3 AM [10 ␮M dissolved in dimethyl sulfoxide (DMSO)] was added, and cells were incubated 40 min at 37°C in the dark and frequently shaken manually and were collected by centrifugation and resuspended in culture medium without FCS. Control cells were treated with vehicle alone (1% of the final volume). After exposure to conditions similar to those that occur during microgravity, Fluo-3 AM fluorescence was recorded on a linear scale at 530 nm (bandwidth 30 nm) at a flow rate of ⬃1000 cells/s. Mean fluorescence values for 3000 events were registered every 10 s. Changes in mean fluorescence were plotted versus time, and ionomycin (10 ␮g/ml) was used as positive control to set intracellular calcium to 100% [33]. Mitochondrial membrane potential was measured using the fluorescent probe JC-1, as described [34]. JC-1 (dissolved in DMSO) was used at 20 ␮M final concentration, treating the controls with vehicle alone (1% of the final volume). After the treatment, human lymphocytes or isolated mitochondria were washed in PBS, incubated 20 min at 37°C in PBS, and then exposed to conditions similar to those that occur during microgravity. After this treatment, they were analyzed in a FL1/FL2 dot plot (530 nm/570 nm). CCCP (50 ␮M)

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was used as positive control, to set mitochondrial uncoupling to 100%, i.e., mitochondrial membrane potential to 0% [34]. The amount of cytochrome c released into the cytosol of human lymphocytes or into the medium by isolated mitochondria was quantified by ELISA, previously validated for both systems by Western blot analysis [22, 32]. Cell extracts or mitochondria supernatants (25 ␮g protein/well) were prepared as reported [35] and were reacted with anticytochrome c mAb diluted 1:250. GAM-AP immunoglobulins were used as secondary antibody at 1:2000 dilution. Color development of the AP reaction was recorded at 405 nm, using p-nitrophenylphosphate as substrate [22]. The absorbance values of the unknown samples were within the linearity range of the ELISA test, assessed by calibration curves with known amounts of cytochrome c (in the range 0 –500 ng/well).

Statistical analysis Data reported in this paper are the mean (⫾SD) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science, San Diego, CA).

RESULTS Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes Exposure of human lymphocytes to conditions similar to those that occur during exposure of cells to microgravity, created by means of the RPM, led to a time-dependent, apoptotic body formation (Fig. 1A). Detection by ELISA of DNA fragments in the cytosols of cells exposed to the same force of gravity through the RCCS confirmed the cytofluorimetric data (Fig. 1B). Apoptosis was significant (P⬍0.01) already at 24 h (⬃threefold over the control) and reached a level of five- to sixfold over the control 24 – 48 h later (Fig. 1, A and B). In the same time-window, we have previously observed a reduction of mitogenic activation of human lymphocytes exposed to microgravity [11]. Conversely, lymphocytes incubated at 1 g under the same experimental conditions did not show any significant sign of apoptosis (Fig. 1, A and B).

Creating conditions similar to those that occur during exposure of cells to microgravity enhances 5-LOX but not COX activity in human lymphocytes A possible role of the arachidonate cascade in lymphocyte apoptosis was checked by measuring the activity of 5-LOX and COX, which catalyzes the committed steps in the biosynthesis of the most biologically active arachidonate derivatives, LTs and prostanoids [25]. As calcium can stimulate 5-LOX and COX activities [25], it was checked whether conditions similar to those that occur during exposure of cells to microgravity might affect the intracellular calcium levels in lymphocytes, and it was found that this was not the case (Table 1). Instead, the activity of 5-LOX increased remarkably (⬃fourfold over 1 g controls) already 2 h after treatment, and it remained at least threefold higher than that of 1 g controls during the following 70 h (Fig. 2A). The increase in 5-LOX activity under conditions similar to those that occur during exposure of cells to microgravity was paralleled by a 2.5-fold increase in the main product of enzyme activity, LTB4, intracellularly (Table 1) and extracellularly (from 15⫾2 to 39⫾4 pmol 䡠 mg protein–1), http://www.jleukbio.org

Fig. 1. Effect of conditions similar to those that occur during exposure of cells to microgravity on human lymphocyte apoptosis. Conditions similar to those that occur during exposure of cells to microgravity (␮g) induced apoptotic body formation (A) and DNA fragmentation (B) time-dependently, whereas human lymphocytes kept at normal gravity (1 g) did not show indications of apoptosis under the same experimental conditions. Values were expressed as fold over controls (1⫽4⫾1% for apoptotic bodies and 0.300⫾0.030 A405 units for DNA fragmentation). *, P ⬍ 0.01 compared with 1 g controls.

