EXPERIMENT RECORD N° 9307
RESLEM/ROALD-2 - Role of the Endocannabinoid System in human Lymphocytes Exposed to Microgravity/Role of programmed cell death (apoptosis) in the depression of human T-lymphocyte activation in microgravity
  1. 2011 • ISS Increments 29-30
  2. 2011 • ISS "PromISSe"- long-duration mission
Life Sciences:
  • Cell and Molecular Biology
Kubik
Jason Hatton
jason.hatton@esa.int
N. Battista (1), M. Maccarrone (1), C. Rapino (1), M. Di Tommaso (1), M. Bari (2), V. Gasperi (2), A. Finazzi-Agrò (2)
(1)  
Department of Biomedical Sciences
Università degli Studi di Teramo
Piazza Aldo Moro n. 45
64100 Teramo
ITALY
Tel:  
+39(0)861.266875
Fax:  
+39(0)861.266877
e-mail:  
nbattista@unite.it
mmaccarrone@unite.it
(2)  
University of Rome 'Tor Vergata'
Department of Experimental Medicine and Biochemical Sciences
ITALY

AIM
The RESLEM (Role of Endocannabinoid System in human Lymphocytes Exposed to Microgravity) experiment was investigating the gene expression of the proteins involved in the metabolic control of Anandamide (AEA) tone in human lymphocytes exposed to microgravity conditions.

The aim was to determine the role of this lipid in the regulations of immune processes and in the cell cycle under microgravity conditions. In fact, Anandamide is a signal for the cells to make a choice between life and death and it might be a contributor responsible for the immune deficit observed in Space.

SPECIFIC GOALS

1. Determine the role of Anandamide (AEA), a key player in apoptosis, and the elements of the endocannabinoid system in the regulation of immune processes in microgravity.
2. Compare data from the flight experiment with those in simulated microgravity in the random positioning machine (RPM) experiments on ground.

BACKGROUND
Anandamide (AEA) is the main representative of a family of polyunsaturated fatty acid amides and esters, called endocannabinoids. It is the most biologically active endocannabinoid described to date and behaves as partial agonist for type-1 (CB1) and as a weak partial agonist for type-2 (CB2) cannabinoid receptors. Additionally, AEA is a full agonist for vanilloid receptors (TRPV 1), thus being considered a bona fide "endovanilloid". Unlike classical neurotransmitters and neuropeptides, AEA is not stored in intracellular compartments, but is produced on demand by receptorstimulated cleavage of lipid precursors, when and where they are requested. It has been demonstrated that human lymphocytes are able to synthesize "on demand" AEA by a N-acylphosphatidyl-ethanolamine (NAPE)-specific phospholipase D (NAPEPLD) activity and hydrolyze it by Fatty Acid Amide Hydrolase (FAAH). Moreover, also the presence of CB1 and CB2 and TRPV 1 receptors has been demonstrated in these cells. The effect of cannabinoids on immune functions appears to be transient and is activated during infections. This concept is supported by the downregulation of cannabinoid receptor expression when the immune cells are activated.

The precise mechanisms through which cannabinoids mediate immune response is only now beginning to be understood and can be broadly categorised into four pathways:

  • apoptosis,
  • inhibition of proliferation,
  • suppression of cytokine and chemokine production,
  • and induction of T regulatory cells (T regs).

Preliminary evidence that the immunosuppressive effects of AEA might be associated with inhibition of lymphocyte proliferation and induction of programmed cell death has been reported, while growing evidence is being collected, suggesting that AEA might have indeed proapoptotic activity in vitro. The role of the immune system and of apoptosis in helping the organisms to adapt to their specific environments is a relevant issue also for space biology. To date, only one report has demonstrated the engagement of a specific signalling cascade as a cause of lymphocyte apoptosis in micro gravity, i.e., that mediated by lipoxygenase activity. Preliminary results have shown that Jurkat cells exposed to simulated microgravity conditions for 24 hours have an unaltered protein expression of the synthesizing and degrading enzymes involved in AEA metabolism and show a modulated expression of cannabinoid receptors. Therefore, our experiment is aimed at studying the gene and protein expression of the enzymes that regulate the endogenous tone of AEA and of its receptors, in order to provide new insights in the immune response of lymphocytes in space, and to identify the role of AEA as modulator of cell survival and death.

NEED FOR MICROGRAVITY
This project aims at investigating gene expression of the proteins involved in the metabolic control of AEA tone, in order to determine the role of this lipid in the regulation of immune processes and in the cell cycle under microgravity, conditions.
In fact, AEA is a signal for the cells to make a choice between life and death and it might be a contributor responsible for the immune deficit observed in space.

