REGULATION OF PHYSIOLOGICAL SYSTEMS BY NUTRIENTS

Microgravity and Immune Responsiveness: Implications for Space Travel

Andrea T. Borchers, PhD, Carl L. Keen, PhD, and M. Eric Gershwin, MD From the Division of Rheumatology, Allergy and Clinical Immunology, University of

California at Davis School of Medicine, Davis, California, USA

To date, several hundred cosmonauts and astronauts have flown in space, yet knowledge about the adaptation of their immune system to space flight is rather limited. It is evident that a variety of immune parameters are changed during and after space flight, but the magnitude and pattern of these changes can differ dramatically between missions and even between crew members on the same mission. A literature search was conducted involving a total of 335 papers published between 1972 and 2002 that dealt with the key words immune response, microgravity and astronauts/cosmonauts, isolation, gravity, and human health. The data from multiple studies suggested that major discrepancies in outcome are due to methodologic differences. However, the data also suggested major factors that affect and modulate the immune response during space travel. In part at least, these discrepancies can be attributed to method- ologic differences. In addition, a variety of other features, in particular the types and extent of stressors encountered during space missions, are likely to contribute to the variability of immune responses during and after space flight. That stress plays an important role in the effects of space flight on immunologic parameters is suggested by the frequent findings that stress hormones are upregulated during and after space flight. Unfortunately, however, the existing data on hormonal parameters are almost as varied as those on immunologic changes, and correlations between the two datasets have only rarely been attempted. The functional implications of space flight–induced alterations in immune response largely remain to be elucidated, but the data suggest that long-term travel will be associated with the development of immune-compromised hosts. Nutrition 2002;18:889–898. ©Elsevier Science Inc. 2002

KEY WORDS: microgravity, space flight, immunity, stress, stress hormones

INTRODUCTION

Since the earliest flights into space, there has been concern about the effects that exposure to such a different environment could have on immune functions and health. To date, several hundred cosmonauts and astronauts have flown in space, yet knowledge about the adaptation of their immune systems to space flight is still limited. This is partly due to the small number of subjects on each space flight and the enormous variability in the responses of each subject to the space environment. In addition, there is great vari- ation in mission characteristics and as yet little understanding of what, if any, effect many of these characteristics have on immu- nologic parameters. In this review, we summarize existing data on space flight–associated changes in immune cell distribution and function, and we briefly highlight some of the factors known to influence immune function but have yet to be considered in the context of space flight.

The problems faced by researchers attempting animal experi- ments in space are no less daunting than those encountered in research on human astronauts or cosmonauts. It is essentially impossible to create adequate controls for all the different factors space-flown animals are exposed to, the major ones being the acceleration during launch, noise, vibration, substantial tempera- ture fluctuations, microgravity, and acceleration during landing. Not surprisingly, the results from experiments involving space- flown animals are as varied and contradictory as are data obtained

from astronauts and cosmonauts, so only selected examples are included in this review.

PHENOTYPIC DATA

Leukocyte and Lymphocyte Subsets

Leukocytosis is one of the most consistent findings in cosmonauts and astronauts on the day of landing after short- and long-duration flights.1–4 As summarized in Table I, this is mostly due to a dramatic increase in polymorphonuclear leukocytes (PMNs), and changes in the absolute numbers and percentages of lymphocytes and lymphocyte subsets differ considerably between studies and between individual missions analyzed within the same study.

Possible Reasons for Altered Lymphocyte Distribution

In space-flown rats, the changes observed in peripheral lympho- cyte and lymphocyte subset distribution are as varied as those in astronauts and cosmonauts. In addition, the effects of space flight on the percentage of these cell types and subsets seems to depend on the lymphoid tissue examined.5–7 The differential effect of space flight on leukocyte distribution in different organs may be mediated in part by changes in adhesion molecules. Tissue-specific alterations in the expression of adhesion molecules were reported in Fischer 344 rats after a 10-d space flight.6 The proportion of cells positive for LFA-1� and � increased in the spleen but decreased in lymph nodes. L-selectin expression was lower in spleen and lymph nodes of space-flown animals, but this difference achieved significance only with the HRL-3 antibody (not the HRL-2 antibody) and only compared with animal experimental

Correspondence to: M. Eric Gershwin, MD, Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, TB 192, Davis, CA 95616, USA. E-mail: megershwin@ucdavis.edu

Nutrition 18:889–898, 2002 0899-9007/02/$22.00 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved. PII S0899-9007(02)00913-9

module (AEM)–housed rats, not with the vivarium-housed ground controls. Expression in AEM-housed ground controls was signif- icantly higher than in vivarium controls. Intracellular adhesion molecule-1 (ICAM-1) was higher on splenocytes of flown animals than those of vivarium controls but lower on lymph node cells, with the latter not achieving significance.

In astronauts, the expression of Mac-1 (CD11b, a subunit of integrin CR3 that binds extracellular matrix proteins) on purified neutrophils was increased 3 d before launch (L-3), but decreased on the day of re-entry (R�0) compared with L-10 and R�3.4 In contrast, L-selectin (CD26L) expression was upregulated only on R�0, and CD11a levels did not change. Binding of neutrophils to tumor necrosis factor-� (TNF-�)–stimulated human umbilical vein endothelial cells (HUVECs) was significantly enhanced on L-4 and R�0 compared with L-10 and R�3. Such increased adhesion and adhesion molecule expression might be expected to result in decreased levels of circulating neutrophils, because enhanced binding of neutrophils to the endothelium would have prevented their release from the marginal pool. Instead, neutrophilia was observed in these astronauts. A possible explanation is that the increased postflight epinephrine levels induced changes in endo- thelial cells that prevented neutrophil binding.

