Impairment of antigen-specific cellular immune responses under simulated microgravity conditions

Summary Microgravity has been implicated to play a role in the observed immune dysfunction of astronauts and cosmonauts after either short-term or long-term space travel. These reports, together with studies describing increased levels of microorganisms in the space cabin environment suggest potential risk for in-flight incidences of infectious diseases. In order to understand the mechanism underlying these immune defects, it is important to have a ground-based model that would reliably mimic the effects of microgravity on antigen-specific immune function. We tested the utility of the rotating wall vessel (RWV) technology developed at NASA as a model system because in the RWV the culture medium and the cells rotate synchronously with the vessel, thereby creating simulated microgravity conditions. We compared the RWV to the conventional tissue culture flask (T-flask), for culturing immune precursor cells with cytotoxic T lymphocyte (CTL) activity against synthetic viral peptides. We observed a significant loss of antigen-specific CTL activity in RWV cultures, but not in those from the T-flask, irrespective of the peptide immunogen used for inducing the primary immune response in different mouse strains. Loss of CTL activity in RWV cultures coincided with a significant reduction in CD8⁺ cells as well as CD4⁺ cells and DEC205⁺ dendritic cells, suggesting adverse effects of RWV culturing on both the effector and accessory cells for the loss of antigen-specific CTL function. These results provide a strong parallel to the reported defects in cell-mediated immunity during space travel and strongly support the utility of the RWV technology as an effective ground-based model for identifying key steps in immune cell dysfunction related to microgravity.     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

Variable lymphocyte responses in rats after space flight.

Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.     

Evaluation of cell culture flasks designed for experiment under altered gravity-vector conditions.

Cell culture flasks applicable for altered gravity conditions, such as centrifugation, clino-rotation or microgravity in space, were manufactured for trial. The flask has flat polystyrene surface for monolayer culture and gas-permeable film window on the opposite face. The space in-between consists the culture chamber to be filled with liquid medium. To reduce the water loss and bubble formation in the culture fluid, another gas permeable window was placed on top to form a space where distilled water may be filled. The double-decker culture flask can be used for both space and ground-based experiments in common.     

Uridine phosphorylase from Novikoff rat hepatoma cells: purification, kinetic properties, and its role in uracil anabolism

Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330-fold from Novikoff rat hepatoma cells by ammonium sulfate precipitation, DEAE-Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 microM, KmRib-1-P = 88 microM, KmUrd = 16 micron, and KmPi = 130 microM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 microM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose-1-phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N-(phosphonacetyl)-L-aspartate ("PALA"), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-1-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.     

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.     

Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase

Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.     

Lymphocyte movements and interactions in microgravity

Cell-cell contacts and the formation of aggregates play an important role in the mitogen induced in-vitro activation of lymphocytes. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space. Direct evidence was obtained for the first time in an experiment performed on a sounding rocket flight, where the movements and interactions of free-floating, non activated cells could be observed in real time in microgravity. In an experiment performed on the IML-2 mission in July 1994, the movements and interactions of human lymphocytes during activation with the mitogen Con A were studied in real time in microgravity.     

Effect of simulated microgravity on the production of IL-12 by PBMCs

During space flight immunity is altered. This phenomenon is partly due to the microgravity condition itself. Our earlier space experiments (INTERFERON) indicated that microgravity has a significant effect at the cellular level. In our subsequent terrestrial studies we applied the Rotating Cell Culture System (RCCS) developed by NASA to mimick microgravity on ground. Previously we reported that human peripheral blood mononuclear cells (PBMCS) respond to simulated microgravity conditions with elevated tumor necrosis factor-alpha (TNF-alpha) production. We extended our investigations to the production of interleukin (IL)-12 under modelled microgravity conditions by separated PBMCs. In simulated microgravity we found significantly elevated level of secreted IL-12 compared to static, standard tissue culture conditions. Following a maximum of TNF-alpha production at 24 hours, the peak of IL-12 production was observed at 48 hours after the start of the experiment. Our results suggest that simulated microgravity favors the establishment of a Th1 type cytokine response.     

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

Sodium-dependent nucleoside transport in mouse lymphocytes, human monocytes, and hamster macrophages and peritoneal exudate cells.

