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1 The “Immune Problem” in Space as a Cellular “Gravity Problem”

Cells of the immune system are exceptionally sensitive to microgravity. During the first Spacelab missions in the year 1983 the pioneering discovery from Cogoli and co-workers - that isolated human lymphocytes failed to proliferate after several days in microgravity - provided the first strong evidence of cell sensitivity to long-term reduced gravity exposure (Cogoli et al. 1984). Follow-up experiments clearly verified the depression of lymphocyte proliferation activation after mitogenic stimulation in long-term microgravity (Cogoli 1996).

Immunological problems of spaceflight were already described since the first Apollo missions, when more than half of the astronauts suffered from bacterial or viral infections (Hawkins and Zieglschmid 1975). Also in crew members of Skylab and Soyuz, a reduced reactivity of blood lymphoid cells has been observed (Konstantinova et al. 1973; Kimzey 1977), whereas recent studies described a reactivation of the varicella zoster virus (VZV), a latent nervous system virus, in astronauts (Cohrs et al. 2008; Mehta et al. 2004), an alarming observation for long-term space missions. Because of the obvious and severe effects on the human immune system, serious concerns arose whether spaceflight-associated immune system weakening ultimately precludes the expansion of human presence beyond Earth’s orbit (Guéguinou et al. 2009) (see also Chaps 9-12, 15-16). Therefore, to understand the cellular and molecular mechanisms of how gravity changes can influence immune cell functions has become an urgent need. For the future of human space flight, we should know which cellular and molecular mechanisms will provide therapeutic or preventive targets for keeping the immune system of astronauts alive during long-term space missions.

The gravity field may act on a cell directly or indirectly. In the experimental systems used, direct influences of altered gravity are more pronounced in vitro, while indirect influences are more apparent in the living organisms (Tairbekov 1996). Therefore, in vitro experiments with living human immune cells in microgravity conditions, such as on board of parabolic flights, sounding rockets, satellites, or the International Space Station (ISS), are providing an ideal platform to elucidate the underlying cellular and molecular mechanisms. In contrast to the logistic limitations of the International Space Station (ISS) and other space-based research platforms, parabolic flights provide frequent and repeated access to microgravity and therefore allow replication and modification of experiments within a reasonable time frame, which are not only characteristics, but rather requirements, of modern biomedical research. Thus, access to space is an instrument to elucidate long-term and functional effects of microgravity, whereas ultrashort, initial and primary effects and mechanisms are amenable by short-term-microgravity provided by parabolic flight maneuvers. In vivo and in vitro experiments can be performed on board of an aircraft which is weightless when it is flying on a Keplarian trajectory, described as an unpropelled body in ideally frictionless space subjected to a centrally symmetric gravitational field. For extensive in vitro experiments with living cells of the human immune system, we recently developed an experimental system which allows for large-scale cell culture experiments with living mammalian cells on board of the parabolic flight aircraft Airbus A300 ZERO-G (Paulsen et al. 2010) and for small scale, but frequent experiments on board of the military fighter aircraft Northrop F-5E (Studer et al. 2010). Due to the fact that cells of the immune system are obviously influenced by altered gravity, its gravisensitive nature render these cells also an ideal biological model in the search for general gravisensitive mechanisms in mammalian cells (Fig. 14.1).

Whereas it has been supposed that the most of the cellular effects of microgravity or of simulated weightlessness by the clinostat model may be attributed to the generalized unspecific reaction of a cell to external influence (Kondrachuk and Sirenko 1996), recent findings support a more specific nature of the cellular reactions (Paulsen et al. 2010). The ability of cells to sense, to interpret, and to respond to mechanical forces are increasingly recognized as playing predominant roles in a variety of relevant cellular functions (Orr et al. 2006; Ingber 2003a) such as cell migration, growth, mitosis, and differentiation (Engler et al. 2006). The mechanisms by which the cells detect mechanical forces could lead to an idea how cells may sense gravitational forces. In the last decade, much progress has been made in understanding the response of cells to physical forces (Vogel and Sheetz 2006; Hoffman and Crocker 2009). However, a clear concept how human cells are sensing gravity is missing and research trying to identify and to understand gravity-sensitive mechanisms in human cells is still at the beginning of a long road.

