ROle of Apoptosis in Lymphocyte Depression (ROALD)
Biology: Molecular and cell biology
M. Maccarrone (1), N. Battista (2), V. Gasperi (2), M. Ranalli (2), M. Bari (2), A. Finazzi-Agro (2), M. Cogoli-Greuter (3), I. Walther (4), A. Cogoli (4), M. Meloni (5), P. Pippia (5)
|(1)||Department of Biomedical Sciences|
Università degli Studi di Teramo
Piazza Aldo Moro n. 45
|Tel: ||0039 (0) 861 266875|
|Fax: ||0039 (0) 861 266877|
|(2)||Department of Experimental Medicine and Biochemical Sciences|
University of Rome Tor Vergata
|(3)||Space Biology Group|
ETH Zurich - Eidgenössische Technische Hochschule
|Tel: ||0041 (0) 44 633 77 58|
|Fax: ||0041 (0) 44 633 14 19|
|(4)||University of Sassari|
|||M. Maccarrone, N. Battista, M. Meloni, M. Bari, G. Galleri, P. Pippia, A. Finazzi-Agro, (2003), "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", Journal of Leukocyte Biology, 73:472-481. 2003.|
|||N. Battista, M.A. Meloni, M. Bari, N. Mastrangelo, G. Galleri, C. Rapino, E. Dainese, A. Finazzi-Agrò, P. Pippia, M. Maccarrone, (2012), "5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment", The FASEB Journal, 26, 5, doi: 10.1096/fj.11-199406, pp. 1791-1798.|
|||M. Maccarrone, M. Bari, N. Battista, A. Finazzi-Agrò, (2001), "The catalytic efficiency of soybean lipoxygenase-l is enhanced at low gravity", Biophysical Chemistry, 90, pp. 97-101.|
|||M. Maccarrone, N. Battista, M.A. Meloni, M. Bari, G. Galleri, P. Pippia, A. Cogoli, A. Finazzi-Agrò, (2003), "Simulated microgravity induces apoptosis in human lymphocytes by a 5-lipoxygenase-mediated mechanism", Journal of Biological Research, LXXIX, pp. 63-64.|
|||M. Maccarrone, N. Battista, M. Bari, A. Finazzi-Agrò, (2002), "Lipoxygenase and apoptosis in microgravity", Journal of Gravitational Physiology, 9, 1, pp. 241-244.|
|||M. Maccarrone, A. Finazzi-Agrò, (2001), "Enzyme activity in microgravity: a problem of catalysis at the water-lipid interface?", FEBS Letters, 504, 1, pp. 80.|
|||M. Maccarrone, A. Finazzi-Agrò, (2001), "Microgravity increases the affinity of lipoxygenases for free fatty acids", FEBS Letters, 489, 2, pp. 283.|
|||M. Maccarrone, M. Bari, T. Lorenzon, A. Finazzi-Agrò, (2000), "Altered gravity modulates prostaglandin H synthase in human K562 cells", Journal of Gravitational Physiology, 7, 2, pp. P61-62.|
|||M. Maccarrone, M. Bari, A. Finazzi-Agrò, (1999), "Lipid peroxidation and polyamine metabolism in K562 cells subjected to altered gravity", Journal of Gravitational Physiology, 6, 1, pp. P25-26.|
|||N. Battista, C. Rapino, V. Gasperi, A. Finazzi-Agrò, M. Maccarrone, (2007), "Effect of RNAlater on lipoxygenase activity and expression, and immune cell apoptosis: opening the gate to the "ROALD" experiment aboard the Space Shuttle", Journal of Gravitational Physiology, 14, pp. 131-132.|
|||M. Maccarrone, A. Finazzi-Agrò, (2003), "The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis", Cell Death and Differentiation, 10, doi:10.1038/sj.cdd.4401284, pp. 946-955.|
ROle of Apoptosis in Lymphocyte Depression (ROALD) will determine the role of programmed apoptosis (cell death) in loss of T-lymphocyte (white blood cells originating in the thymus) activity in microgravity.
Various aspects of the apoptotic process will be assessed, using human T-lymphocytes, by analyzing gene expressions of metabolites of reactive oxygen species and membrane properties.
Experiments performed during spaceflight clearly show that several cellular processes, such as oxidative metabolism, growth rate, signal transduction and gene expression, are modified under conditions of weightlessness. For example, these alterations are associated with atrophy in heart, muscle and bone. 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, and have been associated with:
- altered distribution and downregulation of protein kinase C,
- reduced expression of interleukin-2 and its receptor,
- microtubule anomalies and growth retardation, and
- altered cytoskeletal gene expression.
It has been suggested that reduced growth response in lymphocytes during spaceflight might be linked to apoptosis, based on morphological anomalies and cDNA microarray analysis of space-flown human lymphoblastoid (Jurkat) cells. Though 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. In this context, 5-Lipoxygenase (5-LOX) plays a central role in interleukin-2 expression and activation of human lymphocytes, and is involved in the initiation of programmed death (apoptosis) triggered by several stimuli in different human cells. 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 (approximately 10 - 2g) directly enhances the catalytic efficiency of pure lipoxygenase, up to approximately 4-fold over the ground (1g) controls. In this project, we plan to ascertain whether or not space conditions might induce apoptosis in human lymphocytes through a 5-LOX-mediated pathway. The possible involvement of mitochondrial alterations in the apoptotic program will be also investigated.