Mouse splenocytes and hamster peritoneal exudate cells (PEC), including macrophages, were shown to contain a predominantly Na(+)-dependent and inhibitor (6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside, NBMPR)-resistant transport system for adenosine and other nucleosides. Adenosine (1 microM) was transported about equally in mouse thymocytes and human monocytes from peripheral blood by a Na(+)-dependent system and the NBMPR-sensitive facilitated diffusion system. Hamster PEC also transported inosine, tubercidin, formycin B, uridine, and thymidine in a NBMPR-insensitive manner. With the exception of formycin B, all nucleosides were phosphorylated intracellularly to varying degree, adenosine being almost fully phosphorylated. During the time course of routine experiments (30 s) formycin B was concentrated twofold over external medium levels (1 microM) without any drop-off in the transport rate. On the basis of metabolic studies it was estimated that uridine and tubercidin were also transported against a concentration gradient. Inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, and the pyrimidines uridine, thymidine, and cytidine (all 100 microM) inhibited transport of adenosine and inosine about 50-100%, while 3'-deoxyinosine showed weak inhibitory action. Transport of thymidine was strongly inhibited by nucleosides except by 3'-deoxyinosine. The Na(+)-dependent, active, and concentration transport system appears to be a feature of many immune-type cells, and its presence offers particular conceptual possibilities for the therapy of infections located in these cells.     

Purine and pyrimidine salvage in a clonal Drosophila cell line

A new culture medium, ZH1%, for Drosophila cell lines is described in which treatment of cells with the folate antagonist methotrexate (MTX) leads to overt double auxotrophy for both purines and pyrimidines. Using this medium the response of a clonal Drosophila cell line, KcAlo, to various purines and pyrimidines was measured. Guanosine was found to balance the toxicity of adenosine (AR), and vice versa. In addition AR toxicity was shown to be strongly dependent on the pH of the culture medium. Using ZH1% supplemented with MTX + thymidine (TdR) or with MTX + inosine (HR) several purines or pyrimidines, respectively, were tested for their capacity to support cell proliferation. This provided evidence for pathways for the salvage of thymine, TdR, thymidylic acid, adenine (A), AR, and HR. Bromodeoxyuridine and fluorodeoxyuridine are very active and specific TdR analogues inhibiting Drosophila cell proliferation. Of the many purine analogues tested the only potent growth inhibitor with demonstrable pathway specificity (antagonizable by A only) was methylpurine.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Effect of hindlimb suspension simulation of microgravity on in vitro immunological responses

The effects of microgravity on the immune system are largely unknown, but understanding such effects becomes increasingly important as space exploration continues and mission duration increases. Reductions in postflight human T cell reactivity to mitogens is well documented. Similar results have been obtained using a clinostat as an in vitro model of microgravity. In this study, a rat tail suspension model of weightlessness was used to examine in vitro lymphocyte proliferation in response to mitogens. Experiments were designed to uncover potential deficits in events related to proliferation including cell surface protein and IL-2 receptor (IL-2R) expression, interleukin-2 (IL-2) production, and accessory cells. Suspension of rats for 1 week led to a significant depression in [3H]thymidine incorporation by mitogen-stimulated peripheral blood lymphocytes (PBL) but only a small decrease in the proliferation of lymph node lymphocytes and splenocytes. There were no changes in the percentages of cells expressing CD4, CD5, CD8 or immunoglobulin. Moreover, no changes in IL-2 production or IL-2R expression were observed. More esterase-positive macrophages were detected in all lymphatic tissues of suspended rats, but there was no corresponding increase in the percentage of cells bearing the macrophage markers OX41 or OX42. This increase in the number of macrophages may be related to the observed suppression of lymphocyte proliferation. The tissue specificity of the decrease in mitogen activation indicates that there may be a compartmentalized response in the rats tested in the hindlimb suspension model.     

Saccharomyces cerevisiae URH1 (Encoding Uridine-Cytidine N-Ribohydrolase): Functional Complementation by a Nucleoside Hydrolase from a Protozoan Parasite and by a Mammalian Uridine Phosphorylase

Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.     

Influence of simulated microgravity on the longevity of insect-cell culture

Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth. © 1993 Wiley-Liss, Inc.     

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.