Investigation Of Funtional Change And Mechanism Of T Lymphocytes In Lung Under A Simulated Microgravity Condition

Objective: The present study was conducted to investigate the functional changes of T lymphocytes under a simulated microgravity condition and delineate the mechanism of these changes. We investigated T cells' dysfunctions under simulated microgravity and studied the PKC-mediated signaling pathway and Ras-dependent ERK signaling pathway, the two key events in activation of T cells. We also studied the functional role of leptin as a modulator of lymphocyte and the mechanism behind the change of the two signaling pathways under the simulated microgravity.Methods: 1. Lung T lymphocytes were isolated from rats. In this regard, the Rotary Cell Culture System(RCCS) technology developed at NASA employed to create the microgravity conditions. We conducted a series of experiments, comparing the RCCS cell culturing to the conventional cell culturing. 2. The conventional cultured cells were divided into five groups: control, ConA, leptin, ConA+ leptin. Cells cultured in RCCS were divided into six groups: control, ConA, leptin, ConA+ leptin, ConA+PMA. 3. The cells PKC activity was determined by using Promega's non-radioactive PKC assay system; IL-2 was measured by enzyme-linked immunosorbent assay kits; the observation of expressions of NF-κB, c-fos in cells was performed to detect by immunohistochemical staining; the activation markers CD25, CD71 were analyzed by flow cytomery; proliferation assays of T lymphocytes were determined by MTT assay; Western blot analyses were performed to detect the expression of phosphorylated ERK, total ERK.Results: 1. In the normal gravity environment with a constant acceleration of 9.8 m/s~2, the PKC activity of T lymphocytes and the percentage of NF-kB activated T cells have increased during cells exposed to ConA, compared to control group(P<0.01). IL-2 production, the activation markers CD25, CD71, the percentage of c-fos, proliferation assays, the expression of phosphorylated ERK have increased, too. 2. The PKC activity, the percentage of NF-kB, c-fos, IL-2 production, the activation markers CD25, CD71, the proliferation assays, the expression of phosphorylated ERK have decreased under simulated microgravity conditions in RCCS. 3. We added PMA to ConA cultures of T cells in RCCS, PMA was able to restore some of the PKC activity, the percentage of NF-κB, c-fos, IL-2 production, the activation markersCD25, CD71, the proliferation assays, the expression of phosphorylated ERK compared to ConA group (P<0.01). 4. In conventional cultures, we failed to find the enhancement of the PKC activity with leptin alone, the percentage of NF-kB, c-fos, IL-2 production, the activation markers CD25, CD71, proliferation assays, the expression of phosphorylated ERK compared to ConA group (P<0.01). however, leptin has been shown to enhance cognate T cell response. 5. In cultures under simulated microgravity, all indices decreased, not only with leptin alone but also with leptin and ConA together, which is different from ConA group and leptin and ConA groups under the normal gravity condition (P<0.01). 6. In simulated microgravity cultures, all indices decreased, Compared with the ConA and PMA groupsConclusions: 1. We found abrogation of T cells cultured in the RCCS.2. With the addition of PMA to the ConA- RCCS cultures, the suppression of ConA-activation T cells could be relieved a little. 3. Leptin is known to have no effect on the activation of T lymphocytes. However, Leptin was found to be a good potentiator of activation effect of ConA. 4.