Journal of Applied Physiology

Selected Contribution: Effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

Michael J. Pecaut, Gregory A. Nelson, Luanne L. Peters, Paul J. Kostenuik, Ted A. Bateman, Sean Morony, Louis S. Stodieck, David L. Lacey, Steven J. Simske, Daila S. Gridley


There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34+ population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.

  • microgravity
  • immune system
  • T cells
  • B cells
  • natural killer cells
  • stem cells

because of present-day extended stays on the International Space Station, potential Mars or Moon missions, and even a budding space tourism industry, it is becoming imperative that we understand the effects of spaceflight on human health. Studies in both rat models and humans have shown that the spaceflight environment can influence a number of physiological and immunological parameters. Changes in total body mass (1, 7, 8, 12, 21, 24, 42, 52, 54), thymus and spleen mass (12, 42, 45), circulating corticosterone (7, 32, 34,54), mitogen-induced proliferation (7, 31-33, 36, 37,47), cytokine production and reactivity (4, 13, 31, 32,35, 37, 48, 49), and lymphocyte (Lymph) subpopulations (2,23, 42, 46-48) have all been reported.

There are at least three aspects of the spaceflight environment that may be responsible for changes in immune parameters: mission-related psychological stress, low-dose/low-dose rate radiation, and changes in inertial condition (i.e., microgravity) (42, 50). Our laboratory has already reported results of studies with C57BL/6 mice characterizing the effects of low-dose γ-ray (18, 27,41), proton (14, 15, 25-27, 40), iron, and silicon ion (19) radiation on immunity. Furthermore, our laboratory has collected preliminary data on the effects of hypogravity [Space Transport System (STS)-77 (42)] and hypergravity (17) on immune parameters, which suggest that the influences of these environmental factors are, in at least some respects, similar. At worst, they could interact synergistically and dramatically reduce immune efficacy (16, 50).

On December 5, 2001, the space shuttle Endeavor (STS-108) launched on a 12-day mission flying mice (C57BL/6J) for the first time for the duration of a shuttle mission. Pregnant mice flew on STS-90 but were euthanized early in the mission with only tissues from neonatal animals analyzed. The primary goal of this study was to examine spaceflight as a model for disuse-induced osteopenia in mice and to test the osteoclast inhibiting protein osteoprotegerin for the biotechnology company Amgen (Thousand Oaks, CA). Nonosseous tissues from the placebo-treated groups are being examined to explore the potential of space-flown mice as models for other biomedical disorders.

This paper is the first of two describing the results of the immunological assessment. Herein, we focus on population distributions in the spleen, bone marrow, and, to a limited extent, blood. Part II of this series covers body and lymphoid organ masses, cytokine expression, and activated T- and natural killer (NK)-cell populations (20). By providing this initial characterization, future investigations utilizing the wild-type C57BL/6 mouse model should be facilitated, allowing both the confirmation of the data presented here and the expansion of the current body of spaceflight research to include genetically modified strains (i.e., transgenics or knockouts), which typically have a mixed genetic background that includes the C57 strain. Furthermore, it is our hope that the data presented here make clear the necessity of characterizing the effect of spaceflight environment on the ability of the immune system to respond to an immunogenic challenge. Suboptimal response could result in overwhelming infection and possibly even death, as well as economic loss due to mission failure.


Animals, housing conditions, and specimen collection.

Female C57BL/6J mice were ordered from Jackson Laboratory (Bar Harbor, ME) and delivered to the National Aeronautics and Space Administration (NASA) Life Sciences Support Facility (hangar L) at the Cape Canaveral Air Force Station, Florida, at 43 days of age. The mice were housed in vivarium cages at payload turnover until the age of 64 days, and standard rodent chow was replaced with food bars at 51 days of age. Food bars (American Institute of Baking Teklad no. TD 97071, Manhattan, KS) and water were provided ad libitum throughout the mission.

Animal enclosure module (AEM) hardware was provided by the NASA Ames Research Center (Moffett Field, CA) for both flight and ground simulation controls. This hardware has a housing density that is within the guidelines recommended by the National Institutes of Health with ∼13.5 in.2 floor area per mouse in a ground configuration. In flight, there was a constant flow of air (∼0.25 ft./s) in the AEM to move waste toward an exhaust filter.