2 Gravisensitivity in Cells of the Immune System

Several investigations evidence alterations in signal transduction in lymphocytes. In this type of cells, microgravity affected the protein kinase C (Hatton et al. 2002; Schmitt et al. 1996), whereas delivery of first activation signal, patching, and capping of conA-binding membrane proteins occurred normally (Cogoli et al. 1992). These findings suggest the existence of gravisensitive cellular targets upstream from PKC (protein kinase C) and downstream from the TCR (T cell receptor)/CD3, where the lipid-raft-associated membrane-proximal signalosome complex is located. Gene expression analysis of T cells subjected to simulated microgravity revealed an alteration of several signal moduls, in particular NF-kB and MAPK-signalling (Boonyaratanakornkit et al. 2005). Also the expression of the early oncogenes c-fos, c-myc, and c-jun is inhibited (summarized in Braeucker et al. 2002). Gravisensitive mechanisms have been recently suggested at the chromatin (Paulsen et al. 2010) and epigenetic level (Singh et al. 2010). In Jurkat T cells, enhanced phosphorylation of the MAP kinases ERK-1/2, MEK, and p38 and inhibition of nuclear translocation of NF-kB were the predominant responses to simulated weightlessness (Paulsen et al. 2010). In contrast, nonstimulated myelocytic U937 cells responded to simulated weightlessness with enhanced overall tyrosine phosphorylation and activation of c-jun, whereas PMA (12-O-Tetradecanoylphorbol-13-acetate)-stimulated U937 cells responded with reduced tyrosine phosphorylation and reduced activation of c-jun, compared with 1 g controls (Paulsen et al. 2010).

In other studies, gravisensitivity of pro- and anti-apoptotic pathways has been reported in human mononuclear cells (Bakos et al. 2001), human ML-1 thyroid-carcinoma cells (Kossmehl et al. 2002), and astrocytes (Uva et al. 2002a). On the molecular level, microgravity induced fas, p53, and bax and reduced bcl-2 (Kossmehl et al. 2002; Nakamura et al. 2003; Ohnishi et al. 1999). Interestingly, the expression of fas was elevated in Jurkat-T-cells during space flights of the shuttle missions STS (Space Transportation System)-80 and STS-95 (Cubano and Lewis 2000), suggesting an enhanced fas-fasL-mediated apoptosis of immune cells. During a 14-day space flight (SLS-2-mission) an accumulation of p53 has been found in keratinocytes and myocytes, indicating that central regulatory molecules of nuclear signal transduction and cell cycle are influenced by gravity (Ohnishi et al. 1999). In fact, p53 protein was phosphorylated in Jurkat T cells after 20 s in real microgravity (Paulsen et al. 2010). The diminished proliferative response of T cells upon stimulation during microgravity could also be caused by a reduced expression of IL-2 receptor (Schwarzenberg et al. 1999; Walther et al. 1998), resulting in an impairment of positive regulatory feedback loops. Overall, a decreased capacity of T-cells for the production of cytokines is a prominent effect of microgravity on leukocytes (Cogoli and Cogoli-Greuter 1997).

Microgravity also impaired monocyte function: During the spacelab-mission SLS-1 monocytes lost their capability of secreting IL-1 (Cogoli 1993) and of expressing IL-2-receptor (Hashemi et al. 1999). However, the molecular mechanisms are not identified. Examination of gene expression of monocytes under real microgravity demonstrated significant changes in gene induction associated with differentiation of monocytes into macrophages (Hughes-Fulford et al. 2008). Kaur et al. (2005) investigated monocytes isolated from astronauts before and after a ­mission and compared the results with control groups. They found a reduction of phagocytosis and a reduced oxidative burst- and degranulation-capacity. Meloni et al. (2006) recently demonstrated that simulated weightlessness leads to massive alterations in the cytoskeleton of monocytes, which in turn influences motility and recently revealed during an ISS experiment a severe reduction in the locomotion ability of monocytic cells in microgravity (Meloni et al. 2008) (see also Chap. 12). Importantly, LFA-1 (Lymphocyte function-associated antigen 1) and ICAM-1 (intercellular adhesion molecule-1) adhesion protein expression – important to tether the antigen presenting cell (APC) to the lymphocyte that is activated and that proliferates in response to the antigen presented and other (co-) activation pathways – seemed also to be sensitive to microgravity, whereas their interaction is not altered (Meloni et al. 2008). It seems that not all cell types of the immune system are as ­sensitive to reduced gravity: In vitro studies with natural killer cells in simulated weightlessness and in real microgravity on board of the ISS revealed that neither cytotoxic effects nor interferon production is altered in microgravity (Buravkova et al. 2004).

