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APO-1 protein is increased in spaceflown lymphocytes (Jurkat)☆
Experimental Gerontology 35 (2000) 389 – 400
Fas/APO-1 protein is increased in spaceflown lymphocytes (Jurkat)夞 Luis A. Cubano, Marian L. Lewis* Department of Biological Sciences and Microgravity Biotechnology Laboratory, University of Alabama in Huntsville, Wilson Hall Room 360, Huntsville, AL 35899, USA Received 13 December 1999; received in revised form 22 February 2000; accepted 22 February 2000
Abstract Human lymphocytes flown on the Space Shuttle respond poorly to mitogen stimulation and populations of the lymphoblastoid T cell line, Jurkat, manifest growth arrest, increase in apoptosis and time- and microgravity-dependent increases in the soluble form of the cell death factor, Fas/APO-1 (sFas). The potential role of apoptosis in population dynamics of space-flown lymphocytes has not been investigated previously. We flew Jurkat cells on Space Transportation System (STS)-80 and STS-95 to determine whether apoptosis and the apparent microgravity-related release of sFas are characteristic of lymphocytes in microgravity. The effects of spaceflight and groundbased tests simulating spaceflight experimental conditions, including high cell density and low serum concentration, were assessed. Immunofluorescence microscopy showed increased cell associated Fas in flown cells. Results of STS-80 and STS-95 confirmed increase in apoptosis during spaceflight and the release of sFas as a repeatable, time-dependent and microgravity-related response. Ground-based tests showed that holding cells at 1.5 million/ml in medium containing 2% serum before launch did not increase sFas. Reports of increased Fas in cells of the elderly and the increases in spaceflown cells suggest possible similarities between aging and spaceflight effects on lymphocytes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Fas; Apoptosis; Spaceflight; Lymphocytes; Jurkat
1. Introduction Mammalian cells subjected to conditions of spaceflight and the microgravity environment of space, manifest a number of alterations in structure and function. Among the most 夞 This work was supported by NASA Grants NAG2-985, NCC8-132, and NASA Graduate Student Research Program Grant 97-GSRP-076. * Corresponding author. Tel.: ⫹1-256-890-6553; fax: ⫹1-256-890-6376. E-mail address: [email protected] (M.L. Lewis) 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 9 0 - 5
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notable changes in cells flown on the Space Shuttle are reduced growth activation and decline in growth rate in the total population (Tipton et al., 1996; Cogoli, 1996; Lewis et al., 1998). Other changes include chromosomal aberrations (Yang et al., 1997), inhibited locomotion (Pellis et al., 1997), altered cytokine production (Cogoli and Cogoli–Greuter, 1997; Chapes et al., 1992), changes in PKC distribution (Schmitt et al., 1996), and increased apoptosis (Lewis et al., 1998). Human lymphocytes respond poorly to mitogenic stimulation in microgravity (Cogoli, 1996) and cells of the lymphoblastoid T cell line, Jurkat, are growth arrested (Lewis et al., 1998). Changes similar to those reported for space flown cells are also seen in lymphocytes of aging individuals. These include chromosomal damage (Bolognesi et al., 1999), cell cycle changes (Arbogast et al., 1999; Quadri et al. 1998), altered cytokine production (Paganelli, 1994), differences in PKC distribution (Fulop et al., 1995), and increases in apoptosis and Fas (Aggarwal and Gupta 1998; Potestio et al., 1998; Seishima et al., 1996, Phelouzat et al., 1997). Considerable research over the past three decades has been devoted to characterizing space-related immune cell alterations, yet mechanisms remain largely unknown (Sonnenfeld, 1998). In Jurkat cells flown on Space Shuttle mission STS-76, we noticed an increase in number of apoptotic cells and microgravity-related increase in the cell death factor, soluble Fas/APO-1 (sFas) compared to ground controls (Lewis et al., 1998). We undertook the present study using Jurkat cells flown on Space Shuttle missions STS-80 and STS-95 to investigate the possibility that apoptosis may be a factor in negative population growth response of lymphocytes in microgravity and to further study the increase of Fas in spaceflown cells. The effect of spaceflight-related culture conditions, including high population density and serum reduction to retard cell growth before launch, on apoptosis and sFas release were also characterized. Results are significant to the understanding of cell growth and aging during spaceflight and present new evidence that microgravity affects apoptosis in space-flown lymphocytes. Apoptosis, a Greek word meaning falling away, is the process of cellular suicide triggered by internal (Evan and Littlewood, 1998; McKenna et al., 1998) or external signals (Guchelaar et al., 1997, Schulze–Osthoff et al., 1998). It is characterized by DNA fragmentation (Wyllie, 1980), loss of mitochondrial membrane potential (Susin et al., 1998), cytoskeletal disruption, cell shrinkage, membrane blebbing, and the breakdown of the cell into apoptotic bodies (Cohen, 1993; Squier et al., 1995; Thompson, 1995). Apoptosis in human lymphocytes is commonly mediated by Fas/APO-1 and Fas ligand (Fas-L). Fas/APO-1 (Oehm et al., 1992), also designated as CD95 (Moller et al., 1994), is a member of the tumor necrosis family (TNF) expressed on the surface of various cell types. Fas/APO-1 contains a single membrane spanning region and transduces a death signal when it binds to Fas Ligand (Fas-L), making it the protein responsible for the induction of apoptosis (Nagata and Golstein, 1995). Recently, a variety of Fas proteins produced by alternative splicing and lacking the transmembrane region have been reported (Cascino et al., 1995; Cascino et al., 1996). These are designated as soluble Fas (sFas). These proteins have the ability to bind Fas-L before it binds to Fas (Lee et al., 1998) and have been identified as a possible protection mechanism against Fas-Fas-L induced apoptosis (Cheng et al., 1994). The ability of tumor cells, such as Jurkat, to produce sFas could serve to protect them from apoptosis (Owen–Schaub et al., 1995). The present study was conducted to further characterize the apparent microgravityrelated release of sFas into the medium of Jurkat cells flown on the Shuttle. Results from STS-80 and STS-95 confirm that apoptosis in Jurkat cells is increased during spaceflight and the increased production of Fas is microgravity environment-related. Ground-based
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tests show that Shuttle flight-related cell culture conditions, including lowered serum concentration and cell culture densities characteristic of these experiments do not induce increases in apoptosis or Fas. This information significantly advances our understanding of cellular response during spaceflight and supports the concept that lymphocyte growth and aging are affected in the microgravity environment.