Because these animals for which data are reported here were the controls for a larger study investigating the effectiveness of a pharmaceutical countermeasure for spaceflight-induced osteoporosis, each animal received a 180-μl subcutaneous injection of calcein (20 mg/kg; C-0875, Sigma Chemical, St. Louis, MO) in a sterile PBS vehicle at payload turnover (∼22 h before launch). All mice were 64 days old when injections were administered, with ground control groups age and weight matched on a 2-day delay from flight animals.

The mice included in the immunological assessment were assigned to one of three groups (n = 12/group): flight (Flt), vivarium (Viv) ground controls, and simulated flight controls (AEM). AEM ground controls were kept in the Orbiter Environmental Simulator (OES) room at hangar L. The purpose of the OES was to mimic shuttle temperature, humidity, and CO2 concentration. The parameter of primary modeling concern was CO2, which averaged >3,000 ppm for STS-108, nearly 10 times the level of a well-ventilated terrestrial room. The Viv ground controls were housed in the hangar L animal care facility with standard atmospheric conditions.

After 12 days of flight (11 days 20 h) in the middeck of space shuttle Endeavor (flight STS-108/UF-1), mice were weighed and euthanized by isoflurane inhalation, cardiac puncture, and exsanguination. Euthanization and dissection began ∼3.5 h after landing and were completed within 5 h of landing. Processing of ground control mice occurred 2 days later with the same procedures.

Approximately one-third of the spleen was placed into sterile screw cap tubes containing 1 ml complete RPMI-1640 medium and kept on wet ice. Bone marrow was flushed from the right humerus with 1 ml cold complete RPMI-1640 medium and kept on wet ice. Both tissues were shipped on wet ice from hanger L to the Loma Linda University laboratories and analyzed within 30 h of euthanasia. Whole blood was collected in syringes by cardiac puncture at the time of euthanasia, placed immediately into K2-EDTA-coated microhematocrit tubes (Becton-Dickinson), and shipped to Jackson Laboratory for analysis. Samples were analyzed ∼46 h after euthanasia.

The Institutional Animal Care and Use Committees of the University of Colorado, Amgen, Ames Research Center, and Kennedy Space Center reviewed and approved the protocols for this study. Approval was also obtained from the Loma Linda University Institutional Animal Care and Use Committees for the transfer of mouse blood and tissues.

Splenocyte and bone marrow assessment using the ABC Vet Hematology Analyzer.

Spleen and bone marrow were processed and evaluated as previously described by using the ABC Vet Hematology Analyzer (Heska, Waukesha, WI) (15, 18, 26, 41). Parameters characterized included granulocyte (Gran), eosinophil (Eos), monocyte/macrophage (Mono), and Lymph counts and percentages.

Hematology using the Advia 120 analyzer.

After arrival at Jackson Laboratory, whole blood samples were diluted 1:4 in murine PBS (10 mM NaCl, 155 mM KCl, 10 mM glucose, 1 mM MgCl2, 2.5 mM KHPO4, pH 7.4), and complete blood counts were determined by using the Advia 120 multispecies whole blood analyzer (Bayer, Tarrytown, NY). Results reported here include counts and percentages of peripheral Gran, neutrophils (Neut), basophils (Baso), Eos, Lymph, and monocytes.

Flow cytometry analysis of bone marrow and spleen.

Immunophenotyping was carried out by using a four-color FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) (17, 40,41). Monoclonal antibodies labeled with FITC, R-phycoerythrin, allophycocyanin, or peridinin chlorophyll protein were directed against specific markers on various cell populations. Data were collected on all events until 10,000 cells were counted in a CD45 (30-F11) vs. side-scatter Lymph gate.

For basic Lymph phenotyping, we first gated on the Lymph population on a CD45 vs. side scatterplot. A two-tube custom-conjugate mixture (Pharmingen, San Diego, CA) with antibodies allowed characterization of the following populations: CD45+/CD3+(145–2C11), mature T cells; CD45+/CD3+/CD4+ (RM4–5), T helper (Th) cells; CD45+/CD3+/CD8+(53–6.7), T cytotoxic (Tc) cells; CD45+/B220+ (RA3–6B2), B cells; and CD45+/NK1.1+ (PK136), NK cells.

