Daila S. Gridley, Michael J. Pecaut, Lora M. Green, Glen M. Miller, Melba
L. Andres, Anna L. Smith, Radha Dutta-Roy, Dong Won Kim, Tamako A. Jones,
Deborah K. Murray, Xiao Wen Mao, and Gregory A. Nelson
Radiation Biology Program, Department of Radiation Medicine; Loma Linda
University and Medical Center, Loma Linda, CA 92354
Introduction. Altered gravity, radiation, and psychological stress experienced during long-term missions in space can all induce profound changes in the immune system. The combination of these various factors may result in significantly impaired defense mechanisms against infectious agents (e.g. viruses and bacteria), removal of debris and products of cells that are normally dying in the body every day, and surveillance against aberrant cells, including those that may be neoplastic. Our previous studies employing whole-body irradiation (g-rays, protons, 56Fe26), at doses within the range that might be expected during extended residence in space, have shown that numerous changes occur in immune system integrity and function. The major goal of the present study was to evaluate immunological changes over time due to hyper-gravitational stress. Investigations will be conducted in the near future to determine the influence of the combination of total-body irradiation and altered gravity on immune system responsiveness to challenge.
Materials and Methods. Young adult female C57BL/6 mice (n = 105) were acclimatized for 1 week and exposed to hypergravity at 2G and 3G for various lengths of time; in-room and rotation control groups were included. Assays were performed on days 1, 4, 7, 10, and 21 after initiation of centrifugation. The specific aim was to determine if hypergravity would have a significant effect on the following parameters in the animals: a) body, spleen, thymus, lung, and liver masses; b) leukocyte, red blood cell (RBC), and thrombocyte counts, and hemoglobin and hematocrit levels in blood; c) ability of white blood cells (WBC) to proliferate in response to (and without) a stimulating agent; d) distribution of lymphocyte subpopulations in blood and spleen as determined by 4-color flow cytometry; and e) apoptosis of splenocytes based on annexin V binding and quantified by laser scanning cytometry. The data were analyzed using one- and two-way analysis of variance (ANOVA), Tukey's pair-wise multiple comparison test, and linear regression. A p value of <0.05 was selected to indicate significance, and r2 ³ 0.6 to indicate linearity.
Results. Decreasing body, spleen, thymus, and liver masses were noted with increasing gravity (p<0.001); the effect was linearly dependent on gravity for body, spleen and thymus mass on day 4 post-loading onto the centrifuge. The length of time on the centrifuge also had a significant effect (p<0.001). Lung mass was affected primarily by time.
Spontaneous DNA synthesis by leukocytes was less affected in blood than in spleen. In blood, the length of time on the centrifuge, but not acceleration level, had a significant effect (p=0.003), whereas in the spleen both gravity (p<0.05) and time (p<0.001) played a role. On days 1 and 4, blood and spleen leukocytes from the 3G group had the lowest levels of DNA synthesis. However, there were no linear dependencies on gravity in these measurements.
Response of T and B lymphocytes to mitogenic stimulation was affected by both gravity and time (p's<0.001); there were no linear dependencies on acceleration. Splenic lymphocytes from mice in either the 2G or 3G groups generally had lower stimulation indices (SI) compared to the in-room (1G) control group. However, the rotation controls exhibited the greatest depression in SI values on days 4, 7, and 10 of centrifugation.
Analysis of blood using a veterinary analyzer indicated that numbers of total WBC, lymphocytes, monocytes, and granulocytes, and hemoglobin concentration were significantly affected by both acceleration (p<0.05) and time (p<0.05), whereas the numbers of RBC and platelets and hematocrit, were affected only by time (p<0.001). Linear dependence on gravity was seen on day 4 in numbers of lymphocytes and monocytes. Similar analyses of spleen leukocytes, also showed that WBC counts and the three major leukocyte types were significantly affected by both gravity (p<0.001) and time (p<0.001). Linear dependence on gravity was seen on day 4 in WBC, lymphocyte, macrophage, and granulocyte numbers.
Flow cytometry indicated that peripheral CD3+ T, CD4+ T helper (Th), CD8+ T cytotoxic (Tc), CD19+ B, and NK1.1+ NK cells were greatly affected by gravity (p<0.001) and time (p<0.005). Linear dependence on gravity was seen on day 1 in B cell and on day 4 in T, Th, and Tc cell numbers. Similarly, splenic T, Th, Tc, B, and NK cell counts were greatly affected by gravity (p<0.005) and time (p<0.001), while linear dependencies on gravity were found on day 4 in the T, B and NK cells and on day 10 in the T, Th, and Tc cells. Generally, in both blood and spleen, increasing acceleration resulted in decreasing cell numbers.
Quantification of spleen cells undergoing apoptosis determined by the level of annexin V binding revealed that apoptosis increased as a function of increasing acceleration. The control (1G) spleen cells had a 5.5+/-1% level of apoptosis, whereas spleen cells from mice maintained at 2G had a 20% increase in the level of annexin binding (7.9+/-0.6) at days 1, 4 and 7. The level of apoptosis declined at 10 days and was below basal at day 21. The cells from mice at 3G had a 2-fold increase in apoptosis over control levels at days 1 and 4 (12% of population). The levels of apoptotic cells declined at days 7 & 10, and by day 21 the levels were below basal (3.5%). Thus, some of the loss of splenocytes in the accelerated mice was due to induced apoptosis.
Conclusions. The data demonstrate that blood and especially spleen leukocytes become profoundly depleted during the first week of gravitational stress. The findings are similar to what we have observed after whole-body irradiation. It is not yet known whether the effects of combined irradiation and altered gravity will result in additive or synergistic immunodepression. Furthermore, since a linear relationship was found to exist between some of the measurements and the level of gravity, it may be possible to predict the status of certain aspects of the immune system at <1G (microgravity). In future studies we hope to expand these findings and combine hypergravity with low-dose rate whole-body irradiation. These investigations will include immune challenge with relevant bacterial and viral antigens.
Acknowledgements. This study was supported by Cooperative Research Agreement NCC9-79, the NASA/Ames Research Center and the Chan Shun Foundation.