3 Cell Migration and Cytoskeletal Architecture in Altered Gravity

Cell migration is an essential characteristic of life. Multicellular organisms must be motile to obtain nourishment, evade being eaten in their own right, respond to environmental changes, and reproduce. Likewise, unicellular organisms such as Paramecium or Loxodes must dynamically respond to fluctuations in ever-changing surroundings to assure survival. However, cell migration is also an essential characteristic of many normal and abnormal biological processes within the human organism including embryonic development, defense against infections, wound healing, and tumor metastasis (Lauffenburger and Horwitz 1996; Horwitz and Parsons 1999). Neutrophil granulocytes demonstrate the body’s first line of host defense by recognizing and killing microorganisms. Neutrophil locomotion is integral for immune effector function, because the cells have to leave the blood vessels and navigate to places of infection and injury to fulfil their main task of phagocytosis. Returning astronauts of spaceflight missions exhibited a strong increase of neutrophil granulocytes immediately after landing (Kaur et al. 2004) and neutrophil chemotactic assays showed a tenfold decrease in the optimal dose–response after landing (Stowe et al. 1999). In previous studies, changes in gravity demonstrated an inhibition of lymphocyte locomotion through type I collagen (Pellis et al. 1997; Sundaresan et al. 2002), and culture of human bone marrow CD34+ cells using NASA’s rotating wall vessels resulted in a decreased migration potential (Plett et al. 2004). An altered movement in microgravity was shown for leukocytes and Jurkat T cells, too (Cogoli-Greuter et al. 1996; Sciola et al. 1999), whereas the underlying signal transduction mechanisms are still illusive. On the other side, T cells become more motile after being cultured in 10 g hypergravity (Galimberti et al. 2006).

The cytoskeleton is responsible for giving a cell its shape and for generating the forces required for cell motility. It is an internal network of at least three types of cytosolic fibers: actin filaments, microtubules, and intermediate filaments. Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly polymerized and depolymerized within cells to invoke cellular motility, tissue formation, and repair (Feldner and Brandt 2002; Lee and Gotlieb 2002). Actin dynamics are considered to be the major component of the cytoskeleton responsible for cell motility. It has been shown to be essential for the migration of T lymphocytes as well as neutrophil granulocyte migration, a conclusion readily assumed as actin-depolymerizing drugs inhibit cellular motility (Hofman et al. 1999; Verschueren et al. 1995). In contrast, an intact microtubule network does not appear to be required for neutrophil migration, because microtubule-disrupting drugs such as colchicine even induce the migration of neutrophils (Niggli 2003), probably by inducing changes in the actin network.

Multiple investigators have reported that this complex network of fibers is sensitive to environmental factors such as microgravity and altered gravitational forces (Schatten et al. 2001). Several studies demonstrate modifications of the actin and microtubule cytoskeleton in microgravity. Already a few minutes of weightlessness affected the cytoskeleton of lymphocytes, astrocytes, neurons, and glial cells, disorganizing microtubules, intermediate filaments, and microfilaments (Uva et al. 2002b, 2005; Roesner et al. 2006). Morphological differences of both the microtubule and actin components of the cytoskeleton have been observed in cells grown in real and simulated microgravity (Uva et al. 2002b; Lewis et al. 1998). Hughes-Fulford (2003) reported that actin reorganization responded to the gravity level.