For stem cell quantification, a CD45 vs. side scatterplot, which separates lineages based on cytoplasmic complexity, was used. As bone marrow cells mature, they express increasing density of CD45. Lymph generally exhibit the brightest CD45 staining and are low on side scatter (nongranular). A gate was placed on the population that represented the proportion of leukocytes with blast characteristics (%blasts). Blasts are generally low on side scatter and exhibit dim CD45 compared with mature cells such as the Lymph. Within this gate, cell populations that labeled positively with two hematopoietic stem cell markers, Ly-6A/E (E13–161.7) and CD34 (RAM34), were examined. Three overlapping populations were quantified (CD34+, Ly-6A/E+, and CD34+/Ly-6A/E+).

Analysis was performed by using CellQuest software version 3.1 (Becton Dickinson). The number of cells for bone marrow Lymph population was obtained as follows: number of cells in population/ml = number of leukocytes/ml × percentage of the population. Because only a portion of each spleen was shipped to Loma Linda University for analysis, these data are presented only as percentages.

Statistical analysis.

The results were analyzed by using one-way ANOVA and Tukey's pairwise multiple-comparison test. A P value of <0.05 was selected to indicate significant differences among groups, whereas aP < 0.1 indicated a trend. These analyses were performed by using SigmaStat software, version 2.03 (SPSS, Chicago, IL).


Leukocyte subpopulations in the peripheral blood.

There were no significant differences noted in the peripheral white blood cell (WBC) count among groups (Table1). Assessment of five separate leukocyte subpopulations showed no significant effects on Lymph and Gran counts. However, a significant decrease in monocyte numbers compared with AEM, but not Viv, ground controls was observed.

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Table 1.

Effect of flight condition on blood and bone marrow leukocyte counts

Although there was a significant flight condition dependency in the proportion of Lymph (P < 0.05, Fig.1), post hoc Tukey analysis indicated that there were no differences between Flt and either ground control. There was, however, a significant increase in Lymph proportions for the AEM mice compared with Viv controls. Similarly, there was a trend for a flight condition effect on the proportion of circulating monocytes (P = 0.063). Post hoc analysis suggested that this trend was due to a decrease in the Flt animals compared with AEM controls (P = 0.053). There were no significant changes in the proportion of circulating Gran, or in any Gran subpopulation (Neut, Baso, or Eos) (Table 2).

Fig. 1.

Effect of flight condition on leukocyte subpopulations. A: lymphocytes; B: granulocytes;C: monocytes. Flt, flight group; AEM, animal enclosure module simulated flight control group; Viv, vivarium ground control group. Values are means ± SE. Post hoc Tukey's test:a P < 0.005 andb P < 0.001 vs. AEM;c P < 0.05 andd P < 0.005 vs. Viv.

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Table 2.

Effects of flight condition on circulating granulocyte populations

Because some of the blood samples did not survive the shipping process, the relatively small sample sizes made blood data from the different groups less likely to be statistically significant. Unlike the other tissues, samples from only four of the Flt animals underwent hematological analysis. Similarly, AEM and Viv ground controls included only nine and five samples, respectively.

Leukocyte subpopulations in the spleen.

As shown in Fig. 1, there were highly significant effects of flight condition on proportions of splenic Lymph (P < 0.001) and Gran (P < 0.001). There was a flight-induced increase in Lymph percentages, with a corresponding decrease in Gran compared with both AEM and Viv controls. However, there were no changes noted in monocyte percentages. A similar lack of flight-induced change was noted in the Eos (data not shown for all groups); the percentages ranged from 1.05 ± 0.07% for AEM to 1.18 ± 0.07% for Flt.

Leukocyte subpopulations in the bone marrow.

The flight effect was even more pronounced in the bone marrow differentials than in the spleen. Although there were no effects on cell counts (Table 1), there were highly significant effects of flight condition on Lymph (P < 0.005), Gran (P < 0.001), and monocyte (P < 0.005) proportions (Fig. 1). There were increases found in the Flt animals for both Lymph and monocyte proportions compared with AEM and Viv controls. There were corresponding decreases noted in Flt Gran percentages compared with AEM, but not Viv, ground controls. AEM controls were also significantly greater than Viv controls in this measure. Similar changes occurred in the Eos proportions (Flt = 3.66 ± 0.11%, AEM = 4.05 ± 0.10%, Viv = 3.62 ± 0.10%,P < 0.01 for effect of flight condition); the Flt and Viv groups were both significantly lower than the AEM mice,P < 0.05 for both.

Flow cytometric analysis of Lymph in the spleen.