2. Materials and methods 2.1. Cell culture The human lymphoblastoid cell line, Jurkat clone E6 –1, was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were certified free of mycoplasma contamination by the ATCC. Culture medium consisted of RPMI 1640 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% heat inactivated fetal, bovine serum (FBS) (Summit Biotechnology, Santa Ana, CA, USA) 2 mM glutamine, 1 mM sodium pyruvate, 1 ml/100 of 100 ⫻ nonessential amino acids, and penicillin and streptomycin, 100 units and 100 g/ml, respectively (Life Technologies, Grand Island, NY, USA) and 12.5 mM HEPES buffer (Sigma, St. Louis, MO, USA). Cells, cultured in our laboratory from the ATCC frozen stocks, were used at passage levels three through eight for all tests and flight experiments. 2.2. Hardware Flight and ground-based control hardware for the experiments flown on STS-80 and STS-95 consisted of Bioprocessing modules (BPMs) assembled in our laboratory. BPMs are comprised of four syringes interconnected by tubing via a four-way valve. Polypropylene filter units (Advantec MFS, Inc., Pleasanton, CA, USA) with a Millipore HTTP, 0.4 micron Isopore® membrane (Millipore, Bedford, MA, USA), placed between the valve and the cell syringes allowed separation of cells and culture medium before fixing the cells in microgravity. Manual operation permitted mixing of fluids when an astronaut aligned the BPM valve position and pushed appropriate syringe plungers. The BPMs differed primarily from the hardware used previously on STS-76 (Lewis et al., 1998; Hatton et al., 1998) in that the BPMs accommodated approximately six to ten times more volume than the STS-76 experiment hardware. 2.3. Experimental details In preparation for flight on STS-80 and STS-95, 1.5 million cells/ml were suspended in medium containing 2% FBS and duplicate syringes in each BPM were loaded with 3 ml of the suspension. A third syringe was loaded with 4 ml of medium containing FBS calculated to give a final concentration of 10% when injected into the syringes containing cells. The fourth syringe was loaded with 4 mls of 3% formalin in Dulbecco’s phosphate buffered saline. After loading, BPMs were maintained at 20°C before launch (approximately 28 h) and until an astronaut initiated the experiments on orbit by increasing the temperature to 37°C and injecting 2 ml of activator (medium containing 22% FBS) into each of the syringes containing cells to increase the FBS concentration to 10% to stimulate cell growth. Experiments were activated at 6 (STS-80) and 4.5 (STS-95) h after launch.
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At selected times after activation, cells were filtered from the medium and a 2 ml volume of 3% formalin was injected into each cell syringe. The cells for evaluation of morphologically detectable apoptosis, and culture medium for assay of sFas were stored at 4°C (STS-80) until processing in the laboratory after flight. The medium from STS-95 was stored frozen at ⫺80°C except for the 4 h sample, which was stored unfrozen. For STS-80, cells were fixed immediately and at 4, 24, 48, and 75 h after activation. On STS-95, cells where fixed at 4 and 48 h after activation and medium was collected at 4, 24, and 48 h after activation. 2.4. Apoptosis analyses Cell-free culture medium was tested for sFas protein by an enzyme-linked immunosorbent assay (ELISA) according to methodology and reagents supplied with kits purchased from Oncogene Sciences (Uniondale, NY, USA). The sFas concentration in units/ml was determined from triplicate optical density readings for each sample in duplicate compared to the Oncogene Sciences kit standard diluted in wells of a 96 well plate and read on a Bio–Tek EL 340 microplate reader (Bio–Tek, Winooski, VT, USA). Deltasoft Soft II software for the Bio–Tek Microplate Reader interfaced with a Macintosh computer was used to generate the sFas concentration and standard error of the mean (SEM) for each sample. A unit is define by the kit supplier as the amount of sFas present in 104 cells/ml in cultures of HuT 78 cells. The sensitivity of the assay is 0.05 units/ml. Visualization of Fas in formalin-fixed cells was carried out by immunofluorescence microscopy. Cells were incubated with anti-Fas antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h followed by rinsing and incubation with Texas Redconjugated second antibody (Molecular Probes, Eugene, OR, USA) for 1 h. Cell associated Fas was visualized using a Nikon Labophot microscope equipped with epifluorescence. Formalin fixed cells were evaluated for apoptosis by staining with Hoechst 33253 (Molecular Probes, Eugene, OR, USA) to visualize apoptotic bodies. Cells were distributed by use of a Cytospin 3 (Shandon Lipshaw, Pittsburgh, PA, USA) onto BSA coated coverslips and permeabilized with Triton X 100 (Boehringer, Indianapolis, IN, USA) for 3 min. Cells were then incubated at room temperature with 25 g/ml of Hoechst in phosphate buffered saline for 5 min and the bright blue stained nuclei were counted using a Nikon Labophot microscope equipped with epifluorescence and a 364 nm filter. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid operator bias and re-count of the same cells. Data are expressed as the mean and SEM of the percent of cells in apoptosis calculated from more than 300 cells on each of at least two slide preparations for each sample.
3. Results 3.1. Morphologically detectable apoptosis Counts of cells stained with Hoechst 33253 showed that approximately 9% to 10% of the flown Jurkat cell populations were apoptotic at all time points sampled on both STS-80 (Fig. 1) and STS-95 (Fig. 2). The 0 h baseline of approximately 2% apoptosis (Fig. 1) was
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Fig. 1. Morphologically detected apoptosis in Jurkat cells flown on STS-80. Cells were stained with Hoechst 33253 as described in Section 2. Data represent the average of a minimum of 300 cells/slide for each condition evaluated on at least two slide preparations per sample. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid re-counting the same cells. Error bars represent the SEM of two or more counts of 300 each for each sample.
similar to that of cells cultured under standard, optimal laboratory conditions. Apoptosis in ground controls, handled by the same procedures and timeline as flight, did not exceed the STS-80 value of 6.4% at 4 h after serum concentration and temperature were increased. At 4 h after activation of cells on STS-80, apoptosis was significantly increased in both flight and ground controls (Fig. 1). This was followed by a decrease at 24 and 48 h and an increase again at 75 h as the culture aged and nutrients were depleted. In flown samples, the number of apoptotic cells at 24 and 48 h was more than three times higher than for ground controls. After 75 h, apoptosis in flown and ground populations increased, however; flown cells had approximately 1.7⫻ more apoptotic cells than ground controls. For cells flown on STS-95, the incidence of apoptosis at 4 and 48 h (Fig. 2) was almost identical to STS-80. The maximum number of apoptotic cells in ground controls did not exceed 3.6%, whereas flown cell populations were 9.3% apoptotic at 48 h. 3.2. Increase in cell associated Fas during Spaceflight Cells fixed with formalin 4 h after growth stimulation showed no cell associated Fas in either flight or ground controls (Fig. 3a and b). The number of Fas positive cells was increased in flown samples and at 48 h, almost all of the flown cells were positive (Fig. 3c). At the same time point, only a few ground control cells were positive for Fas (Fig. 3d).