4 Learning About Graviperception in Unicellular Systems

Unicellular systems were and are frequently used as model systems to analyze and understand the influence of gravitational forces on the cellular level. Common subjects of study are ciliates like Paramecium and Loxodes, and flagellates like the algae Euglena (reviewed in Hemmersbach and Braeucker 2002; Haeder et al. 2005). Ciliates and flagellates are particularly interesting, because they can show positive and negative gravitaxis (movement in the same or opposite direction as the gravity vector) and gravikinesis (altered swimming velocity) due to their swimming properties and have the advantage of convenient experimental handling and observation (Planel et al. 1981, 1982; Machemer et al. 1991; Haeder et al. 1996; Hemmersbach et al. 1998; Hemmersbach and Haeder 1999). In these model organisms two different mechanisms of graviperception evolved: (1) in Loxodes gravity is sensed by specific statocyst-like organelles filled with barium sulfate (Müller vesicles) (Penard 1917; Rieder 1977; Fenchel and Finlay 1986); (2) in Euglena and Paramecium density differences between cytoplasm and extracellular medium activate mechanosensitive ion channels (Hemmersbach et al. 1998, 1999; Lebert and Haeder 1996; Lebert et al. 1997). This induces different signal transduction cascades where calcium, cAMP, calmodulin, and phosphorylation processes play an important role (Hemmersbach and Haeder 1999; Haeder et al. 2005; Streb et al. 2002). The sensitivity for gravity differs in Paramecium (0.35 g; Hemmersbach et al. 1996, 1998), Euglena (0.16 g and 0.12 g; Haeder et al. 1996, 1997), and Loxodes (<0.15 g; Hemmersbach et al. 1998). This was shown by using a slow rotating centrifuge microscope (NIZEMI) in microgravity and identifying the acceleration threshold inducing graviresponse. Below these thresholds, protists were unable to sense gravity and lost their typical gravity-based directed movements (Hemmersbach-Krause et al. 1993). These results were independent of the previous exposure to microgravity up to 12 days, although cells underwent several division cycles. Besides the described effects on gravity-based orientation, other effects can be observed due to microgravity in protists like Paramecium, e.g. an increased cell growth rate, increase in cell volume, decrease in total cell protein content, and lower cell calcium content (Planel et al. 1981, 1982; Planel 2004). The research on effects of microgravity on microorganisms will be of common future interest, since they represent an essential component of biological life support systems during long-term spaceflights.

5 The Question of Sensing Gravitational Forces in Human Cells

The gravitational force acting on a cell or a subcellular or molecular structure is equal to the mass of the structure and the acceleration toward the center of Earth. Despite the multitude of observed gravity-dependent effects on the cellular level, there is no clear idea how human cells may sense gravity. In general, direct gravisensing (e.g., by specialized cells as parts of a gravisensing organ) and indirect gravisensing, in which cells without specialized gravity detectors are affected by the acceleration, have to be distinguished (Albrecht-Buehler 1991). It is also important to mention, to which extent the direction and the amplitude of gravity is sensed. According to basic laws of physics, the weight of a single normal-sized cell seems too small compared with other cellular forces to allow them the distinction between up and down (Albrecht-Buehler 1991). In this context, thermal and mechanical noise within cells seem too high in relation to the change of force due to the change from normogravity to microgravity (Klopp et al. 2002). But since the weight of the surrounding environment is much larger, cells may be able to sense certain environmental changes caused by gravity and thus may sense indirectly at least the amplitude of gravitational forces (Albrecht-Buehler 1991).