There were flight-dependent proportional differences noted in the major splenic Lymph subpopulations as measured by flow cytometry (Fig.2). The proportions of CD3+ T and B220+ B cells were both significantly influenced by flight condition (both P < 0.05). T-cell proportions significantly decreased, whereas B cells increased, in the AEM, but not Viv, ground controls. There were no changes in NK1.1+ NK cell proportions.

Fig. 2.

Effect of flight condition on T-, B-, and natural killer (NK)-cell subpopulations. A: CD3+ T cells.B: B220+ B cells. C: NK1.1+ NK cells. Values are means ± SE. Post hoc Tukey's test: a P < 0.05 andb P < 0.001 vs. AEM;c P < 0.001 vs. Viv.

As shown in Fig. 3, the proportion of CD3+/CD4+ Th cells was also significantly influenced by flight condition (P < 0.001). Post hoc Tukey's test indicated that Flt Th-cell percentages were significantly lower than those for both ground controls. In contrast, there were no significant effects of flight condition on CD3+/CD8+ Tc-cell proportions. This led to a reliable decrease in the CD4-to-CD8 ratio in Flt animals compared with both ground controls.

Fig. 3.

Effects of flight condition on T-cell subpopulations.A: CD3+ CD4+ T helper cells.B: CD3+ CD8+ T cytotoxic cells.C: CD4-to-CD8 ratios. Values are means ± SE. Post hoc Tukey's test: P < 0.001 vs.a AEM and vs. b Viv.

Flow cytometric analysis of the bone marrow.

As was noted in the major leukocyte populations, the effects of flight condition were more pronounced on the Lymph populations in the bone marrow than in the spleen, with significant main effects found in T-, B-, and NK-cell percentages (P < 0.001,P < 0.001, and P < 0.05, respectively). However, the differences noted in this compartment were in the opposite direction from that of the spleen. CD3+T-cell percentages were higher in Flt animals compared with both ground controls, with corresponding decreases in the B220+ B cells. There were also increases in the NK1.1+ NK-cell proportions in Flt animals compared with AEM, but not Viv, controls. With the exception of a significant decrease in B-cell counts in Flt mice compared with AEM controls, the changes in bone marrow percentages were not reproduced in their corresponding phenotype counts (Table3).

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Table 3.

Effect of flight condition on bone marrow lymphocyte subpopulations

There were also significant flight effects on the Th- and Tc-cell proportions (P < 0.001 for both subsets). Th- and Tc-cell percentages were higher in Flt animals compared with both ground controls. Therefore, unlike what was found in the spleen, no significant differences were seen in the CD4-to-CD8 ratios. There were strong trends for flight-induced changes in both Th- (P= 0.051) and Tc-cell (P = 0.051) counts (Table 3). Th-cell numbers were significantly higher in Flt animals compared with Viv ground controls. Similarly, there was a strong trend for a Flt vs. Viv difference in Tc-cell counts (P = 0.06).

Hematopoietic activity in the bone marrow also appeared to be dependent on flight condition (Table 4). There were differences in the counts and percentages of the blast population, as well as the CD34+ stem cell subpopulation. Flight condition-dependent changes in the double-positive CD34/Ly-6A/E percentages were also present. There were significant decreases in the blast cell proportions in Flt bone marrow compared with Viv, but not AEM, controls. The increases in the proportion of CD34+cells in Flt animals were significant compared with AEM, but not Viv, controls. There was a trend for a flight condition dependency in the Ly-6A/E+ cell proportions (P = 0.078) that was driven by a slight difference between ground controls (P = 0.063). Significant decreases in the proportion of the blasts, as well as the CD34+ and CD34+/Ly-6A/E+ subpopulations, were found in AEM controls compared with Viv controls. These findings were reflected, although somewhat less significantly, in the cell counts. There were significant flight dependencies in the blast and CD34+ cell numbers. Blast cell counts were significantly lower in Flt animals compared with Viv, but not AEM, controls. However, the flight dependencies in the CD34+ cell counts were driven by a significant decrease in the AEM animals compared with the Viv group. There was also a trend for a decrease in AEM compared with Flt animals (P = 0.069). Finally, the main effect of flight in the double-positive CD34/Ly-6A/E cell counts was reduced to a trend (P = 0.095).

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Table 4.

Effects of flight condition on bone marrow stem cell populations

Habitat temperatures.