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Fig. 2. Morphologically detected apoptosis in Jurkat cells flown on STS-95. Cells were stained with Hoechst 33253 as described in Section 2. Data represent the average of a minimum of 300 cells/slide evaluated on at least two slide preparations per sample. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid re-counting the same cells. Error bars represent the SEM of two or more counts of 300 each for each sample.
3.3. Increase in sFas during Spaceflight Because of crew time and hardware limitations, the medium from two different time points (0 and 4 h; and 4 and 24 h) was pooled after activation in microgravity for the STS-80 experiment. Although not significant because of pooling the samples, the concentration of soluble Fas in the medium of cells flown on STS-80 seemed to increase over time from 0 to 24 h (Fig. 4). Due to a hardware anomaly, the 48-h sample for STS-80 was lost. For STS-95, the concentration of sFas significantly increased between 24 and 48 h (Fig. 5), thus confirming the time- and microgravity-dependent increase in sFas as previously reported (Lewis et al., 1998). Ground control values for sFas were negative for both STS-80 and STS-95 with the exception of the 0.2 units at the 4 h time point for the STS-95 ground control. The negative values for sFas (Figs. 4 and 5) represent absorption values below the lowest concentration of the standard. The software (Delta Soft II) program used to compare test values to the standard to obtain test sample concentrations extrapolates values into the negative range. 3.4. Effect of serum concentration and cell density on sFas release To retard cell growth until activation of the experiment in space, cells were held in medium containing 2% FBS at 20°C for approximately 28 h total before launch and before activation of the cells on orbit. The potential effect of serum concentration on release of sFas was evaluated in ground-based experiments by maintaining approximately 1.5
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Fig. 3. Cell associated Fas in ground and flight cells. Formalin-fixed cells were evaluated for presence of cell associated Fas by immunofluorescence microscopy as described in Section 2. At 4 h, both flown (a) and ground control (b) cells were negative for Fas. By 48 h, almost all of the flight cells were positive (c) as shown by the arrows. Conversely, only one of the five ground control cells stained positive for Fas (d).
million Jurkat cells/ml at 20°C in the laboratory for 24 h in medium containing 0%, 2%, and 10% FBS. At all serum concentrations, sFas values were negative showing that serum concentration did not affect release of sFas in the culture medium. The spaceflight experiments were set up at a concentration of approximately 1.5 million cells/ml to have a sufficient number of cells for post-flight analyses. To determine if cell density could affect the amount of sFas released into the medium, cell cultures were initiated in the laboratory at 1.2 million/ml and allowed to grow without the addition of fresh medium for 72 h. The cells grew to a density of more than 4 million/ml. Viability dropped sharply from 98% at 24 h to 45% at 72 h. Fig. 6 shows that the number of apoptotic nuclei were within the normal range of approximately 2% to 3% at 24 h but increased to 8% by 72 h as the viability declined at the high cell density. Soluble Fas levels remained at approximately zero, as expected at 0 to 24 h but increased to almost 1 unit when the culture became overgrown by 72 h.
4. Discussion The present study was initiated to address whether increased apoptosis and a timedependent, microgravity-related, release of the cell death factor, soluble Fas/APO-1 (sFas), reported previously (Lewis et al., 1998) are characteristic responses of lymphocytes to conditions of spaceflight and microgravity. Results confirmed an increase in sFas in medium of Jurkat cells flown on the Shuttle and define this as a repeatable, time- and microgravity-related response of these cells. Furthermore, this study also confirms that apoptosis, one of the characteristics of immunoscenescence (Phelouzat et al., 1996), is increased during space flight. High cell population density and low serum concentration in the medium were evaluated in ground-based studies to determine the effect on sFas because these were
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Fig. 4. Soluble Fas in cell-free medium of STS-80 flight and ground control cells. Because of the design of the spaceflight experiment due to hardware constraints, the medium samples taken at 0 and 4 h and 4 and 24 h were pooled. The Fas concentration was determined from triplicate optical density readingings for each sample in duplicate compared to the standard as described in Section 2. Error bars represent the SEM calculated by a Deltasoft Soft II program for the Bio–Tek Microplate Reader interfaced with a Macintosh computer to generate the Fas concentration and SEM. Though not statistically significant, medium from 0/4 h and 4/24 h showed an increase in the amount of sFas as time in microgravity increased. All ground control samples were negative for sFas. (Values were extrapolated into the negative range by the Delta Soft II software used to compare test values to the standard to obtain test sample concentrations).
conditions to which cells were subjected during the flight experiments. For serum concentrations tested (0%, 2%, and 10%), the sFas concentration in medium was essentially zero. Thus, holding cells for approximately 28 h in medium containing 2% serum before launch and until activation on orbit had no effect on sFas release. In ground-based tests, high cell density culture resulted in a slight, but not statistically significant, increase in the total concentration of sFas in the culture medium over a period of 0 to 72 h (Fig. 6). At 72 h, the culture was in decline as indicated by decreased viability (data not shown), and an increased number of cells undergoing apoptosis (Fig. 6). The increase in the level of sFas in medium of space-flown cells is therefore not a result of high culture density but rather some other factor, possibly radiation or microgravity per se, characteristic of the microgravity environment. Owen–Schaub et al. (1995) suggest that tumor cells may produce sFas as a means of protecting against apoptosis. Based on results of STS-80 and STS-95, it does not seem that sFas protects the cells from apoptosis during spaceflight because the percentage of apoptotic cells remained at 9 to 10% throughout yet sFas levels in the medium increased with time in microgravity. Longer duration experiments in microgravity in which cells are maintained by adding fresh medium are needed to assess whether sFas is correlated with recovery of the population from apoptosis. Holding cells in low-serum medium at low temperature (20°C) may have predisposed
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Fig. 5. Amount of sFas in cell-free medium of STS-95 flight and ground controls. Flight samples taken at, 24 and 48 h showed a time-dependent and microgravity-related increase in sFas. The sFas levels in ground controls did not increase with time. The Fas concentration was determined from triplicate optical density readings for each sample in duplicate compared to the standard as described in Section 2. Error bars represent the SEM calculated by a Deltasoft Soft II program for the Bio–Tek Microplate Reader interfaced with a Macintosh computer to generate the Fas concentration and SEM. The Delta Soft II software used to compare test values to the standard to obtain test sample concentrations extrapolates values into the negative range.