5.1 Sensing Stress as Changes in the Gravitational Environment

The environment, which is sensed locally (Choquet et al. 1997), consists of cells and of the intercellular matrix. In microgravity, force-induced breakage of cell–cell- or cell–matrix-adhesion sites could be reduced. The mechanical properties of the matrix have predominant effects on different cell functions (Discher et al. 2005). The forces sufficient to activate such kind of cellular response are very low (10pN for 1 s, Jiang et al. 2006). Adhesion-mediated signalling provides cells with information about multiple parameters of their microenvironment, including mechanical characteristics (Bershadsky et al. 2006). During the last decade, several molecules and mechanisms of adhesion-mediated signalling have been identified, mostly in endothelial cells, where the response to fluid shear stress alters and regulates several cellular functions (Engler et al. 2006). In endothelial cells, integrins are crucially involved in endothelial mechanosensing of shear stress (Shyy and Chien 2002). Immunoglobulin family adhesion receptor PECAM-1 directly transmits mechanical force and, in cooperation with VE-cadherin and VEGFR-2, mediates the response of confluent endothelial cells to shear flow (Tzima et al. 2005). Interestingly, modulation of the expression of surface adhesion molecules such as ICAM-1 has been reported as the consequence of long-term microgravity (Buravkova et al. 2005; Romanov et al. 2001). Additionally, early molecular mechanisms responsible for gravity sensing of endothelial cells involve caveolae and Caveolin-1 phosphorylation (Spisni et al. 2006). Importantly, the appropriate formation and function of the immunological synapse between T cells and antigen-presenting cells require a well-defined spatial orientation of membrane adhesion molecules ICAM-1 and LFA-1 (Mossman et al. 2005). Therefore it is possible that integrin-mediated force transduction renders the immunological synapse a gravisensitive site.

5.2 Cellular Mechanosensory System

If the cells are able to sense gravitational forces by adhesion to the extracellular matrix, the question about the responsible mechanosensory system arises. Paradigms of cellular mechanosensing have been reviewed by Orr et al. (2006). One possibility is that the entire actin network serves as a mechanosensor. The folding state of cytoskeleton-associated proteins, which creates or masks binding sites for other proteins, depends on the strains in the actin network (reviewed in Vogel and Sheetz 2006). Forces applied to or taken from the actin network could be therefore transduced in altered binding of signal proteins to the cytoskeleton or the gain or loss of enzyme function. Consequently, microgravity may reduce the force inside the actin network, which could be then transduced into a certain biochemical signal by cytoskeleton-associated proteins. According to the tensegrity model (Fuller 1961), the whole cell is a prestressed structure (Ingber 1993, 2003b), with tensions generated by the actin–myosin network, by cellular force through focal adhesions (Choquet et al. 1997; Tamada et al. 2004), by cell–cell adhesions and polymerization of cytoskeletal elements (Wang et al. 2001, 2002; Wang and Stamenovic 2000; Stamenovic et al. 2002). A cell would not maintain its shape stability under load without a preexisting stress or prestress in the mechanical elements (Ingber 2003b). During gravitational unloading of the cell, intracellular forces induced by the prestress could be altered and transduced into a biochemical response. Force-induced changes of protein conformation and exposure of cryptic binding sites for signal proteins have been described as a possible method of mechanotransduction (reviewed in Vogel and Sheetz 2006). Unfolding of proteins with tandem-repeat domains can occur with applied forces on the order of 50–200 pN, as demonstrated by single-molecule experiments with actinin (Rief et al. 1999), filamin (Furuike et al. 2001), and spectrin (Rief et al. 1999). Tandem-repeat sequences are found in most extracellular matrix (ECM) proteins and many proteins that link the integrins to the cytoskeleton. Therefore, cells could hypothetically “measure” strain by integrating the number of unfolded domains (Hoffman et al. 2007). Many molecules are stabilized by disulphide bonds and their redox state can be therefore sensitive to force (Vogel and Sheetz 2006). In cross-linked molecules, only a small alteration in force could readily cause extensive conformational changes, suggesting binding partners of cross-linking proteins as potential signal proteins in mechanotransduction, such as including heat shock proteins, protein kinase C, Ral A (ras-related protein A), PIP2 (Phosphatidylinositol 4,5–bisphosphate), PIP 3 (Phosphatidylinositol (3,4,5)–trisphosphate), PI3-kinase (Phosphatidylinositol 3–kinase), MEKK1 mitogen-activated protein kinase kinase 1 (as reviewed in Otey and Carpen 2004; Stossel et al. 2001), and regulatory proteins for the Rho GTPases (Mammoto et al. 2007; Ohta et al. 2006). Interestingly, Rho kinase has been found to regulate the intracellular micromechanical response of adherent cells (Kole et al. 2004) and small G proteins are discussed having a significant role in mechanotransduction (Burridge and Wennerberg 2004). A special class of mechanosensing mechanisms (Orr et al. 2006) is represented by myosins. In most myosin classes, mechanical load alters ADP release. Myosin mechanosensing is best demonstrated by myoIC during adaptation in hearing (LeMasurier and Gillespie 2005). Enzymatic activity can be regulated in response to mechanical force by opening of enzymatic cleavage sites. Fibronectin, for example, has a partially cryptic disulphide isomerase (Langenbach and Sottile 1999) and a cryptic metalloprotease activity (Schnepel and Tschesche 2000), but it is not known whether these activities can be regulated by force (Vogel and Sheetz 2006). It has been demonstrated that application of a force to fibronectin binding induced rapid local src activation forming a directional wave propagated away from the stimulation site along the plasma membrane (Wang et al. 2005). Structural motifs that could change conformation over a range of mechanical forces and could therefore exhibit mechanosensory functions are diverse (Bershadsky et al. 2006; Martinac 2004; Vogel and Sheetz 2006). Considering the myriad of multidomain cross-linking proteins in different cells and subcellular localizations, it is likely that mechanoresponse is a highly specific and specialized process and not a general and nonspecific phenomenon.