whereas 5-LOX protein content was not affected (Table 1). Unlike 5-LOX, 12-LOX and 15-LOX were not affected by conditions similar to those that occur during exposure of cells to microgravity, and after 2 h or 48 h of treatment, their activity remained the same as in the untreated controls (320⫾30 pmol 12-HPETE 䡠 min–1 䡠 mg protein–1 and 730⫾70 pmol 15HPETE 䡠 min–1 䡠 mg protein–1, respectively). Also, COX activity did not change under the same conditions (Fig. 2B); neither changed the level of its main product PGE2 (Table 1). Therefore, only the 5-LOX branch of the arachidonate cascade appears to be up-regulated in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity. The role of 5-LOX in apoptosis induced by creating conditions similar to those that occur during exposure of cells to

microgravity was further investigated by treating intact lymphocytes with specific enzyme inhibitors, chemically unrelated to each other, such as ETYA [28] and CA [36]. Also, inhibitory anti-LOX mAb [19], electrotransferred inside the cells, were used to further validate the data. The results obtained with each compound are summarized in Table 2. It shows that ETYA and CA inhibited 5-LOX activity at 2 h and apoptotic body formation at 48 h (10 –20% of the controls). The same effects were observed in lymphocytes containing inhibitory anti-LOX mAb (Table 2), and nonimmune mouse serum, electrotransferred into the cells under the same experimental conditions, was ineffective (not shown). Moreover, incubation of intact lymphocytes with 10 ␮M ETYA or 40 ␮M CA for 2 h reduced the intracellular content of LTB4 to 15% and 20%, respectively, of the vehicle-treated controls (100%⫽50⫾5 pmol 䡠 mg protein–1; P⬍0.01 vs. controls). Conversely, MK886, an inhibitor of the 5-LOX activating protein (FLAP), which “presents” the arachidonate substrate to the enzyme [37], reduced 5-LOX activity and lymphocyte apoptosis to 65–70% of the controls (Table 2) as did MAFP, a selective phospholipase A2 (PLA2) inhibitor [38]. Moreover, the intracellular calcium chelator EGTA-AM [39] reduced 5-LOX activity and lymphocyte apoptosis to 30% and 40% of the controls, respectively, whereas the p38 MAPK inhibitor SB203580 [40] and the COX inhibitor indomethacin [41] were ineffective (Table 2). Finally, apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity required new mRNA transcription and protein synthesis, as shown by the ability of actinomycin D and cycloheximide to block apoptotic body formation, whereas 5-LOX activity was independent of new gene expression (Table 2). Unlike human lymphocytes, U937 cells subjected to the same environment did not show significant increases in apoptotic bodies or DNA fragments, which were ⬃1.3- and ⬃1.6-fold the controls after 48 h (not shown). It is interesting that 5-LOX activity could not be detected in these lymphoma cells, corroborating a previous report [23].

Creating conditions similar to those that occur during exposure of cells to microgravity reduces mitochondrial membrane potential and induces cytochrome c release Conditions similar to those that occur during exposure of cells to microgravity led to a time-dependent decrease in mitochonTABLE 1. Levels of Intracellular Calcium, 5-LOX Protein, LTB4, and PGE2 in Human Lymphocytes Subjected to Conditions Similar to Those that Occur during Exposure of Cells to Microgravity Parameter (% of control) [Ca2⫹]a 5-LOX proteinb LTB4 contentc PGE2 contentd

Time of exposure 2h

48 h

120 ⫾ 12 100 ⫾ 10 250 ⫾ 25* 100 ⫾ 10

115 ⫾ 12 110 ⫾ 10 210 ⫾ 22* 110 ⫾ 10

a 100% ⫽ 6 ⫾ 1% of Calcium rise induced by ionomycin; b 100% ⫽ 0.450 ⫾ 0.050 A405 units; c 100% ⫽ 50 ⫾ 5 pmol 䡠 mg protein⫺1; d 100% ⫽ 60 ⫾ 5 nmol 䡠 mg protein⫺1; * P ⬍ 0.01 compared with 1 g controls.