There was an inconsistency in the temperatures of the different habitats. Differences existed between Flt and AEM temperatures, despite very similar temperature profiles between the space shuttle middeck (23.8 ± 1.4°C, 28.6°C maximum, 18.4°C minimum) and the OES (23.9 ± 1.4°C, 28.7°C maximum, 18.3°C minimum). For the three Flt units, temperatures were consistently above 30°C (Flt 101: 31.8 ± 2.3°C, 35.7°C maximum, 22.4°C minimum; Flt 102: 33.6 ± 2.2°C, 37.3°C maximum, 22.7°C minimum; Flt 103: 32.7 ± 2.3°C, 37.3°C maximum, 22.7°C minimum). Maximum temperatures (>36°C) occurred briefly after launch (∼4 h) and when the cabin was depressurized (14.7–10.2 psi for ∼24 h) on flightday 5 for an extravehicular activity (“space walk”) from the space shuttle middeck.

For the three AEM units, temperatures were consistently <28°C (AEM 001: 26.5 ± 1.5°C, 31.0°C maximum, 21.6°C minimum; AEM 002: 25.3 ± 1.5°C, 28.6°C maximum, 19.6°C minimum; AEM 104: 24.6 ± 1.5°C, 28.6°C maximum, 18.4°C minimum). Temperatures were taken within the AEM cages at a location close to the exhaust filter for both Flt and AEM conditions. Temperatures were not taken within the cages of the Viv mice, but were taken within the animal holding room (27.5 ± 1.0°C, 29.9°C maximum, 23.7°C minimum). Air in the vivarium cages (with a microisolator top) is stagnant, whereas air within the habitat hardware is constantly moving.


Due to large differences in launch vehicle, mission profile, flight hardware, ground controls, and animal models, data from flight experiments have sometimes been inconsistent and difficult to characterize. Despite these limitations, it is generally accepted that changes in inertial condition (i.e., hyper- or hypogravity), as well as other factors associated with spaceflight, can have a significant impact on a number of immune parameters.

The data show no significant differences in peripheral WBC counts after STS-108, although the values appeared to be slightly lower in Flt animals compared with ground controls. Because this is the first mouse study in which these parameters have been quantified, there are no other reports against which the present data can be directly compared. However, this is similar to results from some past flights using the rat model. Depending on the flight, peripheral WBC counts have either remained constant (28, 29) or decreased (2, 7,23). Due to the constraints of this study, we were unable to obtain accurate splenic cell counts. However, others have reported decreases in total splenocyte counts after flight (22,38). Furthermore, rat splenocytes labeled with the pan-leukocyte antibody OX-1 (different from the clone used here) either decreased (48) or remained constant (49) after flight.

Several studies have found that spaceflight causes neutrophilia. In rats, circulating Neut proportions consistently increased (7, 23,28, 29), with a corresponding decrease in Lymph (2, 6, 7,23, 28, 29). Eos counts have been shown to decrease (2), while proportions remain constant (28,29). However, after STS-108, there were no significant effects of flight condition on the proportion or number of either the circulating Gran subpopulations (Neut, Baso, or Eos) or Lymph. The discrepancies are likely due to the choice in animal models. However, the lack of statistical significance reported here may also be due to the small sample size available for analysis. The decreases in peripheral monocyte counts in the Flt animals compared with AEM controls is consistent with most (2, 6, 7, 23), but not all (7, 28, 29), flights using other animal models.

In contrast to the blood, there were flight-induced increases in splenic Lymph proportions, with corresponding decreases in Gran. Furthermore, there were no significant changes in the splenic monocyte populations after STS-108. Although there appears to be nothing in the literature to confirm or contrast these findings, such a change would be consistent with a phenotypic shift between the splenic and blood compartments (42).

Flow cytometric analysis of splenocytes indicated that the spaceflight environment can influence distinct Lymph phenotypes. Specifically, the proportion of splenic T cells decreased, whereas B cells increased. Past flights characterizing splenic T-cell percentages in rat models are somewhat contradictory, with decreases (42), no changes (22, 38), or increases (23, 48, 49) all reported after spaceflight. Slightly less contradictory findings have been reported in B-cell percentages, which have been shown to either decrease (22) or remain unchanged (2, 23,38) after spaceflight.

