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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 21, 2005 as doi:10.1096/fj.04-3122fje. |
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* Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania, USA; and
Department of Biomedical Engineering, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA
1 Correspondence: Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut St., 210 Stratton Hall, Philadelphia, PA 19104, USA. E-mail: bwr24{at}drexel.edu
SPECIFIC AIMS
Immune function is suppressed in space flight, demonstrated by reduced mitogen-stimulated proliferation of postflight astronaut peripheral blood mononuclear cells (PBMCs). Although less consistent, the production of interleukin (IL) -2 and interferon (IFN) -
/ß and -
by stimulated T cells also appear to be reduced. Astronauts regain immune function 3 days upon return to Earth, but this recovery has not been clearly demonstrated after exposure of PBMCs to modeled microgravity (MMG). PBMCs cultured for 48 h in MMG using the high aspect ratio vessel (HARV), a type of rotating wall vessel (RWV) bioreactor, were subsequently recovered in stationary ground conditions for up to 120 h. Specific aims were to characterize the recovery of phytohemagglutinin (PHA) -stimulated proliferation and functional cytokine production after exposure to MMG.
PRINCIPAL FINDINGS
1. Suppressed proliferation of PBMCs in MMG
Forty-eight h exposure to MMG by HARV-type RWV completely inhibited PHA-stimulated proliferation of PBMCs to the level of unstimulated stationary controls (P<0.001). In further characterizing the kinetics of this suppression, MMG inhibited proliferation by 50% at 6 h (P<0.05) and completely by 24 h (P<0.001). Loss of proliferation response to mitogen was not due to an increase in cellular damage or death in HARV-treated samples compared with stimulated stationary controls, concluded from trypan blue exclusion and treatment with ethidium monoazide (EMA), which will permeate dead cells for detection by flow cytometry.
2. Recovery of proliferation after exposure to MMG
After culturing PBMCs in HARVs or stationary conditions for 48 h, cells were resuspended at an equal concentration of 1 x 106 cells/mL in media replenished with phytohemagglutinin (PHA). Full recovery of the proliferation response after exposure to MMG was gradually achieved over 72 h of recovery (Fig. 1
). At 24 h of recovery, PHA-stimulated proliferation of HARV cultures was 8% of stimulated stationary control, which was not considered statistically different from that of unstimulated control. By 48 h, the proliferative response of PBMCs previously exposed to MMG increased to 75% of stimulated stationary control (P<0.05), indicating 75% (i.e., partial) recovery. Full recovery was observed at 72 h, in agreement with published data of postflight studies of astronauts. Additional experiments were conducted to characterize proliferation from 0–24 and 72–120 h after exposure to 48 h MMG. PHA-stimulated stationary cultures reached maximum proliferation at 48 h, while PHA-stimulated proliferation of cells recovered after 48 h in HARV peaked at 72 h, indicating a delayed, but not statistically reduced, nonspecific mitogen-stimulated proliferation of PBMCs after exposure to MMG (see Fig. 3A
).
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3. Recovery of activation marker expression after exposure to modeled microgravity
Flow cytometry confirmed that recovered cells did not differ in the distribution of T cell subtypes (CD4+ vs. CD8+) compared with stimulated stationary controls. After stimulation with PHA, T cells not exposed to MMG maximally expressed CD69 at 6 h and CD25 at 24 h. In contrast, T cells previously exposed to 48 h in MMG began CD69 expression at 6 h, but maintained expression until a maximum point at 24 h. Maximal expression of CD25 then followed at 48 h poststimulation.
4. Recovery of cytokine production after exposure to modeled microgravity
ELISA
IL-2 detected in supernatant was reduced by 
80% in HARVs cultured for 24 h (P<0.05) and was not detectable in any cultures at 48 h or more. Detection of IFN- in MMG, while not significantly reduced at 24 h, was indistinguishable from stationary controls at 48 h. IL-2 in supernatants increased from 0–24 h of PHA-stimulated recovery after 48 h exposure to MMG. IL-2 in supernatants from recovered HARV samples was significantly greater than from stationary samples at 8, 12, and 20 h of recovery (Fig. 2
A, P<0.05). IFN- was nearly constant from 0–120 h of recovery and did not differ between stimulated stationary and HARV cultures.
