1. An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes. Techniques currently used for the isolation of plasmamembranes include zonal or density gradient centrifugation, the ripflip method developed for the microscopic observation of small pieces of the cytoplasmic side of plasma membranes, and methods based on the adhesion of negatively charged cells to a positively charged surface such as polylysine-coated polyacrylamide or glass beads. A method to isolate large quantities of directly accessible cytoplasmic surface of the plasma membranes suitable for both microscopy and biochemical analysis was developed. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water. Optimal conditions were established for all preparation steps, including polylysine coating, cell adhesion, and membrane washing, ensuring both high purity and high yield of the membrane preparation. This method allows (i) the creation of isolated plasma membranes without chemical (high-salt) or mechanical (vortexing or sonication) treatments, (ii) direct lipid extraction on glass plates, and (iii) the ability to study both biochemical and structural properties of isolated plasma membranes. Methyl-b-cyclodextrin (MbCD)1 is used to alter the cholesterol content of cells and, in particular, the cholesterol content of the plasma membrane. The cholesterol content of cellular membranes and the relationships between cholesterol-enriched domains and physiological function is an active area of research. Although questions remain unanswered with regard to detailed kinetics, dependence on composition and temperature, and specific and nonspecific effects, exposure of cells to MbCD reduces cellular cholesterol. However, reduction in cellular cholesterol following MbCD treatment might not quantitatively reflect the changes in plasma membrane cholesterol content. Having developed a method to isolate plasma membranes in quantities suitable for biochemical examination, MbCD cholesterol depletion evaluated using intact cells and their plasma membranes were compared. MbCD treatment extracted cholesterol from the plasma membrane of HAB2 cells, a hemagglutinin-expressing fibroblast cell line, and intact HAB2 cells in a temperature-dependent way, and the reduction in plasma membrane cholesterol content was not proportional to the decrease observed using intact cells. Nearly complete removal of plasma membrane cholesterol can be achieved by extraction at physiological temperature (37 _C). At 4 _C, MbCD extraction of cholesterol from the plasma membrane is less. These data indicate that one cannot predict the loss of cholesterol from the plasma membrane based on the loss determined from intact cells. Treatment with 10 mM MbCD for 30 min at 37 _C did not deplete cholesterol from all membrane fractions but essentially depleted all of the cholesterol in the plasma membrane. The roles of cholesterol in membrane heterogeneity/domains (rafts vs. nonrafts) and the association and function of proteins to specialized domains are of considerable interest. MbCD is often used to selectively deplete cholesterol from low- and high-density membrane fractions. Not only are MbCD concentration and exposure time important parameters in perturbing the cholesterol content of the membrane, but also extraction temperature is critical. Having a method to isolate and evaluate biochemical quantities of pure plasma membrane will benefit those studies where cholesterol perturbation needs to be minimized. The combined effects of MbCD concentration, exposure time, and temperature extraction now can be evaluated easily. 2. Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. We tested the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(/) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. We hypothesize that BMSC-mediated cytotoxicity of lymphocytes involves the FasL-activated apoptotic machinery. FasL is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) family. FasL interacts with its receptor, Fas (CD95/APO-1) and triggers a cascade of subcellular events culminating in apoptotic cell death. FasL and Fas are key regulators of apoptosis in the immune system. In addition, FasL is expressed by cells in immune-privileged sites, such as cancer cells, neurons, eyes, cytotrophoblasts of the placenta, and reproductive organs. In neurons, FasL expression specifically protects against T cell-mediated cytotoxicity. The discovery that FasL is also expressed by a variety of tumor cells raises the possibility that FasL may mediate immune privilege in human tumors. Activated T cells expressing Fas are sensitive to Fas-mediated apoptosis. Thus, up-regulation of FasL expression by tumor cells may enable tumorigenesis by targeting apoptosis in infiltrating lymphocytes. In the present work, we show that BMSCs can mediate immunosuppressive activity by FasL-induced killing of activated lymphocytes. Thus, BMSCs have properties of immuneprivileged cells. 3. 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.
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