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release remained ⱖfourfold higher than that of 1 g controls in the following 64 h (Fig. 3B). The effect at 8 h was prevented by ETYA, CA, mAb, MK886, MAFP, EGTA-AM, SB203580, indomethacin, and the other compounds listed in Table 2, in a way that fully resembled their ability to inhibit 5-LOX activity (Table 2). In particular, 10 ␮M cyclosporin A, a selective inhibitor of the mitochondrial permeability transition pore [42], did not affect 5-LOX activity, cytochrome c release, or apoptosis (Table 2), suggesting that opening this pore did not contribute to lymphocyte death induced by conditions similar to those that occur during exposure of cells to microgravity. As the release of cytochrome c can trigger an apoptotic caspase cascade [35, 43], we tested the effect of inhibitors of caspase-3 and caspase-9 on apoptosis. Table 2 shows that the caspase-3 inhibitor Z-DEVD-FMK or the caspase-9 inhibitor Z-LEHDFMK, each used at 50 ␮M, lowered, to 25–30% of the control cells, apoptosis induced in human lymphocytes by conditions similar to those that occur during exposure of cells to microgravity without affecting 5-LOX activity.

5-LOX and its products uncouple isolated mitochondria and lead to cytochrome c release Treatment of mitochondrial suspensions with pure 5-LOX within a concentration range previously found to be physiological for the 15-isozyme [44] dose-dependently increased the uncoupling of mitochondria (up to fourfold over the controls) and the release of cytochrome c (up to threefold), and ETYA and anti-LOX mAb fully prevented these effects of 5-LOX TABLE 2. Effect of Various Compounds on 5-LOX Activity, Cytochrome c Release, and Apoptotic Body Formation in Human Lymphocytes Subjected to Conditions Similar to Those that Occur during Exposure of Cells to Microgravity

Fig. 2. Effect of conditions similar to those that occur during exposure of cells to microgravity on 5-LOX and COX activity in human lymphocytes. The activity of 5-LOX (A) and COX (B) was measured in cells exposed to conditions similar to those that occur during exposure of cells to microgravity (␮g) or normal gravity (1 g) for different periods of time. Values were expressed as fold over controls (1⫽1650⫾160 pmol 䡠 min–1 䡠 mg protein–1 for 5-LOX and 800⫾75 nmol O2 䡠 min–1 䡠 mg protein–1 for COX). *, P ⬍ 0.01 compared with 1 g controls.

drial membrane potential, i.e., increase in mitochondrial uncoupling, which was ⬃5.5-fold over the controls after 4 h and remained ⱖfourfold over the controls in the following 68 h (Fig. 3A). Conversely, 1 g controls did not show any significant change in mitochondrial membrane potential in the same timewindow (Fig. 3A). Western blot analysis (not shown) demonstrated that anticytochrome c mAb specifically recognized a single immunoreactive band of ⬃15 kDa in lymphocyte saps, corroborating our previous observations on other human cells [22]. These antibodies were used to quantify cytochrome c release into cell cytosol by ELISA [22]. Conditions similar to those that occur during exposure of cells to microgravity led to a ⬃fivefold increase in cytochrome c release after 8 h, and cytochrome 476

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Compound Vehicle ETYA (10 ␮M) CA (40 ␮M) mAb (200 ␮g) MK886 (10 ␮M) MAFP (5 ␮M) EGTA-AM (50 ␮M) SB203580 (30 ␮M) Indomethacin (10 ␮M) Actinomycin D (10 ␮g/ml) Cycloheximide (10 ␮g/ml) Cyclosporin A (10 ␮M) Z-DEVD-FMK (50 ␮M) Z-LEHD-FMK (50 ␮M)

5-LOX activity (%)

Cytochrome c release (%)

Apoptotic bodies (%)

100 10 ⫾ 2* 15 ⫾ 2* 20 ⫾ 2* 70 ⫾ 7** 68 ⫾ 7** 30 ⫾ 3* 90 ⫾ 9 100 ⫾ 10 100 ⫾ 10 100 ⫾ 10 95 ⫾ 10 100 ⫾ 10 100 ⫾ 10

100 20 ⫾ 2* 25 ⫾ 2* 20 ⫾ 2* 72 ⫾ 7** 70 ⫾ 7** 33 ⫾ 3* 95 ⫾ 10 100 ⫾ 10 100 ⫾ 10 100 ⫾ 10 95 ⫾ 10 100 ⫾ 10 100 ⫾ 10

100 20 ⫾ 2* 20 ⫾ 2* 25 ⫾ 3** 65 ⫾ 7** 70 ⫾ 7** 40 ⫾ 4* 90 ⫾ 9 100 ⫾ 10 20 ⫾ 2 25 ⫾ 2 90 ⫾ 9 30 ⫾ 3* 25 ⫾ 3*