them to apoptosis and may account for the increase in number of apoptotic cells in samples 4 h after activation (Figs. 1 and 2). It is unlikely that these factors alone account for the 9% to 10% apoptosis in flown cultures at 24 and 48 h compared to ground controls that were less than 4% apoptotic. Messman and Pittman (1998) showed that PC12 cells cultured in medium without serum could be rescued if the serum was added back. The increase in morphologically detectable apoptosis at 4 h (Figs. 1 and 2) may be due to cells already committed to die due to serum reduction. Ground control cells seemed to recover after increasing serum to 10% because the number of apoptotic cells at 24 and 48 h decreased to baseline levels. However, approximately 10% of the cells in microgravity continued to exhibit apoptosis indicating that serum reduction is not responsible for apoptosis in microgravity. In space, the cells do not respond to growth activation, i.e., they do not grow when serum is added and the temperature is raised to 37°C, whereas the ground controls increase by a factor of 1.6 in the same time (Lewis et al., 1998) Thus in space, sFas does not seem to be associated with increased growth activation. Soluble Fas increases were more notable after 48 h in microgravity and were higher than ground controls, which responded to growth activation but were essentially negative for sFas. We have found that use of the Jurkat cell line provides a stock of consistently growing, easily cultured, readily available cells thus reducing variables associated with use of fresh peripheral lymphocytes or blood bank buffy coat preparations. By using Jurkat cells, we were also able to show that results from STS-80 and STS-95 were consistent with our results from STS-76 flown in different hardware. For all tests and flight experiments cells
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Fig. 6. Effect of cell density on apoptosis and sFas. The percentage of apoptotic nuclei and sFas in culture medium were evaluated as described in Section 2. Counts of cells stained with Hoechst 33253 for visualization of apoptotic bodies showed a significant increase in apoptosis as time in culture and culture density increased. Though the concentration of sFas in the medium increased to 1.3 units/ml by 72 h, this value was less than that for the flown cultures at 48 h (Fig.5).
were cultured in our laboratory and used at passage levels three through eight from the ATCC frozen stocks to minimize culture to culture variability. And for all flight experiments, the cells were suspended in culture medium at 1.5 million cells/ml to ensure consistency among experiments. Jurkat cells have been flown on a number of Space Shuttle missions (Lewis et al., 1998; Hatton et al., 1998; Limouse et al., 1991; Schmitt et al., 1996). The cells are easily and consistently growth-stimulated by increasing the serum concentration from 2% to 10%. Increased cell death and Fas in the membrane of cells (Phelouzat et al., 1997) has been related to the aging process. Criswell–Hudak (1991) proposed looking at aging as an adaptation and comparing it to the adaptations that occur during spaceflight to obtain information about aging. Our results with space-flown Jurkat cells show that these cells are an excellent model for investigating cell aging under gravity-altered conditions. Our data now add new information to expand the current similarities (Hughes–Fulford, 1991; Czeisler et al., 1991) between aging and spaceflight. The increase in amounts of cell associated Fas in immune cells in space and in the elderly (Aggarwal and Gupta, 1998; Potestio et al., 1998; Seishima et al., 1996), further suggests that spaceflight may contribute to premature aging of lymphocytes.
Acknowledgments The authors express their appreciation to the crews of STS-80 and STS-95 and especially to Astronauts John Glenn and Scott Parazynski whose expertise resulted in
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100% success of on-orbit operations of our experiments on STS-95. We also express our appreciation to the support facility at KSC for the help in the pre-launch phase of the experiments. Thanks to Dr E. H. Piepmeier for facilitating the immunofluorescence assays and photography. We acknowledge C. A. Yancey and K. L. Murphy for technical and administrative support throughout the experiments. References Aggarwal, S. & Gupta, S. (1998). Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl-2, and Bax. J Immunol 160, 1627–1637. Arbogast, A., Boutet, S., Phelouzat, M. A., Plastre, O., Quadri, R., & Proust, J. J. (1999). Failure of T lymphocytes from elderly humans to enter the cell cycle is associated with low Cdk6 activity and impaired phosphorylation of Rb protein. Cell Immunol 197, 46 –54. Bolognesi, C., Lando, C., Forni, A., Landini, E., Scarpato, R., Migliore, L., & Bonassi, S. (1999). Chromosomal damage and ageing: effect on micronuclei frequency in peripheral blood lymphocytes. Age Ageing 28, 393–397. Cascino, I., Fiuccci, G., Papoff, F., & Ruberti, G. (1995). Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol 154, 2706 –2713. Cascino, I., Papoff, F., Eramo, A., & Ruberti, G. (1996). Soluble Fas/Apo-1 splicing variants and apoptosis. Front Biosci 1, d12–18. Chapes, S. K., Morrison, D. R., Guikema, J. A., Lewis, M. L., & Spooner, B. S. (1992). Cytokine production by immune cells in space. J Leuk Biol 52, 104 –110. Cheng, J., Zhou, T., Liu, C., Shapiro, J., Vrauer, M., Kiefer, M., Varr, P., & Mounts, J. (1994). Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263, 1759 –1762. Cogoli, A. (1996). Gravitational physiology of human immune cells: a review of in vivo, ex vivo, and in vitro studies. J Gravitational Physiol 3, 1–9. Cogoli, A. Cogoli–Greuter, M. (1997). Activation and proliferation of lymphocytes and other mammalian cells in microgravity. Adv Space Biol Med 6, 33–79. Cohen, J. (1993). Apoptosis. Immunology Today 14, 126 –130. Criswell–Hudak, B. (1991). Immune response during space flight. Exp Gerontol 26, 289 –296. Czeisler, C., Chiasera, A., & Duffy, J. (1991). Research on sleep, circadian rhythms and aging: applications to manned spaceflight. Exp Gerontol 26, 217–232. Evan, G. & Littlewood, T. (1998). Apoptosis. Sci 281, 1317–1321. Fulop, T. Jr., Leblanc, C., Lacombe, G., & Dupuis, G. (1995). Cellular distribution of protein kinase C isozymes in CD3-mediated stimulation of human T lymphocytes with aging. FEBS Lett 375, 69 –74. Guchelaar, H., Vermes, A., Vermes, I., & Haanen, C. (1997). Apoptosis: molecular mechanisms and implications for cancer chemotherapy. Pharm World Sci 19, 119 –125. Hatton, J., Lewis, M., Roquefeuil, S., Chaput, D., Cazenave, J., & Schmitt, D. (1998). Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity. J Cell Biochem 70, 252– 67. Hughes–Fulford, M. (1991). Altered cell function in microgravity. Exp Gerontol 26, 247–256. Lee, S., Kim, S., Lee, J., Shin, M., Dong, A., Na, W., Park, Q., Kim, K., Kim, C., Kim, S., & Yoo, N. (1998). Detection of soluble Fas mRNA using in situ reverse transcription-polymerase chain reaction. Lab Invest 78, 453– 459. Lewis, M., Reynolds, J. L., Cubano, L. A., Hatton, J. P., Lawless, B. D., & Piepmeier, E. H. (1998). Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat). FASEB J 12, 1007–1018. Limouse, M., Manie, S., Konstantinova, I., Ferrua, B., & Schaffar, L. (1991). Inhibition of phorbol ester-induced cell activation in microgravity. Exp Cell Res 197, 82– 86. McKenna, S., McGowan, A., & Cotter, T. (1998). Molecular mechanisms of programmed cell death. Adv Biochem Eng Biotechnol 62, 1–31. Messman, C. A., Pittman, R. N. (1998). Asynchrony and commitment to die during apoptosis. Exp Cell Res 238, 389 –398. Moller, P., Koretz, K., Leithauser, F., Bruderlein, S., Henne, C., Quentmeier, A., & Krammer, P. (1994). Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 57, 371–377.