Fig. 14.1
figure 1_14

T-cell activation in 1g (static) culture versus simulated microgravity (clinorotation); Photo credit to Clarence Sams (NASA Johnson Space Centre) and Mayra Nelman-Gonzalez (Wyle)

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5.3 Force-Sensitive Ion Channels

Mechanosensitive ion channels respond to mechanical stimuli with a change in their conductive state. Either the mechanosensitive channel senses directly alterations of the lipid bilayer, or transmit forces of the cytoskeleton or the extracellular matrix via a physical connection. Ion-channel involvement in mechanosensing is well described in prokaryotic systems (Martinac 2004). However, prokaryotic ion channels are relatively force-insensitive and only open at high tensions that are approaching the lytic tensions for the lipid bilayer (Vogel and Sheetz 2006), whereas typical membrane tensions in animal cells are a thousand fold lower than the activating bacterial tensions (Sheetz 2001). Membrane channels, for example, the PKD2 Ca2+ channel and inner rectifier K+  channel in eukaryotes, are activated upon stretching (Martinac 2004), leading to channel opening, ion flux, and most probably to the recruitment and activation of downstream signalling molecules. In this context, cellular responses to forces in bone and cartilage are probably the consequence of out-of-plane forces on channel–cytoskeleton linkages (Haut Donahue et al. 2004). Possible cellular gravisensing mechanisms and gravisensitive cellular molecules and functions are summarized in Table 14.1.

Table 14.1 Cells of the immune system are exceptionally sensitive to microgravity
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6 Conclusion

Taken together, cellular gravisensing may not result from a direct activation of a single gravisensing molecule. Instead, gravitational stress and forces may be sensed by an individual cell in the context of altered extracellular matrix mechanics, cell shape, cytoskeletal organization, or internal prestress in the cell–tissue matrix (Ingber 1999).

The development of cellular mechanosensitivity and mechanosensitive signal transduction was probably an evolutionary requirement to enable our cells to sense their extracellular matrix and their individual microenvironment. However, mechanosensitive mechanisms were designed to work under the condition of 1 g, but never had the possibility to adapt and adjust their reaction to conditions below 1 g. Therefore it is possible that the same mechanisms, which enable human cells to sense and to cope with mechanical stress, are potentially dangerous in microgravity. It is a major challenge to find out if our cellular machinery is able to live and to work without gravity force or if our cellular architecture will keep us dependent on the gravity field of Earth. With the completion and utilization of the International Space Station and with mission plans to Moon and Mars during the first half of our century, astronautics has entered the era of long-term space missions. Such long-term missions represent a challenge never experienced before: Small or even marginal medical problems could easily evolve to substantial challenges, which could possibly endanger the entire mission. Since crew performance is the crucial factor during space missions and since evacuation or exchange of the crew is impossible during interplanetary flights, to elucidate the underlying mechanism of limiting factors for human health and performance in microgravity, such as for the immune system, and to identify and test potential counteractive interventions is an urgent need. Therefore, identification of gravisensitive cellular reactions will also help to understand the molecular mechanisms of disturbed immune cell function in space in order to identify, to test, and to provide new targets for therapeutic or preventive intervention related to the immune system of astronauts during long-term space missions.