The values refer to measurements made 2 h (5-lipoxygenase activity), 8 h (cytochrome c release), or 48 h (apoptotic bodies) after exposure to conditions similar to those that occur during exposure of cells to microgravity and are expressed as percentage of vehicle-treated cells (100%⫽7350⫾730 pmol 䡠 min⫺1 䡠 mg protein⫺1 for 5-LOX activity, 1.250⫾0.080 A405 units for cytochrome c release, and 22⫾3% for apoptotic bodies). Treatment of human lymphocytes at 1 g with every compound listed did not significantly affect cell death under the same experimental conditions (at 1 g, 100%⫽1980⫾190 pmol 䡠 min⫺1 䡠 mg protein⫺1 for 5-LOX activity, 0.280⫾0.030 A405 units for cytochrome c release, and 4⫾1% for apoptotic bodies). * P ⬍ 0.01 and ** P ⬍ 0.05 compared with vehicle-treated controls.

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ⱖthreefold higher than in the untreated controls during the following 3.5 h (Fig. 4B). Moreover, 5-HPETE and 9-HPOD, the main hydroperoxides generated by 5-LOX from arachidonic [eicosatetraenoic (ETE)] and linoleic (octadecadienoic) acid, respectively [20], dose-dependently increased cytochrome c release from mitochondria, reaching an ⬃threefold maximum at a concentration of 1 ␮M (Fig. 4C).

DISCUSSION

Fig. 3. Mitochondrial uncoupling and cytochrome c release in human lymphocytes subjected to conditions similar to those that occur during exposure of cells to microgravity. Cells exposed to conditions similar to those that occur during exposure of cells to microgravity (␮g) showed a time-dependent, mitochondrial uncoupling (A) and cytochrome c release (B), whereas lymphocytes kept at normal gravity (1 g) under the same experimental conditions did not. Values were expressed as fold over controls (1⫽5⫾1% of the uncoupling induced by CCCP for mitochondrial uncoupling and 0.250⫾0.040 A405 units for cytochrome c release). *, P ⬍ 0.01 compared with 1 g controls.

(Fig. 4A). Conversely, cyclosporin A was ineffective (Fig. 4A), suggesting that the permeability transition pore did not contribute to mitochondrial membrane disruption by 5-LOX. Remarkably, conditions similar to those that occur during exposure of cells to microgravity enhanced the effect of 5-LOX on mitochondrial membrane potential and cytochrome c release (Fig. 4A). Time-course experiments showed that 5-LOX-induced mitochondrial uncoupling and cytochrome c release were significant (P⬍0.01) already after 30 min and remained

Previous experiments from flight- and ground-based model systems clearly indicate unexpected alterations of human lymphocytes, leading to growth retardation and depression of mitogenic activation. However, the mechanism by which microgravity inhibits lymphocyte proliferation remains unknown, although it does not depend on the lack of binding of extracellular signaling molecules [45]. Recently, morphological detection and quantitation of Fas/APO-1 protein have suggested that spaceflight increases apoptosis in human lymphoblastoid (Jurkat) cells [12], and apoptotic death also has been shown to occur in osteoblasts [6] and thyroid carcinoma cells [46] subjected to conditions similar to those that occur during exposure of cells to microgravity. Here, we extend these observations to primary human lymphocytes, showing that these cells undergo apoptosis in a time-window (24 –72 h) compatible with the lymphocyte depression, IL-2/IL-2 receptor down-regulation, microtubule anomalies, and altered cytoskeletal gene expression already reported under spaceflight conditions [7, 11–14]. Most notably, we show that the execution of the death program is based on a temporal chain of events, which starts with the early (within 2 h) activation of 5-LOX and continues with the 5-LOX-dependent damage of mitochondrial integrity (4 h), cytochrome c release (8 h), and caspase activation, ultimately leading to apoptosis (48 h). These events, depicted in Figure 5, were confirmed in a cell-free system based on isolated mitochondria, whereas conditions similar to those that occur during exposure of cells to microgravity failed to induce apoptosis in a model of human immune cells (U937) devoid of 5-LOX. Therefore, it can be proposed that 5-LOX is a “gravity responder” that executes the apoptotic events induced by microgravity in human lymphocytes, perhaps as a result of its ability to directly damage mitochondria. We propose that 5-LOX might be one of the “early factors” regulating the response of lymphocytes to microgravity [14]. In this context, it is noteworthy that a previous study has shown no microgravityinduced stimulation of apoptosis in peripheral blood mononuclear cells or activated T cells, which instead were protected against radiation and activation-associated programmed death [47]. However, the number of differences in the methodology, including the composition of the cell population and most notably the time-window studied, might explain the discrepancy with the present study. Conditions similar to those that occur during exposure of cells to microgravity enhanced the activity of 5-LOX without increasing its protein content (Fig. 2A and Table 1), suggesting that 5-LOX activation does not require new gene expression. Consistently, the inhibition of mRNA transcription by actinomycin D and of the protein synthesis by cycloheximide did not