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Nagata, S. & Golstein, P. (1995). The Fas death factor. Science 267, 1449 –1456. Oehm, A., Behrmann, I., Falk, W., Pawlita, M., Maier, G., Klas, C., Li–Eever, M., Richards, S., Dhein, J., & Trauth, B. (1992). Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen. J Biol Chem 267, 10709 –10715. Owen–Schaub, L., Angelo, L., Radinsky, R., Ware, C., Gerner, T., & Bartos, D. (1995). Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis? Cancer Lett 94, 1– 8. Paganelli, R., Scala, E., Quinti, I., & Ansotegui, I. J. (1994). Humoral immunity in aging. Aging (Milano) 6, 143–150. Pellis, N., Goodwin, T. J., Risin, D., McIntyre, B. W., Pizzini, R. P., Cooper, D., Baker, T. L., & Spaulding, G. F. (1997). Changes in gravity inhibit lymphocyte locomotion through type I collagen. In Vitro Cell Dev Biol Anim 33, 398 – 405. Phelouzat, M., Laforge, T., Arbogast, A., Quadri, T., Boutet, S., & Proust, J. (1997). Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors. Mech Aging Dev 96, 35– 46. Phelouzat, M. A., Arbogast, A., Laforge, T., Quadri, R. A., & Proust, J. J. (1996). Excessive apoptosis of mature T lymphocytes is a characteristic feature of human immune senescence. Mech Ageing Dev 88, 25–38. Potestio, M., Caruso, C., Gervasi, F., Scialabba, G., D’Anna, C., Di Lorenzo, G., Balistreri, C. R., Candore, G., & Romano, G. C. (1998). Apoptosis and ageing. Mech Ageing Dev 102, 221–237. Quadri, R. A., Arbogast, A., Phelouzat, M. A., Boutet, S., Plastre, O., & Proust, J. J. (1998). Age-associated decline in cdk1 activity delays cell cycle progression of human T lymphocytes. J Immunol 161, 5203–5209. Schmitt, D., Hatton, J., Emond, C., Chaput, D., Paris, H., Levade, T., Cazenace, J., & Schaffar. L. (1996). The distribution of protein kinase C in human leukocytes is altered in microgravity. FASEB J 10, 1627–1634. Schulze–Osthoff, K., Ferrari, F., Los, M., Wesselborg, S., & Peter, M. (1998). Apoptosis signaling by death receptors. Eur J Biochem 254, 439 – 459. Seishima, M., Takemura, M., Saito, K., Sano, H., Minatoguchi, S., Fijiwara, H., Hachiya, T., & Noma, A. (1996). Highly sensitive ELISA for soluble Fas in serum: increased soluble Fas in the elderly. Clin Chem 42, 1911–1914. Sonnenfeld, G. (1998). Immune Response in Space Flight. Int J Sports Med 19, S195–S204. Squier, M. K., Sehnert, A. J., & Cohen, J. J. (1995). Apoptosis in leukocytes. J Leukoc Biol 57, 2–10. Susin, S., Zamzami, N., & Kroemer, G. (1998). Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1366, 151–165. Thompson, C. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456 –1462. Tipton, C., Greenleaf, J., & Jackson, C. (1996). Neuroendocrine and immune system responses with spaceflights. Med Sci Sports Exerc 28, 988 –998. Wyllie, A. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556. Yang, T., George, K., Johnson, A. S., Durante, M., & Fedorenko, B. S. (1997). Biodosimetry results from space flight Mir-18. Radiat Res 148, S17–23.
Fas/APO-1 protein is increased in spaceflown lymphocytes (Jurkat)夞 Luis A. Cubano, Marian L. Lewis* Department of Biological Sciences and Microgravity Biotechnology Laboratory, University of Alabama in Huntsville, Wilson Hall Room 360, Huntsville, AL 35899, USA Received 13 December 1999; received in revised form 22 February 2000; accepted 22 February 2000
Abstract Human lymphocytes flown on the Space Shuttle respond poorly to mitogen stimulation and populations of the lymphoblastoid T cell line, Jurkat, manifest growth arrest, increase in apoptosis and time- and microgravity-dependent increases in the soluble form of the cell death factor, Fas/APO-1 (sFas). The potential role of apoptosis in population dynamics of space-flown lymphocytes has not been investigated previously. We flew Jurkat cells on Space Transportation System (STS)-80 and STS-95 to determine whether apoptosis and the apparent microgravity-related release of sFas are characteristic of lymphocytes in microgravity. The effects of spaceflight and groundbased tests simulating spaceflight experimental conditions, including high cell density and low serum concentration, were assessed. Immunofluorescence microscopy showed increased cell associated Fas in flown cells. Results of STS-80 and STS-95 confirmed increase in apoptosis during spaceflight and the release of sFas as a repeatable, time-dependent and microgravity-related response. Ground-based tests showed that holding cells at 1.5 million/ml in medium containing 2% serum before launch did not increase sFas. Reports of increased Fas in cells of the elderly and the increases in spaceflown cells suggest possible similarities between aging and spaceflight effects on lymphocytes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Fas; Apoptosis; Spaceflight; Lymphocytes; Jurkat
1. Introduction Mammalian cells subjected to conditions of spaceflight and the microgravity environment of space, manifest a number of alterations in structure and function. Among the most 夞 This work was supported by NASA Grants NAG2-985, NCC8-132, and NASA Graduate Student Research Program Grant 97-GSRP-076. * Corresponding author. Tel.: ⫹1-256-890-6553; fax: ⫹1-256-890-6376. E-mail address: [email protected] (M.L. Lewis) 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 9 0 - 5
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notable changes in cells flown on the Space Shuttle are reduced growth activation and decline in growth rate in the total population (Tipton et al., 1996; Cogoli, 1996; Lewis et al., 1998). Other changes include chromosomal aberrations (Yang et al., 1997), inhibited locomotion (Pellis et al., 1997), altered cytokine production (Cogoli and Cogoli–Greuter, 1997; Chapes et al., 1992), changes in PKC distribution (Schmitt et al., 1996), and increased apoptosis (Lewis et al., 1998). Human lymphocytes respond poorly to mitogenic stimulation in microgravity (Cogoli, 1996) and cells of the lymphoblastoid T cell line, Jurkat, are growth arrested (Lewis et al., 1998). Changes similar to those reported for space flown cells are also seen in lymphocytes of aging individuals. These include chromosomal damage (Bolognesi et al., 1999), cell cycle changes (Arbogast et al., 1999; Quadri et al. 1998), altered