The inconsistencies in the splenic T- and B-cell results could be due to a number of factors, including flight profile, hardware, and species; all of which were variable across past flights. In addition to the differences in animal model, many of these flights were either Cosmos missions or space shuttle missions that included single- rather than group-housed animals. Handling, timing, and analysis techniques may also affect results. Most previous flight studies have utilized single-color labeling methods. The multicolor cytometric methods used after the STS-77 and STS-108 missions allowed us to characterize the various phenotypes with a greater degree of accuracy. However, despite these improvements in methodology, it is unlikely that a difference in technique would produce opposing results across flights. Because the flights that resulted in similar T- and B-cell shifts [STS-108, STS-77 (42), and STS-57 (22)] utilized nearly identical launch vehicles, flight hardware, and mission durations, discrepancies in Lymph shifts are more likely due to differences in these factors rather than in animal model or analysis technique.

Although we were unable to phenotype the peripheral blood, circulating T-cell percentages have remained constant (23), whereas B-cell percentages and counts consistently decreased (2,23) after flight in the rat model. This suggests that there may have been a phenotypic switch between compartments in the rat, with B cells leaving the blood and entering the spleen. However, further study is needed to confirm this shift in the mouse model.

After STS-108, there was a shift in splenic T-cell subtypes. CD3+/CD4+ Th-cell proportions decreased, whereas CD3+/CD8+ Tc-cell proportions remained constant after flight. This led to a reliable decrease in the splenic CD4-to-CD8 ratio similar to at least one previous flight (42). A reduction in this ratio has frequently been associated with an immunocompromised state. However, without knowing what the circulating CD4+ and CD8+ counts are, it is difficult to make more firm conclusions.

As was seen in the general Lymph populations, there have been a number of flights that did not influence splenic T-cell subset percentages. The proportions of both Th (2) and Tc cells (2, 23,38) have often remained constant. However, there have also been a number of flights that resulted in increases in Th- (22, 38,49) and Tc-cell (22, 48, 49) proportions. In sharp contrast, there has only been one report, other than ours, in which splenic Th-cell percentages have decreased (23). Although CD4-to-CD8 ratios have not typically been reported in the past, we have found similar decreases across two similar flights with two different species (42). The discrepancies between the data presented here and that of past flights is somewhat disconcerting. There does not appear to be any hardware, launch vehicle, or flight duration-dependent rationale that adequately explains these inconsistencies. Furthermore, because we found similar results with two different species, this parameter does not appear to be a driving factor. Flow cytometric technique may have been of primary importance as the CD4+and CD8+ T cells are relatively specific subpopulations. Previously used single-color labeling techniques may not have been exclusive enough (i.e., CD4 can be found on both Th cells and macrophages).

In any case, there appears to be a slight shift in immunological homeostasis away from the cell-mediated branch of adaptive immunity toward humoral mechanisms, as indicated by the phenotypic shift away from T cells toward B cells. Furthermore, as will be discussed in part II of this series (20), the changes noted in phenotype distributions are also consistent with the changes noted in IL-2 expression. Mitogen-induced IL-2 production was significantly lower in Flt mice than in both AEM and Viv ground controls. However, interferon-γ, tumor necrosis factor-α, IL-2, and IL-4 expression did not appear to be influenced by spaceflight. Because the mitogen used specifically stimulated T cells (phytohemagglutinin), it is possible that the generalized T-to-B-cell population shift may be related to decreased capacity for IL-2 expression.

There were no significant effects of flight on NK cell proportions in the spleen. This is similar to what Sonnenfeld et al. (48,49) found in the rat model. However, whereas there was an increase in the proportion of activated NK cells after the present flight (see part II of this series) (20), there have been reports of flight-induced decreases in splenic NK cytotoxicity (31, 44).

As we found no flight effects on the total bone marrow Lymph counts, there is little indication that hematopoietic mechanisms are responding to the changes noted in the spleen and blood. In contrast, although there were no effects of flight on monocyte, Lymph, or Gran counts, there were increases in the Lymph and monocyte proportions, with corresponding decreases in Gran. Because these changes are similar to those found in the spleen and peripheral blood, this is consistent with a shift in leukocyte homeostasis and/or their migration patterns. Rather than compensating for changes in peripheral phenotypes, the bone marrow appears to be inherently involved in these shifts.

Although not directly comparable, this fundamental shift in leukocyte homeostasis is not inconsistent with past experiments investigating hematopoiesis. Although there has been at least one report of an increase in hematopoietic measures (6), the majority of studies suggest that the response of bone marrow to various colony-stimulating factors has either decreased (1,47-49) or remained constant (7, 23, 30, 52) after flight.

