TRIPLELUX-B: Immunological and cellular reactions under influence of microgravity
  1. 2011 • 54th ESA Parabolic Flight Campaign
Life Sciences:
  • Cell and Molecular Biology
A300 ZERO-G Airbus
Patrik Sundblad
P.D. Hansen (1)
Dept. of Ecotoxicology
Franklinstrasse 29
10578 Berlin

The TRIPLELUX-B experiment will contribute to answer the question:
Does the change in gravity have an qualitative and quantitative effect on the phagocytotic activity of mussel haemocytes?

Haemocytes (blood cells of mussels, Figure 1, see attachments) are the primary phagocytotic defence against invading microorganisms and foreign particles. The haemocytes of invertebrate mussels are comparable in their function to macrophages of vertebrates. The decreased activity in the immune system of astronauts is usually observed after long term missions and consequently exposure to microgravity. Whether microgravity can cause these immunological effects is still an open question.

The aim of the TRIPLELUX-B experiment is to investigate under space flight conditions the effects of microgravity related to the potential of isolated Blue Mussel haemocytes to perform phagocytosis as an immune function and to study the universal validity of these innate immune responses.
The haemocytes of mussels are comparable with the human macrophages but their immune system is older than the invention of anti-bodies and the complement immune system.
The endpoint of exposure to microgravity under flight conditions is the production of Reactive Oxygen Species (ROS). The phagocytosis and the formation of ROS are key-immune functions and are present in vertebrate as well in invertebrate species.

60th ESA Parabolic Flight Campaign 2014

Immunological and cellular reactions under influence of microgravity and cosmic radiation - The application of a biosensor with phagocytotic cells
56th ESA Parabolic Flight Campaign

TRIPLELUX-B (Gene, immune and cellular responses to single and combined Space flight conditions)
ISS Increment 23-24

During the TRIPLELUX-B Parabolic Flight experiment the phagocytotic activity of the haemocytes will be stimulated by Zymosan particles. The cells are ingesting the Zymosan and start to produce ROS in an attempt to destroy the particles. By adding Peroxidase and Luminol the activity of the phagocytotic cells becomes visible by luminescence that can be measured quantitatively.
The amount of light (Fig. 4) is directly proportional to the amount of oxy radicals produced by the cells.
The reaction of the cells under standard gravity is carefully validated and investigated under standardized conditions in the laboratory and demonstrated in extended field studies.
In figure 4, the development of luminescence during a run under standard gravity conditions is shown. The mixing of the cells with Zymosan will generate a sharp flash (see operational Flow Chart in Fig. 5). The initial flash is build up by the turbulences and is fading away quickly following by the increase of luminescence because of the increase by phagocytotic activity of haemocytes. This reaction will occur approx. 10 minutes after contact of the haemocytes with the Zymosan particles. The light output is increasing and will come to maximum after approx. 50 to 60 minutes.
During the parabolic flight the cells will be exposed to a repeated change from zero-gravity to nearly doubling of the standard gravity. A change in the potential of the phagocytotic haemocytes to produce ROS will be directly
visible in an decrease or increase of the light output.

The light output from the cells will be measured by a high-sensitive luminometer. The data will be recorded during the flight and plotted on a computer screen. The operational flow chart of the experiment is demonstrated in Fig. 4.
The TRIPLELUX-B parabolic flight experiment rack is in total rated concerning loading as a ‘light’ experiment. The total weight is below 50 kg. All the elements are mounted directly at the supporting rack. The most prominent structure is the experimental containment.
The experimental containment (Fig. 6) contains the cuvette filled with haemocytes and the separate photomultiplier of a luminometer for measuring the light output (Fig. 4).
The Photomultiplier is controlled by a multimeter. The data processing, computing system and the power supply for the whole experiment are mounted at the supporting rack (Fig. 6).

L. Renwrantz, I. Daniel, P.D. Hansen, (1985), "Lectin - Binding to Hemocytes of Mytilus edulis", Developmental and Comparative Immunology, 9, pp. 203-210.
P.D. Hansen, R. Bock, F. Brauer, (1991), "Investigations of phagocytosis concerning the immunological defence mechanism of Mytilus edulis using a sublethal luminescent bacterial assay (Photobacterium phosphoreum).", Comparative Biochemistry and Physiology, 100C, 1/2, pp. 129-132.
C. Blaise, F. Gagné, J. Pellerin, P.D. Hansen, S. Trottier, (2002), "Molluscan shellfish biomarker study of the Saguenay Fjord (Quebec, Canada) with the soft-shell clam Mya arenaria.", Environmental Toxicology, 17, 3, pp. 170-186.
C. Blaise, S. Trottier, F. Gagné, C. Lallement, P.D. Hansen, (2002), "Immunocompetence of bivalve hemocytes by a miniaturized phagocytosis assay", Environmental Toxicology, 17, 3, DOI: 10.1002/tox.10047, pp. 160-169.
P.D. Hansen, (2003), "Biomarkers - Chapter 6", Bioindicators & Biomonitors - Principles, Concepts and Applications, 6, B.A. Markert, A.M. Breure, H.G. Zechmeister, Elsevier, Amsterdam, pp. 203-220.
P.D. Hansen, E. Unruh, (2005), "TRIPLE LUX-B: Phagocytosis in Mussel Hemocytes", Proceedings of the 9th European Symposium on Life Sciences Research in Space / 26th Annual International Gravitational Physiology Meeting.
K. Huber, M. Krötz-Fahning, B. Hock, (2005), "Phagocytosis as a biomarker for stress responses", Journal of Gravitational Physiology, 12, 1, pp. 265-266.
F. Gagné, C. Blaise, M. Fournier, P.D. Hansen, (2006), "Effects of selected pharmaceutical products on phagocytic activity in Elliptio complanata mussels", Comp. Biochem. Physiol. C Toxicol Pharmacol., 143, 2, pp. 179-186.
K. Huber, M. Krötz-Fahning, B. Hock, (2006), "Respiratory burst as a biomarker for stress responses", Protoplasma, 229, pp. 221-224.
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Fig 1: Sucked haemolymph (blood plasma) from a blood sinus of the blue mussel Mytilus edulis and the Haemocytes without and with stretched pseudopodia of phagocytotic active haemocytes.

Fig 2: Freshly collected hemocytes are attaching to the surface of Neubauer chamber starting with the attachment at the surface of the haemocytes and consequently the uptake and phagocytosis of foreign particles (Phase contrast; 200 x magnification).

Fig 3: Hemocytes after phagocytosis of particles stained with a fluorescence dye (FITCyeast cells inside of hemocytes, 200 x).

Fig.4: Development of the luminescence light output after hemocytes and zymosan are mixed. (Nikon P1 Luminometer; 2x106 cells x mL-1).

Fig. 5: Operational Flow Chart of the TRIPLELUX-B Experiment.

Fig 6: Layout and rack of the experimental units: (1) main power supply, (2) data processing and computing system, (3) luminometer and amplifier, (4) power supply (computing system), (5) power socket, (6) multimeter, (7) fuse box, (8) emergency stop button, (9) experimental containment.
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