cytokine production (Paganelli, 1994), differences in PKC distribution (Fulop et al., 1995), and increases in apoptosis and Fas (Aggarwal and Gupta 1998; Potestio et al., 1998; Seishima et al., 1996, Phelouzat et al., 1997). Considerable research over the past three decades has been devoted to characterizing space-related immune cell alterations, yet mechanisms remain largely unknown (Sonnenfeld, 1998). In Jurkat cells flown on Space Shuttle mission STS-76, we noticed an increase in number of apoptotic cells and microgravity-related increase in the cell death factor, soluble Fas/APO-1 (sFas) compared to ground controls (Lewis et al., 1998). We undertook the present study using Jurkat cells flown on Space Shuttle missions STS-80 and STS-95 to investigate the possibility that apoptosis may be a factor in negative population growth response of lymphocytes in microgravity and to further study the increase of Fas in spaceflown cells. The effect of spaceflight-related culture conditions, including high population density and serum reduction to retard cell growth before launch, on apoptosis and sFas release were also characterized. Results are significant to the understanding of cell growth and aging during spaceflight and present new evidence that microgravity affects apoptosis in space-flown lymphocytes. Apoptosis, a Greek word meaning falling away, is the process of cellular suicide triggered by internal (Evan and Littlewood, 1998; McKenna et al., 1998) or external signals (Guchelaar et al., 1997, Schulze–Osthoff et al., 1998). It is characterized by DNA fragmentation (Wyllie, 1980), loss of mitochondrial membrane potential (Susin et al., 1998), cytoskeletal disruption, cell shrinkage, membrane blebbing, and the breakdown of the cell into apoptotic bodies (Cohen, 1993; Squier et al., 1995; Thompson, 1995). Apoptosis in human lymphocytes is commonly mediated by Fas/APO-1 and Fas ligand (Fas-L). Fas/APO-1 (Oehm et al., 1992), also designated as CD95 (Moller et al., 1994), is a member of the tumor necrosis family (TNF) expressed on the surface of various cell types. Fas/APO-1 contains a single membrane spanning region and transduces a death signal when it binds to Fas Ligand (Fas-L), making it the protein responsible for the induction of apoptosis (Nagata and Golstein, 1995). Recently, a variety of Fas proteins produced by alternative splicing and lacking the transmembrane region have been reported (Cascino et al., 1995; Cascino et al., 1996). These are designated as soluble Fas (sFas). These proteins have the ability to bind Fas-L before it binds to Fas (Lee et al., 1998) and have been identified as a possible protection mechanism against Fas-Fas-L induced apoptosis (Cheng et al., 1994). The ability of tumor cells, such as Jurkat, to produce sFas could serve to protect them from apoptosis (Owen–Schaub et al., 1995). The present study was conducted to further characterize the apparent microgravityrelated release of sFas into the medium of Jurkat cells flown on the Shuttle. Results from STS-80 and STS-95 confirm that apoptosis in Jurkat cells is increased during spaceflight and the increased production of Fas is microgravity environment-related. Ground-based
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tests show that Shuttle flight-related cell culture conditions, including lowered serum concentration and cell culture densities characteristic of these experiments do not induce increases in apoptosis or Fas. This information significantly advances our understanding of cellular response during spaceflight and supports the concept that lymphocyte growth and aging are affected in the microgravity environment.
2. Materials and methods 2.1. Cell culture The human lymphoblastoid cell line, Jurkat clone E6 –1, was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were certified free of mycoplasma contamination by the ATCC. Culture medium consisted of RPMI 1640 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% heat inactivated fetal, bovine serum (FBS) (Summit Biotechnology, Santa Ana, CA, USA) 2 mM glutamine, 1 mM sodium pyruvate, 1 ml/100 of 100 ⫻ nonessential amino acids, and penicillin and streptomycin, 100 units and 100 g/ml, respectively (Life Technologies, Grand Island, NY, USA) and 12.5 mM HEPES buffer (Sigma, St. Louis, MO, USA). Cells, cultured in our laboratory from the ATCC frozen stocks, were used at passage levels three through eight for all tests and flight experiments. 2.2. Hardware Flight and ground-based control hardware for the experiments flown on STS-80 and STS-95 consisted of Bioprocessing modules (BPMs) assembled in our laboratory. BPMs are comprised of four syringes interconnected by tubing via a four-way valve. Polypropylene filter units (Advantec MFS, Inc., Pleasanton, CA, USA) with a Millipore HTTP, 0.4 micron Isopore® membrane (Millipore, Bedford, MA, USA), placed between the valve and the cell syringes allowed separation of cells and culture medium before fixing the cells in microgravity. Manual operation permitted mixing of fluids when an astronaut aligned the BPM valve position and pushed appropriate syringe plungers. The BPMs differed primarily from the hardware used previously on STS-76 (Lewis et al., 1998; Hatton et al., 1998) in that the BPMs accommodated approximately six to ten times more volume than the STS-76 experiment hardware. 2.3. Experimental details In preparation for flight on STS-80 and STS-95, 1.5 million cells/ml were suspended in medium containing 2% FBS and duplicate syringes in each BPM were loaded with 3 ml of the suspension. A third syringe was loaded with 4 ml of medium containing FBS calculated to give a final concentration of 10% when injected into the syringes containing cells. The fourth syringe was loaded with 4 mls of 3% formalin in Dulbecco’s phosphate buffered saline. After loading, BPMs were maintained at 20°C before launch (approximately 28 h) and until an astronaut initiated the experiments on orbit by increasing the temperature to 37°C and injecting 2 ml of activator (medium containing 22% FBS) into each of the syringes containing cells to increase the FBS concentration to 10% to stimulate cell growth. Experiments were activated at 6 (STS-80) and 4.5 (STS-95) h after launch.