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affect 5-LOX activity (Table 2). Instead, its inhibition by EGTA-AM (Table 2) suggests that calcium ions are important for 5-LOX activation, in keeping with previous findings that these ions stimulate enzyme activity and favor the binding of 5-LOX to cell membranes [48]. Remarkably, in a model of programmed death of immune cells, calcium elevation has been shown to occur a few minutes after the application of the proapoptotic stimulus [22], and this might hold true also for microgravity-induced apoptosis. However, the limited amount of lymphocytes available did not allow measurement of intracellular calcium in time-course experiments. More recently, activation of cellular 5-LOX by different stimuli has been shown to depend on enzyme phosphorylation by p38 MAPK [40]. However, the lack of effect of SB203580 (Table 2) seems to rule out a role of this kinase in regulating 5-LOX activation induced by conditions similar to those that occur during exposure of cells to microgravity. It should be recalled that 5-LOX can oxygenate arachidonic ETE acid to an intermediate 5-HPETE, able to damage mitochondria and induce apoptosis [17]. This activity requires the release of ETE from lipid bilayers, catalyzed by PLA2, and the “presentation” of ETE to the active site of 5-LOX by FLAP [37]. Here, we show that the FLAP inhibitor MK886 and the PLA2 inhibitor MAFP only partially (⬃30%) inhibited 5-LOX activity in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity, whereas 5-LOX inhibitors ETYA, CA, or mAb almost completely blocked it (Table 2). These findings suggest that most of 5-LOX activity (⬃70%) in lymphocytes exposed to conditions similar to those that occur during exposure of cells to microgravity was not based on the oxygenation of membrane-derived ETE, and indeed, LTB4, the end-product of this pathway, was increased less than 5-LOX (2.5- vs. fourfold) in treated cells (Fig. 2A and Table 1). Therefore, it can be suggested that fatty acids bound to mitochondrial membranes represent an alternate substrate of 5-LOX, in line with previous observations that 5-LOX oxygenates these organelles [17]. Conversely, 12- and 15-LOX also can damage mitochondrial membranes [17], and their products can induce apoptosis in human cells [30]. However, creating conditions similar to those that occur during exposure of cells to microgravity does not affect 12- or 15-LOX activity in human lymphocytes, suggesting that these isozymes are not involved in the cellular sensitivity to weightlessness. Moreover, it should be recalled that each of the 5-LOX inhibitors used

here, widely used to block the cellular activity of this enzyme [17, 37, 49], also might have an alternate action: ETYA is an arachidonic acid analog that might affect multiple arachidonic acid-metabolizing enzymes, CA might inhibit glutathione Stransferase, and MK886 might inhibit PLA2 [17, 37, 50]. These

4 Fig. 4. Effect of 5-LOX and its products on isolated mitochondria. (A) Dose-dependence of mitochondrial uncoupling and cytochrome c release on the amount of pure barley 5-LOX after 2 h of incubation. Inhibitors of 5-LOX ETYA (10 ␮M), mAb (200 ␮g), and inhibitor of permeability transition pore cyclosporine A (CycA; 10 ␮M) were added to mitochondrial suspensions at the same time as 5-LOX. The effect of conditions similar to those that occur during exposure of cells to microgravity (␮g) on the mitochondrial alterations induced by 5-LOX (100 units) was also checked. None of the inhibitors or conditions similar to those that occur during exposure of cells to microgravity affected mitochondrial parameters when used alone. (B) Time-dependence of mitochondrial uncoupling and cytochrome c release upon incubation with 5-LOX (100 units/test). (C) Effect of the 5-LOX products 5-HPETE and 9-HPOD on cytochrome c release after 2 h of incubation. *, P ⬍ 0.01; **, P ⬍ 0.05 compared with untreated controls; @, P ⬍ 0.01 compared with 5-LOX (100)treated samples.