Flow cytometric analysis of bone marrow stem cell populations gives credence to a shift in leukocyte hematopoiesis. Although the results indicate that there were flight-induced decreases in the overall blast population, CD34+ parameters increased compared with AEM, but not Viv, controls. No significant differences, however, were seen in the Ly-6A/E+ population. This is of interest because CD34/Ly-6A/E+ cells are part of a self-renewing hematopoietic stem cell population (39), suggesting that, if there is indeed a change in hematopoietic mechanisms, it occurs while the cells are differentiating into more mature phenotypes.

The proportion of CD34/Ly-6A/E double-positive cells was also shown to be dependent on flight condition. Although post hoc analysis indicated that there were no significant differences across groups, this population is relatively small to begin with, and even a limited change could have large downstream consequences. Furthermore, as this population includes hematopoietic stem cells involved in the recovery of short-term multilineage bone marrow after lethal irradiation (39), changes could be of critical importance after a particularly large solar particle event.

In contrast to the general leukocyte distribution, flight-induced changes in bone marrow Lymph subpopulations were very different from, and in some cases opposed, those of the spleen. T-cell proportions increased with a corresponding decrease in B cells. Also, both Th- and Tc-cell percentages increased in the bone marrow after flight. As a result of this simultaneous increase, there were no significant differences noted in the CD4-to-CD8 ratio. There were also flight-induced increases in the NK-cell proportions. These phenotypic shifts in bone marrow Lymph are similar to previous reports (48). Together, these results suggest that, whereas hematopoietic mechanisms appear to be encouraging the leukocyte shift toward monocytes and Lymph, this cannot be said for all of the different Lymph phenotypes.

For some of these analyses, there were significant differences between AEM and Viv ground controls. Although this was the case for the proportion of circulating Lymph, most of the differences between ground controls were found in the analysis of the bone marrow (%Lymph, %Gran, %Eos, %blast, %CD34, number of CD34, and %Ly6/CD34). Similarly, Flt animals were different from one or the other of the ground control groups more often in the bone marrow (%Lymph, %Gran, %Eos, %blast, %CD34, and number of CD34 stem cells) than in blood (number of monocytes) or spleen (%T and %B cells). Most of these parameters were found to be significantly lower in Flt animals compared with AEM, but not Viv, controls. This suggests that animals in the ground AEMs may have been more stressed than even the Flt animals.

As stated previously, all mice in this study were treated with calcein before flight to enable the characterization of bone growth. A more detailed discussion of the effects of this label is included in part II of this series (20). However, calcein has a relatively short half-life (3) and is cleared from circulation within a few hours (43). In vivo studies strongly indicate that calcein has no effects on leukocyte population distribution or function (5, 10, 11, 53). Hence, we expected no significant effect of calcein on the parameters measured in this study.

A source of potential differences between flight and ground immune parameters may be the elevated temperature of the flight AEMs. NASA Ames Research Center personnel have run a test since the STS-108 mission and have identified the problem to be caused by recirculation of air within and between hardware units. As a result, some of the heat generated by the airflow exhaust fans in the AEMs increased the temperature of the intake air stream. The relative configuration and airflow characteristics of the ground AEM group were sufficiently different from those of the Flt group that waste heat was not recirculated and the inlet temperature more closely matched the room temperature. Similar elevated temperatures in flight have occurred previously with rats flown in the AEM habitats. For example, on STS-41, temperatures in flight AEMs peaked at 36°C (9, 51), and on STS-72 temperatures peaked at 35°C (unpublished data). Average temperature profiles were not published for these missions. Although temperature data have been published for very few flights, unusually high temperatures have not occurred on all flights (STS-77: 28.6 ± 1.0°C, 31.3°C maximum, 25.6°C minimum) (6).

There are several factors, different for each group, that impact the ability of mice to reject metabolic heat, the real physiological driver of temperature. These differences make it very difficult to simply rerun a set of Viv to mimic the high temperatures experienced by the Flt mice. First of all, in microgravity, buoyancy-driven convective heat flow does not exist (hot air rising because it is less dense than cooler air). Second, the forced-air convection (∼0.25 ft./s to move waste toward the exhaust filter) of the hardware (Flt and AEM groups) causes convective rejection of metabolic heat to the degree that a slight “wind chill” effect (not characterized for mice) may effectively reduce the “temperature” slightly. Finally, the temperature within the vivarium cages (Viv group) is most likely much higher than the temperatures of the animal holding rooms because the air within the vivarium cages is stagnant due to the greatly restricted flow through the microisolator cage tops.