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At selected times after activation, cells were filtered from the medium and a 2 ml volume of 3% formalin was injected into each cell syringe. The cells for evaluation of morphologically detectable apoptosis, and culture medium for assay of sFas were stored at 4°C (STS-80) until processing in the laboratory after flight. The medium from STS-95 was stored frozen at ⫺80°C except for the 4 h sample, which was stored unfrozen. For STS-80, cells were fixed immediately and at 4, 24, 48, and 75 h after activation. On STS-95, cells where fixed at 4 and 48 h after activation and medium was collected at 4, 24, and 48 h after activation. 2.4. Apoptosis analyses Cell-free culture medium was tested for sFas protein by an enzyme-linked immunosorbent assay (ELISA) according to methodology and reagents supplied with kits purchased from Oncogene Sciences (Uniondale, NY, USA). The sFas concentration in units/ml was determined from triplicate optical density readings for each sample in duplicate compared to the Oncogene Sciences kit standard diluted in wells of a 96 well plate and read on a Bio–Tek EL 340 microplate reader (Bio–Tek, Winooski, VT, USA). Deltasoft Soft II software for the Bio–Tek Microplate Reader interfaced with a Macintosh computer was used to generate the sFas concentration and standard error of the mean (SEM) for each sample. A unit is define by the kit supplier as the amount of sFas present in 104 cells/ml in cultures of HuT 78 cells. The sensitivity of the assay is 0.05 units/ml. Visualization of Fas in formalin-fixed cells was carried out by immunofluorescence microscopy. Cells were incubated with anti-Fas antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h followed by rinsing and incubation with Texas Redconjugated second antibody (Molecular Probes, Eugene, OR, USA) for 1 h. Cell associated Fas was visualized using a Nikon Labophot microscope equipped with epifluorescence. Formalin fixed cells were evaluated for apoptosis by staining with Hoechst 33253 (Molecular Probes, Eugene, OR, USA) to visualize apoptotic bodies. Cells were distributed by use of a Cytospin 3 (Shandon Lipshaw, Pittsburgh, PA, USA) onto BSA coated coverslips and permeabilized with Triton X 100 (Boehringer, Indianapolis, IN, USA) for 3 min. Cells were then incubated at room temperature with 25 g/ml of Hoechst in phosphate buffered saline for 5 min and the bright blue stained nuclei were counted using a Nikon Labophot microscope equipped with epifluorescence and a 364 nm filter. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid operator bias and re-count of the same cells. Data are expressed as the mean and SEM of the percent of cells in apoptosis calculated from more than 300 cells on each of at least two slide preparations for each sample.
3. Results 3.1. Morphologically detectable apoptosis Counts of cells stained with Hoechst 33253 showed that approximately 9% to 10% of the flown Jurkat cell populations were apoptotic at all time points sampled on both STS-80 (Fig. 1) and STS-95 (Fig. 2). The 0 h baseline of approximately 2% apoptosis (Fig. 1) was
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Fig. 1. Morphologically detected apoptosis in Jurkat cells flown on STS-80. Cells were stained with Hoechst 33253 as described in Section 2. Data represent the average of a minimum of 300 cells/slide for each condition evaluated on at least two slide preparations per sample. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid re-counting the same cells. Error bars represent the SEM of two or more counts of 300 each for each sample.
similar to that of cells cultured under standard, optimal laboratory conditions. Apoptosis in ground controls, handled by the same procedures and timeline as flight, did not exceed the STS-80 value of 6.4% at 4 h after serum concentration and temperature were increased. At 4 h after activation of cells on STS-80, apoptosis was significantly increased in both flight and ground controls (Fig. 1). This was followed by a decrease at 24 and 48 h and an increase again at 75 h as the culture aged and nutrients were depleted. In flown samples, the number of apoptotic cells at 24 and 48 h was more than three times higher than for ground controls. After 75 h, apoptosis in flown and ground populations increased, however; flown cells had approximately 1.7⫻ more apoptotic cells than ground controls. For cells flown on STS-95, the incidence of apoptosis at 4 and 48 h (Fig. 2) was almost identical to STS-80. The maximum number of apoptotic cells in ground controls did not exceed 3.6%, whereas flown cell populations were 9.3% apoptotic at 48 h. 3.2. Increase in cell associated Fas during Spaceflight Cells fixed with formalin 4 h after growth stimulation showed no cell associated Fas in either flight or ground controls (Fig. 3a and b). The number of Fas positive cells was increased in flown samples and at 48 h, almost all of the flown cells were positive (Fig. 3c). At the same time point, only a few ground control cells were positive for Fas (Fig. 3d).
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Fig. 2. Morphologically detected apoptosis in Jurkat cells flown on STS-95. Cells were stained with Hoechst 33253 as described in Section 2. Data represent the average of a minimum of 300 cells/slide evaluated on at least two slide preparations per sample. Counts for determining the percentages of apoptotic cells were performed in blind studies according to laboratory standard procedures using a specific field advancement pattern to avoid re-counting the same cells. Error bars represent the SEM of two or more counts of 300 each for each sample.
3.3. Increase in sFas during Spaceflight Because of crew time and hardware limitations, the medium from two different time points (0 and 4 h; and 4 and 24 h) was pooled after activation in microgravity for the STS-80 experiment. Although not significant because of pooling the samples, the concentration of soluble Fas in the medium of cells flown on STS-80 seemed to increase over time from 0 to 24 h (Fig. 4). Due to a hardware anomaly, the 48-h sample for STS-80 was lost. For STS-95, the concentration of sFas significantly increased between 24 and 48 h (Fig. 5), thus confirming the time- and microgravity-dependent increase in sFas as previously reported (Lewis et al., 1998). Ground control values for sFas were negative for both STS-80 and STS-95 with the exception of the 0.2 units at the 4 h time point for the STS-95 ground control. The negative values for sFas (Figs. 4 and 5) represent absorption values below the lowest concentration of the standard. The software (Delta Soft II) program used to compare test values to the standard to obtain test sample concentrations extrapolates values into the negative range. 3.4. Effect of serum concentration and cell density on sFas release To retard cell growth until activation of the experiment in space, cells were held in medium containing 2% FBS at 20°C for approximately 28 h total before launch and before activation of the cells on orbit. The potential effect of serum concentration on release of sFas was evaluated in ground-based experiments by maintaining approximately 1.5
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Fig. 3. Cell associated Fas in ground and flight cells. Formalin-fixed cells were evaluated for presence of cell associated Fas by immunofluorescence microscopy as described in Section 2. At 4 h, both flown (a) and ground control (b) cells were negative for Fas. By 48 h, almost all of the flight cells were positive (c) as shown by the arrows. Conversely, only one of the five ground control cells stained positive for Fas (d).
million Jurkat cells/ml at 20°C in the laboratory for 24 h in medium containing 0%, 2%, and 10% FBS. At all serum concentrations, sFas values were negative showing that serum concentration did not affect release of sFas in the culture medium. The spaceflight experiments were set up at a concentration of approximately 1.5 million cells/ml to have a sufficient number of cells for post-flight analyses. To determine if cell density could affect the amount of sFas released into the medium, cell cultures were initiated in the laboratory at 1.2 million/ml and allowed to grow without the addition of fresh medium for 72 h. The cells grew to a density of more than 4 million/ml. Viability dropped sharply from 98% at 24 h to 45% at 72 h. Fig. 6 shows that the number of apoptotic nuclei were within the normal range of approximately 2% to 3% at 24 h but increased to 8% by 72 h as the viability declined at the high cell density. Soluble Fas levels remained at approximately zero, as expected at 0 to 24 h but increased to almost 1 unit when the culture became overgrown by 72 h.