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Fig. 5. Model of microgravity-induced apoptosis in human lymphocytes. Creating conditions similar to those that occur during exposure of cells to microgravity enhances, in a calcium (Ca2⫹)-dependent manner, 5-LOX activity, which is directly associated to decreased mitochondrial potential (⌬⌿), increased cytochrome c (cyt c) release, and caspase activation, ultimately leading to apoptosis. The conversion by 5-LOX of arachidonic (ETE) acid released from membranes through PLA2 into 5-HPETE can contribute to mitochondrial damage, and the FLAP is needed for this conversion. The model suggests a central role for 5-LOX in microgravity-induced apoptosis and also is based on the effect of specific inhibitors (EGTA-AM, ETYA, CA, MK886, and MAFP) and inhibitory mAb on their targets along the apoptotic pathway.

actions might contribute to the sensitivity of intact cells to conditions similar to those that occur during exposure of cells to microgravity and indirectly affect 5-LOX activity. Indeed, it should be recalled that MK886 is inactive in broken cells or with purified 5-LOX [37]; therefore, the observation that intact cell treatment with this inhibitor led to reduced 5-LOX activity in a cell-free assay seems to suggest indirect effects of this compound. Overall, the mechanism by which conditions similar to those that occur during exposure of cells to microgravity enhance 5-LOX remains elusive, as investigations regarding the effect of microgravity on enzyme catalysis are also still very scant [18, 51]. However, it is noteworthy that in vitro the catalytic efficiency of soybean LOX-1, which is prototypal for

structural and kinetic studies on LOXs from different species [37], has demonstrated recently to be enhanced directly by microgravity [18]. This effect might be a result of a better dispersion in water of the substrates of 5-LOX, leading to an easier formation of enzyme-substrate complexes [52]. Also, the interaction of 5-LOX with cytoskeletal proteins [53] might play a role in the formation of the enzyme-substrate complex under microgravity, based on reaction-diffusion processes such as those involved in microtubule self-organization [54]. The availability of calcium ions might favor these interactions [48, 53]. Conversely, the possibility that the “tensegrity” paradigm [2] might supplement reaction-diffusion processes [54] in the sensitivity of enzyme reactions to microgravity remains to be

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explored. At any rate, our results clearly show that conditions similar to those that occur during exposure of cells to microgravity enhance 5-LOX activity in human lymphocytes. 5-LOX activation appears to be the critical event in apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity, as suggested by the effect of several compounds on 5-LOX and apoptosis (Table 2). In this context, it seems of particular interest that 5-LOX directly uncouples mitochondria leading to cytochrome c release and that 5-HPETE generated by 5-LOX through PLA2 and FLAP contributes to ⬃30% of mitochondrial damage (Table 2). The effect of 5-LOX and its main products on isolated mitochondria (Fig. 4) strongly supports this conclusion. These findings are in keeping with a function for LOXs in programmed organelle degradation, based on the ability of the 15-isozyme to make pore-like structures in the lipid bilayer [44]. Calcium might favor the release of mitochondrial cytochrome c [55], whereas opening the mitochondrial-permeability transition pore [42] does not seem to contribute to 5-LOX-induced mitochondrial damage (Fig. 4A) nor to apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity (Table 2), as suggested by the lack of effect of cyclosporin A. The release of cytochrome c is indeed a converging point in apoptotic death induced by unrelated stimuli in different cell types [31, 43, 55]. Its release in the cytosol is usually followed by activation of a caspase cascade, initiated by caspase-3 and caspase-9, which are the most proximal members of the proteolytic chain [35, 43]. Caspases are thought to form a proteolytic machinery within the cell, resulting in the breakdown of key enzymes and cellular structures, and to activate DNases responsible for the chromatin degradation seen in apoptosis [35, 43]. Also, apoptosis induced by conditions similar to those that occur during exposure of cells to microgravity appears to be executed through this series of events, as caspase-3 or caspase-9 inhibitors reduced apoptotic body formation to 25–30% of the controls (Table 2). In conclusion, we have demonstrated that human lymphocytes are forced to apoptosis by conditions similar to those that occur during exposure of cells to microgravity, through a pathway based on calcium-dependent 5-LOX activation, mitochondrial membrane disruption, and cytochrome c release, followed by caspase activation and cell death, as summarized in Figure 5.

ACKNOWLEDGMENTS Financial support was provided by Agenzia Spaziale Italiana (contracts ARS-99-39 and I/R/098/00), Rome, Italy. The authors thank Drs. Riccardo Pauselli and Marianna Di Rienzo for their skillful assistance.

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