Therefore, the heat loads for each group (Flt, AEM, and Viv) are inevitably different. The degree to which forced air vs. buoyancy-driven convective forces results in heat rejection is not characterized for mice. However, forced-air convection is likely to remove more heat than buoyancy in this investigation, effectively reducing the heat load for the Flt and AEM groups and raising the heat load for the Viv group. Thus these effects will tend to reduce the differences that would otherwise be predicted based on temperature alone. Still, because of heat load differences, firm conclusions about the effects of the spaceflight environment on any physiological parameter, particularly those as sensitive as immunity, should be viewed with caution.

Another aspect of space experimentation that results in uncontrollable differences is the access to a three-dimensional environment in microgravity (Flt) vs. the two-dimensional floor space for the ground controls (AEM and Viv). Appropriately accounting for the translation from livable floor space to livable volume is not an issue that has been quantified but is inevitably going to impact social behavior and potentially stress. Because corticosterone measurements (a common indicator of stress levels) were not taken, this is purely speculative. We suggest that translating usable volume for the flight animals to more floor space for the ground controls may be important when the design of future spaceflight hardware for animals is considered. This, in addition to the convection discussion, is another example of how it is impossible to change only one variable when removing gravity. The “spaceflight environment” becomes the variable, and it is important to understand the complexity of this parameter.

Despite the described issues, these results suggest that the spaceflight environment can have a considerable influence on every level of immunity, from hematopoiesis to acquired immunity. With all of the changes in immune parameters reported here and elsewhere, regardless of the inconsistency of the data, it is surprising that the question of immune efficacy has not been directly addressed. Specifically, do these changes in immune homeostasis affect the ability to respond to an immunogenic challenge (e.g., bacteria, viruses, and fungi)? Ground-based models for various aspects of the spaceflight environment are currently headed in this direction. For example, we have already begun to characterize the effects of γ-, iron-, and silicon radiation-induced changes on the ability of the immune system to respond to, and develop memory for, a primary and secondary challenge (unpublished observations). Furthermore, plans for a continuation of this work using proton radiation (the most common form of space radiation), as well as hypergravity (as modeled by centrifugation), are in progress. The next logical step would be to expose animals to a relevant challenge while in the spaceflight environment. This type of information should help mission managers formulate a more accurate astronaut risk assessment for long-duration missions.


Many people contributed to the success of this flight: too many to name individually. At Loma Linda University, Tamako A. Jones, Anna L. Smith, Radha Dutta-Roy, Melba L. Andres, Glen M. Miller, and Lora Benzatyan all provided valuable assistance. Mark Rupert from BioServe Space Technologies at the University of Colorado was the payload manager for this flight and was ultimately responsible for coordinating all of the groups involved and making sure all aspects of the experiment were ready for flight. Dr. Beverly Girten coordinated the NASA Ames Research Center team that performed preparation and verification activities of the flight hardware. Katrien Morgan and Gail Clark from Johnson Space Center were the payload integration managers, responsible for coordinating the manifesting, integration, and operations teams along with the formal submittal of much of the paperwork for flight. Several groups at the Kennedy Space Center were responsible for timely payload turnover, physical installation, and removal of the commercial biomedical testing module from the space shuttle in addition to on-site science support. In particular, we thank Ramona Bober for coordinating all animal activities at hangar L. In short, we would like to extend our appreciation for the many people required in performing spaceflight experimentation.

Sources of support include NASA cooperative research agreements NCC9–149 and NCC8–242, National Heart, Lung, and Blood Institute Grant HL-64885, Chan Shun International Foundation, the Department of Radiation Medicine of the Loma Linda University Medical Center, and Amgen.


  • Address for reprint requests and other correspondence: M. J. Pecaut, Dept. of Radiation Medicine, Radiobiology Program, Loma Linda Univ. School of Medicine, Chan Shun Pavilion, Rm. A-1010, 11175 Campus St., Loma Linda, CA 92354 (E-mail:mpecaut{at}

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • First published January 3, 2003;10.1152/japplphysiol.01052.2002


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