Pre-cursor flights
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.

ROle of Apoptosis in Lymphocyte Depression (ROALD)
Experiment on the International Space Station during Expedition 18 in 2008.


PROTOCOL

Biological samples and chemicals:
(i) Biological samples

- Human Peripheral Blood Mononuclear Cells,10 x 106 cells/ml, Preferred 0.9 ml, per sample Minimum 0.8 ml, Maximum 0.1 ml per culture chamber in RPMI-1640 Medium (4 samples x 8 EC´s)

(ii) Reagents and chemicals

- Activator: Concanavalin A (0.15 mg/L, 0. 09 ml/sample (min=0.08-max 0.1 ml) - 4 samples x 8 EC ´s
- Fixative: RNA later (0.9 ml/sample (min=0.8-max=1.0 ml) - 4 samples x 8 EC´s

Number of samples per condition:
4 Experimental samples per condition/time point (A sample is defined as one replicate of a cell+activator+fixativ, combination)

General Experiment Procedure - Overview (Note time/temperature margins are described in the Functional Objectives table in the attachment section):

- Cells prepared at launch site from peripheral human blood or buffy coat
- Cells loaded into EC´s no earlier than L-28 h, H/O no earlier than L-14 h
- Cells to be maintained at +23°C to +37°C during upload until installation in KUBIK
- Experiment installation 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. The experiment termination should not exceed 5 days after launch.
- Five experimental steps after launch
   • One activation
   • Four fixations (0 h, +3 h, +24 h, +48 h) and three freezing (0 h, 3 h, +24 h +48 h) - see Functional Objectives for margins
- Number of replicates for each experimental condition n=4
- Samples fixed with RNAlater solution (T+0 h, T+3 h ,T+24 h, T+48 h), then stored at +4 °C or frozen stowage (-20°C or colder, for days or weeks)
- Ambient download. Please, note that download temperature depends on the stowage temperature on ISS.

• Parameters measured:

- Postflight analysis of fixed samples (see section Results: PLANNED ANALYSIS)
- Temperature & time step recording

• Number of human test subjects: Blood donors on ground at launch site:

- Preferred: 3 blood donors on ground (sample: 450 ml whole blood per donor)
- Back-up: 2 blood donors on ground (sample: 450 ml whole blood per donor)
Please, note that the blood donors at launch site cannot be the same selected for EST.
Note: ESA Medical Board Approval, as well as investigator instituton medical board approval is required for the blood draw procedure.

Ground reference experiment:
No simultaneous ground reference experiment required.
Ground reference experiment performed post-flight using actual time/temperature profile of flight and the flight model.

EXPERIMENT EXECUTION
The RESLEM/ROALD-2  experiment launched onboard of the Soyuz TMA-3M space craft (ISS flight: 29S) on 21 December 2011 13:16 GMT (14:16 CET) from Baikonur.

After docking with the ISS, the experiment hardware was hosted in the centrifuge insert of the Kubik facility, the ESA incubator in the Columbus module of the International Space Station.

The flight set is composed of 8 Experiment Hardware (EH), 1 dummy unit for centrifuge balancing, 1 soft pouch for upload and download of EH. Since the EHs have to be uploaded at controlled temperature (>23°C), an additional soft pouch (BIOKIT) with Phase Change Materials cartridges is used to provide the necessary thermal control during the upload phase.The EH is composed of an Experiment Unit (EU) inserted in one Experiment Container (EC). The experiment container, named KIC, has already been flown in several biological missions (BIO4, SPHINX).

The EH has been labelled as "ROALD-2" due to the commonality with an other experiment already executed on the ISS in 2008.

Each EU contains 4 Culture Chambers, 4 Activator Reservoirs and 4 Fixative Reservoirs. Required actions on the samples are fully autonomous: cell activation and fixation occur by liquids injection by means of a spring controlled piston, the culture chamber volume simultaneously expands due to the displacement of a floating piston.

The sequence of activation and fixation operations is controlled by a timeline loaded on the EU electronics before launch. After experiment execution the 8 EH are installed in MELFI at -80°C before re-entry with Soyuz 28S.The same soft pouch is used to transport the 8 EH during upload and download.