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Bioassays
Detection of functional IL-2 and IFN-/ß were similar to stimulated stationary controls by 24 h after exposure to MMG. Functional IL-2 was greatest in supernatant at 24 h of recovery (Fig. 2B, P
<0.05), while IFN- /ß appeared to increase from 24–72 h, although this was not significant (Fig. 2C
). Accumulated IL-2 and IFN in supernatants from 48 h HARVs and from up to 72 h recovery after removal from HARV are summarized in Fig. 3
B relative to stimulated stationary controls.
CONCLUSIONS AND SIGNIFICANCE
Our studies extend for the first time the realm of an RWV-based investigation beyond the duration of acute MMG exposure and into the recovery phase. Exposure to MMG in the HARV-type RWV bioreactor inhibited PHA-stimulated proliferation of PBMCs, followed by full recovery after 72 h in stationary ground conditions. The experimental focus on PBMCs, either flown in space or exposed to MMG, eliminates in vivo confounders, such as the physical and psychological stress of space flight, suggesting that RWV cell culture is perhaps a suitable means for assessing the direct effects of microgravity on cellular immune function.
We demonstrated partial recovery of PHA-stimulated proliferation of PBMCs by 48 h (75%) and full recovery by 72 h, in agreement with postflight studies suggesting that astronauts fully recover cellular immunity by three days. MMG delayed, but did not significantly reduce, recovery of maximal PHA-stimulated proliferation. We observed a decrease in IL-2 and nonsignificant drop in IFN- at 24 h of exposure to MMG (ELISA). The apparent drop in IFN- was resolved by 48 h in MMG, as reported by Cooper and Pellis (1998), suggesting that IFN production overcomes the effects of MMG. No studies, however, have reported on the recovery of cytokine production after exposure to MMG. Functional IL-2 returned within 24 h and dropped off significantly by 48 h in both stimulated stationary and stimulated HARV cultures. Detection of IL-2 in supernatants in the early stages after exposure to MMG (i.e., less than 24 h), exceeded that of stimulated stationary cultures. This finding suggests a delay in IL-2 consumption in those cultures recovered from MMG compared with those in stimulated stationary condition, further supported by the observed delay in IL-2 receptor expression in these cells (Fig. 3A, B
). Delayed IL-2R expression, then, appeared to predestine the delay in maximal proliferative response observed in PBMCs recovered from 48 h exposure to MMG. IL-2 was not detectable by ELISA in supernatants from any cultures stimulated for 48 h or greater, and the bioassay revealed no functional IL-2 in supernatants from most subjects at 72 h of recovery. Taken together, these results suggest that after early accumulation associated with a relative delay in signaling events leading to receptor expression, IL-2 that was present may have become bound to IL-2 receptors, thus contributing to gradual recovery of proliferation over a 72 h period after exposure to MMG. Conversely, ELISA detected no difference in IFN- in cell culture supernatants for up to 120 h of recovery. Functional IFN-/ß appeared to possibly increase from 24–72 h compared with stationary control. We conclude that the IL-2 that was being produced in cultures recovered from exposure to MMG was also being consumed by proliferating T cells (autocrine), while IFN was not (paracrine), such that detection of functional IFN in the supernatants of activated T cells may be expected to increase as IL-2 decreases.
The effects of increased flight duration on immune recovery and possible associated clinical consequences remain entirely unknown. As a result, continued research must strive to identify countermeasures for microgravity- and stress-induced immunosuppression in flight, design interventions to bolster immunity in space, and develop agents to enhance postflight immune recovery. Such initiatives are of paramount interest to NASA as delineated in the current Bioastronautics Critical Path Roadmap. Notable similarities between microgravity-induced cellular immune suppression and that experienced in aging have led to the suggestion that (modeled) microgravity may serve as a condensed model for the study of long-term age-related immune degradation. Our studies reinforce previously published data suggesting that MMG is an innovative, valuable tool for studying multiple aspects of cellular immune function.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3122fje;
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, 
, =) are relative to stimulated stationary culture at same time points. IL-2 receptor (IL-2R) expression and maximal proliferation response to PHA were delayed in MMG. CD3, pan-marker for T cells; PHA, phytohemagglutinin A; IL-2, interleukin-2; IFN, interferon.