4. Discussion The present study was initiated to address whether increased apoptosis and a timedependent, microgravity-related, release of the cell death factor, soluble Fas/APO-1 (sFas), reported previously (Lewis et al., 1998) are characteristic responses of lymphocytes to conditions of spaceflight and microgravity. Results confirmed an increase in sFas in medium of Jurkat cells flown on the Shuttle and define this as a repeatable, time- and microgravity-related response of these cells. Furthermore, this study also confirms that apoptosis, one of the characteristics of immunoscenescence (Phelouzat et al., 1996), is increased during space flight. High cell population density and low serum concentration in the medium were evaluated in ground-based studies to determine the effect on sFas because these were
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Fig. 4. Soluble Fas in cell-free medium of STS-80 flight and ground control cells. Because of the design of the spaceflight experiment due to hardware constraints, the medium samples taken at 0 and 4 h and 4 and 24 h were pooled. The Fas concentration was determined from triplicate optical density readingings for each sample in duplicate compared to the standard as described in Section 2. Error bars represent the SEM calculated by a Deltasoft Soft II program for the Bio–Tek Microplate Reader interfaced with a Macintosh computer to generate the Fas concentration and SEM. Though not statistically significant, medium from 0/4 h and 4/24 h showed an increase in the amount of sFas as time in microgravity increased. All ground control samples were negative for sFas. (Values were extrapolated into the negative range by the Delta Soft II software used to compare test values to the standard to obtain test sample concentrations).
conditions to which cells were subjected during the flight experiments. For serum concentrations tested (0%, 2%, and 10%), the sFas concentration in medium was essentially zero. Thus, holding cells for approximately 28 h in medium containing 2% serum before launch and until activation on orbit had no effect on sFas release. In ground-based tests, high cell density culture resulted in a slight, but not statistically significant, increase in the total concentration of sFas in the culture medium over a period of 0 to 72 h (Fig. 6). At 72 h, the culture was in decline as indicated by decreased viability (data not shown), and an increased number of cells undergoing apoptosis (Fig. 6). The increase in the level of sFas in medium of space-flown cells is therefore not a result of high culture density but rather some other factor, possibly radiation or microgravity per se, characteristic of the microgravity environment. Owen–Schaub et al. (1995) suggest that tumor cells may produce sFas as a means of protecting against apoptosis. Based on results of STS-80 and STS-95, it does not seem that sFas protects the cells from apoptosis during spaceflight because the percentage of apoptotic cells remained at 9 to 10% throughout yet sFas levels in the medium increased with time in microgravity. Longer duration experiments in microgravity in which cells are maintained by adding fresh medium are needed to assess whether sFas is correlated with recovery of the population from apoptosis. Holding cells in low-serum medium at low temperature (20°C) may have predisposed
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Fig. 5. Amount of sFas in cell-free medium of STS-95 flight and ground controls. Flight samples taken at, 24 and 48 h showed a time-dependent and microgravity-related increase in sFas. The sFas levels in ground controls did not increase with time. The Fas concentration was determined from triplicate optical density readings for each sample in duplicate compared to the standard as described in Section 2. Error bars represent the SEM calculated by a Deltasoft Soft II program for the Bio–Tek Microplate Reader interfaced with a Macintosh computer to generate the Fas concentration and SEM. The Delta Soft II software used to compare test values to the standard to obtain test sample concentrations extrapolates values into the negative range.
them to apoptosis and may account for the increase in number of apoptotic cells in samples 4 h after activation (Figs. 1 and 2). It is unlikely that these factors alone account for the 9% to 10% apoptosis in flown cultures at 24 and 48 h compared to ground controls that were less than 4% apoptotic. Messman and Pittman (1998) showed that PC12 cells cultured in medium without serum could be rescued if the serum was added back. The increase in morphologically detectable apoptosis at 4 h (Figs. 1 and 2) may be due to cells already committed to die due to serum reduction. Ground control cells seemed to recover after increasing serum to 10% because the number of apoptotic cells at 24 and 48 h decreased to baseline levels. However, approximately 10% of the cells in microgravity continued to exhibit apoptosis indicating that serum reduction is not responsible for apoptosis in microgravity. In space, the cells do not respond to growth activation, i.e., they do not grow when serum is added and the temperature is raised to 37°C, whereas the ground controls increase by a factor of 1.6 in the same time (Lewis et al., 1998) Thus in space, sFas does not seem to be associated with increased growth activation. Soluble Fas increases were more notable after 48 h in microgravity and were higher than ground controls, which responded to growth activation but were essentially negative for sFas. We have found that use of the Jurkat cell line provides a stock of consistently growing, easily cultured, readily available cells thus reducing variables associated with use of fresh peripheral lymphocytes or blood bank buffy coat preparations. By using Jurkat cells, we were also able to show that results from STS-80 and STS-95 were consistent with our results from STS-76 flown in different hardware. For all tests and flight experiments cells
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Fig. 6. Effect of cell density on apoptosis and sFas. The percentage of apoptotic nuclei and sFas in culture medium were evaluated as described in Section 2. Counts of cells stained with Hoechst 33253 for visualization of apoptotic bodies showed a significant increase in apoptosis as time in culture and culture density increased. Though the concentration of sFas in the medium increased to 1.3 units/ml by 72 h, this value was less than that for the flown cultures at 48 h (Fig.5).
were cultured in our laboratory and used at passage levels three through eight from the ATCC frozen stocks to minimize culture to culture variability. And for all flight experiments, the cells were suspended in culture medium at 1.5 million cells/ml to ensure consistency among experiments. Jurkat cells have been flown on a number of Space Shuttle missions (Lewis et al., 1998; Hatton et al., 1998; Limouse et al., 1991; Schmitt et al., 1996). The cells are easily and consistently growth-stimulated by increasing the serum concentration from 2% to 10%. Increased cell death and Fas in the membrane of cells (Phelouzat et al., 1997) has been related to the aging process. Criswell–Hudak (1991) proposed looking at aging as an adaptation and comparing it to the adaptations that occur during spaceflight to obtain information about aging. Our results with space-flown Jurkat cells show that these cells are an excellent model for investigating cell aging under gravity-altered conditions. Our data now add new information to expand the current similarities (Hughes–Fulford, 1991; Czeisler et al., 1991) between aging and spaceflight. The increase in amounts of cell associated Fas in immune cells in space and in the elderly (Aggarwal and Gupta, 1998; Potestio et al., 1998; Seishima et al., 1996), further suggests that spaceflight may contribute to premature aging of lymphocytes.
Acknowledgments The authors express their appreciation to the crews of STS-80 and STS-95 and especially to Astronauts John Glenn and Scott Parazynski whose expertise resulted in
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