On 22 December NASA astronaut Dan Burbank set up the KUBIK-3 incubator in front of the European Drawer Rack for experiment processing. He then successfully tested the temperature and centrifuge settings of the KUBIK-3 incubator and the KUBIK-6 incubator (acting as a backup for KUBIK-3) located inside the European Drawer Rack. Both incubators were set to 37 deg C.

The experiment eventually was started following installation of the eight experiment containers (and a dummy experiment container) by ESA astronaut André Kuipers and his colleague Dan Burbank inside the KUBIK-3 incubator on 24 December. A few hours later four 0g and 1g centrifuge experiment containers with samples were removed from KUBIK 3 and placed in one of the European-built MELFI freezers at -95 deg C. Two 0g and 1g centrifuge experiment containers with samples were removed by Kuipers 24 hours later and again placed in MELFI. This process was repeated by Kuipers for the remaining two 0g and 1g centrifuge experiment containers with samples 48 hours after start of the experiment, concluding processing with the KUBIK-3 incubator. Temperature and centrifuge data files were hereafter downlinked to ground.

The cells are supposed to come back to Earth by end of April 2012 on board of Soyuz TMA-22 - ISS flight 28S.

further information:

on the web page of Kayser Italia for RESLEM/ROALD-2

on the web page of Kayser Italia for ROALD

PLANNED ANALYSIS

1) Gene Expression by quantitative RT-PCR (NAPE-PLD, FAAH, CB1, CB2 and TRPV1) -0, 3, 24 h & 48 h timepoints

2) Protein expression by Western blot and ELISA (NAPE-PLD, FAAH, CB1, CB2 and TRPV1) -0, 3, 24 h & 48 h timepoints

RESULTS
The experimental activities, performed in the KUBIK incubator, were stopped at different time points and the experimental containers were stored in the Minus Eighty Degrees Celsius Laboratory Freezer for the ISS (MELFI) facility. The preliminary post-flight analysis, using quantitative real time-polymerase chain reaction (qRT-PCR) and western blot techniques, showed that microgravity alters the expression, at transcriptional and translational level, of the main AEA-synthesizing and -degrading enzymes. The project will disclose a potential engagement of endocannabinoid signalling in lymphocyte apoptosis and immunodepression already documented in space.

[1]  
M. Maccarrone, N. Battista, M.A. Meloni, M. Bari, G. Galleri, P. Pippia, A. Cogoli, A. Finazzi-Agrò, (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, 4, pp. 472-481.
[2]  
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.
[3]  
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.
[4]  
M. Maccarrone, N. Battista, M. Bari, A. Finazzi-Agrò, (2002), "Lipoxygenase and apoptosis in microgravity", Journal of Gravitational Physiology, 9, 1, pp. 241-244.
[5]  
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.
[6]  
M. Maccarrone, A. Finazzi-Agrò, (2001), "Microgravity increases the affinity of lipoxygenases for free fatty acids", FEBS Letters, 489, 2, pp. 283.
[7]  
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.
[8]  
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.
[9]  
N. Battista, M.A. Meloni, 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.
[10]  
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.
[11]  
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.
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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.

Flow Diagram with Functional Objective indicated.

List of Functional Objectives (FO) related to pre-flight / in-flight / post-flight timeline.

The readily packed Experiment Hardware in the soft pouch on 20 December 2011, short before loading into the Soyuz space craft for lift-off on 21 December 13:16 GMT (14:16 CET). Credit: Kayser Italia

The EH has been labelled as ROALD2 due to the commonality with another experiment already executed on the ISS in 2008. Credit: Kayser Italia

The EH is composed of an Experiment Unit (EU) inserted in one Experiment Container (EC), also named KIC. KICs can be integrated with KUBIK, BIOKON, and BIOBOX facilities. Credit: Kayser Italia http://www.kayser.it/index.php/life-science/experiment-containers/kic

List of Functional Objectives (FO) with additional parameters.

ISS030-E-033238 (24 December 2011) European Space Agency astronaut Andre Kuipers, Expedition 30 flight engineer, works with the ESA Role of Apoptosis in Lymphocyte Depression 2 (ROALD-2) experiment in the KUBIK-3 thermostatic container located in the Columbus laboratory of the International Space Station. Credit: NASA/ESA

ISS030-E-033272 (24 December 2011) European Space Agency astronaut Andre Kuipers, Expedition 30 flight engineer, prepares to insert ESA Role of Apoptosis in Lymphocyte Depression 2 (ROALD-2) experiment samples into a Minus Eighty Laboratory Freezer for ISS (MELFI-1) dewar tray located in the International Space Station's Kibo laboratory. Credit: NASA/ESA
 
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