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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.
The concept of microgravity (free-fall) influencing cellular functions in nonadherent cells has not been a part of mainstream scientific thought. Utilizing rotating wall vessels (RWVs) to generate simulated microgravity conditions, we found that respiratory burst activity was significantly altered in nonadherent promyelocytic (HL-60) cells. Specifically, HL-60 cells in simulated microgravity for 6, 19, 42, 47, and 49 d had 3.8-fold fewer cells that were able to participate in respiratory burst activity than cells from 1 × g cultures (P = 0.0011, N = 5). The quantity of respiratory burst products from the cells in simulated microgravity was also significantly reduced. The fold increase over controls in mean fluorescence intensities for oxidative products from cells in microgravity was 1.1 ± 0.1 versus 1.8 ± 0.3 for cells at 1 × g (P = 0.013, N = 4). Furthermore, the kinetic response for phorbol ester-stimulated burst activity was affected by simulated microgravity. These results demonstrate that simulated microgravity alters an innate cellular function (burst activity). If respiratory burst activity is impaired by true microgravity, then recovery from infections during spaceflight could be delayed. Finally, RWVs provide an excellent model for investigating the mechanisms associated with microgravity-induced changes in nonadherent cells.
Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytose Aspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of DC expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.
Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein–Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm3 tubes, and treated with 3 mMn-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated with n-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.
Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75° C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80° C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45° C. After thawing, atrial cells showed 53 ± 5% of the metabolic activity, 84 ± 5% of the number, and 92 ± 2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25° C yielded the best results. The thawed ventricular cells showed 83 ± 5% of the metabolic activity, 91 ± 5% of the number, and 96 ± 2% of the viability of nonfrozen cells.
Using five trophoblast cell lines of different differentiation status, we have shown that trophoblasts could constitutively release the transforming growth factor beta-1 (TGFβ1), but not TGFβ2. Treatment of the human tumorigenic, TL, and BeWo cells with a differentiating agent and a potent protein kinase C activator—the tumor-promoting agent—or the JEG-3 cells with cholera toxin—a potent cyclic adenosine 3′:5′monophosphate (cAMP) inducer—or its analogue 8-bromo-cAMP, potentiates TGFβ production, but the two signaling pathways appear to be mutually exclusive. Surprisingly, the JAR cell line failed to respond to either type of TGFβ activator. Based on reverse transcriptase (RT)-polymerase chain reaction (PCR), it was found that only the JAR cell line expressed messenger ribonucleic acid for decorin, a natural inhibitor of TGFβ, and none of the cell lines had detectable protein expression as determined by immunocytochemical studies. The cell number in cultures with decorin was invariably lesser than in those without decorin under serum-free conditions for all the cell lines tested. These results suggest that TGFβ may act as an autocrine-survival factor for transformed trophoblasts by allowing the cells to survive longer under a microenvironment which is not favorable for growth. Lastly, our results indicate that decorin, acting in a paracrine manner, may also play an important negative regulatory role in the development of transformed trophoblasts by sequestering TGFβ, thereby preventing its action.
The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor α (TGFα), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates. The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys. TGFα messenger ribonucleic acid, evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines. Large amounts of TGFα peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells. RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines). The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates. This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFα found in significant amounts only in the SK-NEP-1 cell line.