The experiment will further our understanding of the mechanisms by which microgravity and spaceflight factors influence immune cell function. In particular the effect on activation of apoptosis has not been explored. Although preparatory experiments can be performed with ground based techniques, such as the Random Positioning Machine (RPM) the effect of microgravity and cosmic radiation for the duration required for this experiment can only be explored using an orbiting space vehicle.
Several cellular processes are modified when cells are placed under conditions of weightlessness. As yet, there is no coherent explanation for these observations, nor it is known which biomolecules might act as gravity sensors. Lipoxygenases generate leukotrienes and lipoxins from arachidonic acid, being responsible for many pharmacological and immunological effects, some of which are known to be affected by microgravity. In the course of the 28th Parabolic Flight Campaign of the European Space Agency we measured the activity of pure soybean lipoxygenase-l on linoleic acid, by a fibre optics spectrometer developed on purpose. It was found that microgravity reduced the apparent Michaelis-Menten constant Km of the enzymatic reaction to one fourth with respect to the 1 g control, whereas the catalytic 8 constant Kcat was unaffected. Consequently, the catalytic efficiency of lipoxygenase-1 (i.e., the Kcat/Km ratio) was approximately 4-fold higher in flight than on ground. This unprecedented finding suggests that lipoxygenase-1 might be a molecular target for gravity.
Parabolic flight experiment on the 28th ESA experiment campaign in 2000
The experiment examined the effect of short duration microgravity on the activity of enzymes which regulate apoptotic processes. The results of these experiments showed that the kinetics of some reactions was modified by micro gravity exposure. A full description of the experiment can be found on the ESA Erasmus experiment archive.
Experiment protocol Biological Samples:
- Purified Human Peripheral Blood Lymphocytes
General Experiment Procedure (Note time/temperature margins are described in the Functional Objectives table):
- Cells prepared at launch site from peripheral human blood or buffy coat.
- Cells loaded into EC's no earlier than L-24 h, H/O no earlier than L-18 h.
- Cells to be maintained at +23°C to + 30°C during upload until installation in KUBIK.
- Experiment activation no later than L+72 h (minimum = ASAP; maximum= 72 h), upper limit is defined by viability of the cells up to the last fixation/termination point. This should not exceed 5 days after launch.
- Five experimental steps after launch:
• One activation;
• Two sample freezings & two fixations then freezing (activation +0 h, + 3 h, + 24 h, +48 h).
- Number of replicates for each experimental condition n =4.
- Samples frozen with no fixation (T+0 h, T+3 h) or samples fixed with RNAlater solution only (T+24 h, T+48 h only), then frozen stowage (-20°C or colder, for days or weeks).
- Post-flight analysis of fixed samples.
- Temperature & time recording.
Number of human test subjects
- Minimum: 4-5 blood donors on ground, up to 450 ml whole blood per donor.
- Desired: 4-5 blood donors on ground up to 450 ml whole blood per donor.
Ground reference experiment(s):
No simultaneous ground reference experiment required.
Ground reference experiment performed postflight using actual time / temperature profile of flight.
further information on the webpage of Kayser Italia
The experiment will be uploaded with the Space Shuttle in passive thermal control unit to maintain the temperature above +25 degrees C to ensure good viability of the samples. Upon arrival to ISS, the ECs are installed in the Kubik incubator, pre-warmed to +37 degrees C. Experiment operations are performed automatically. Once installed in Kubik followed by a short pre-incubation period, the experiment is started by adding activator. A first set of samples is transferred to frozen stowage (-20 degrees C or colder) for 3-hours after activation, while the remaining samples are fixed with RNAlaterTM at 24-hours and 48-hours after activation to determine the time line events of the apoptotic process. Then they are transferred to frozen stowage. Samples must be maintained frozen following completion of the experiment, including during download.
In the post-flight analysis, we tested the following parameters directly correlated to programmed cell death:
- gene expression of calpain and p53;
- 5-LOX protein expression and activity;
- DNA fragmentation.
Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (~3-fold compared to ground-based controls) and cleaved-poly (ADP-ribose) polymerase (PARP) protein expression (~3-fold), as well as mRNA levels of apoptosis-related markers such as p53 (~3-fold) and calpain (~4-fold); these changes were paralleled by an early increase of 5-LOX activity (~2-fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.
In this context, it is worth reminding that AEA (Anandamide), unlike other endocannabinoids, inhibits FAAH activity by promoting the release of arachidonate, to be converted by lipoxygenases into hydroperoxides, which act as competitive inhibitors of FAAH. The observation that all lipoxygenase inhibitors (MK886, caffeic acid), as well as the hydro(pero)xides of AEA generated by lipoxygenase, yield the same effects on FAAH seems to stress the hypothesis that lipoxygenase pathway might regulate FAAH activity. This regulatory mechanism could, thus, have a general validity also under microgravity conditions and could be involved in controlling AEA levels and pro-apoptotic cytokines release in immunodepression.
The experiment was continued in 2011/2012 during the flight on Expedition 29/30 to the International Space Station.
|Model of microgravity-induced apoptosis in human lymphocytes|
|Experiment Concept Diagram: The experiment requires cultivation of lymphocytes (non adherent culture). Activator needs to be added to the cell culture. Cells are then cultivated for a period of time prior to either freezing or addition of fixative followed by freezing.|
|Detailed Experiment Timeline and associated Functional Objectives (FO).|
|Flow Diagram with Functional Objective indicated.|
|List of Functional Objectives (FO) related to pre-flight / in-flight / post-flight timeline.|
Jason Hatton (e-mail: firstname.lastname@example.org)