FUNCTIONAL DATA

Among the functional parameters examined in cosmonauts and astronauts shortly after landing, and occasionally during space- flight, are lymphocyte proliferation, delayed-type hypersensitivity (DTH) responses, antibody production, natural killer (NK) activ- ity, and cytokine responses (reviewed by Cogoli8). Data on lym- phocyte proliferation are available for a large number of crew members and consistently indicate a reduction in the mitogen- induced proliferative response of peripheral blood mononuclear cells (PBMCs). Much more limited and highly variable informa- tion exists for other immune functions.

Decreases in DTH responses have been observed during and after space flight in some cosmonauts and astronauts.9,10 Based on measurements of DTH responses during a total of three missions, involving three, three, and four subjects, respectively, Taylor and Janney10 suggested that cellular immune responses are still de- creasing on flight day (FD) 4, with maximum suppression occur- ring between FD 5 and FD 10. Although this hypothesis is inter- esting, it is important to note that there was a limited number of subjects on each flight, and there was considerable variability in their responses. Interestingly, the scores of two cosmonauts from

TABLE I.

CHANGES IN PERIPHERAL BLOOD LEUKOCYTE NUMBERS OR DISTRIBUTION (STATED AS PERCENTAGE) AFTER SPACE FLIGHT

Cell type Change observed after space flight* n subjects/missions Days of space flight Reference

Polymorphonuclear leukocytes†

Neutrophil number significantly 1 (two-fold) 41/11 3–18 84 Number significantly 1 (5000 � 100 versus 3100

� 100 cells/�L) 30/6 4–5 92

Neutrophil number significantly 1 (�1.5-fold‡) 16/3 8, 9, and 14, respectively 4 Number significantly 1 (�2-fold‡) 19/§ 10–18 1 Neutrophil number significantly 1 (5330 � 1289

versus 2638 � 525 cells/�L; 3821 � 1314 versus 2461 � 525 cells/�L)

5/2 4–7 3

6/3 11–16 Monocytes Number 1 (250 � 280 versus 160 � 20 cells/�L) 30/6 4–5 92

Number 1 or 2 after different missions 16/3 8, 9, and 14, respectively 4 Number �, but significant reduction in the

CD14�/CD16� subset relative to the entire monocyte population

19 for peripheral monocytes, 14 for CD14�/CD16�

monocyte subset§

10–18 1

Number 1 (536 � 60 versus 357 � 69 cells/�L; 437 � 92 versus 332 � 188 cells/�L)

5/2 4–7 3

6/3 11–16 T cells (CD3�) Percentage � (slightly 1 in 6, slightly 2 in 5) 11/2 6 and 8 84

Percentage slightly, but significantly 2 13/§ 10–18 1 CD4� T cells Percentage � (1 in 8, 2 in 3) 11/2 6 and 8 84

Percentage significantly2 (23 � 2 versus 32 � 2%) 30/6 4–5 92 Percentage significantly 2‡ 20/§ 10–18 1

CD8� T cells Percentage � (1 in 5, 2 in 5, � in 1) 11/2 6 and 8 84 Percentage significantly2 (12 � 1 versus 17 � 1%) 30/6 4–5 92 Percentage significantly 2‡ 20/§ 10–18 1

CD4�/CD8� ratio 1 in 7, 2 in 4 11/2 6 and 8 84 1 in 23 of 27 27/4 10–18 1

B cells Percentage � (2 in 7, 1 in 4) 11/2 6 and 8 84 Percentage � 30/6 4–5 92 Percentage � 13/§ 10–18 1

Natural killer cells Percentage 2 (3 � 1 versus 9 � 1%) 10/§ 4–5 92 Percentage � 13/§ 10–18 1

* Day of re-entry versus before launch. † Some investigators provided numbers only for neutrophils, a subcategory of polymorphonuclear leukocytes. ‡ The date are represented graphically, making it difficult to determine actual numbers. § Number of missions not specified. 1, increase; 2, decrease; �, no statistically significant change occurred.

890 Borchers et al. Nutrition Volume 18, Number 10, 2002

long-term missions to the Mir space station markedly dropped approximately 1 wk after each had participated in a series of extended and unscheduled extravehicular activities to perform urgent repairs.9 This observation is consistent with the concept that the subsequent decreases in cellular immune function were stress induced.

Humoral immunity has only rarely been studied in cosmonauts and astronauts. After early Soviet space flights, no significant changes in total immunoglobulins (Ig) and Ig isotypes were ob- served.11 Similar results were reported in a more recent study of Shuttle astronauts.4

NK activity against 3H-uridine–labeled K562 target cells was depressed in a majority of cosmonauts after short (7 to 14 d) and long (65 to 366 d) missions.12–16 Although the decrease in the mean cytotoxicity index of 33 cosmonauts was not significant, in 18 of the 33 subjects it fell below the lower limit of the normal range.14 This depression persisted for at least 1 wk after space missions of longer duration (211 to 237 d). Some of the decrease in NK activity may be attributable to the reduced number and percentage of NK cells generally observed after space flight. There are, however, indications that the ability of individual NK cells to bind to target cells is impaired and that fewer of them exhibit cytotoxic activity after short- and long-duration missions.16

Data on the production of cytokines in cosmonauts and astro- nauts are surprisingly limited given their importance not only in regulating and coordinating immune responses but also in bone remodeling. TNF-�, interleukin-1 (IL-1), and IL-6 are proinflam- matory cytokines elaborated during an acute-phase response. Ob- servation of increased excretion of cortisol and IL-6 and elevated levels of fibrinogen synthesis (an acute-phase protein) early after launch and immediately after landing suggest that these two stresses are associated with an acute-phase response.17,18 It is likely that such a response also occurred in space-flown rats as indicated by elevated serum corticosterone concentrations and increased TNF-�, IL-6, and nitric oxide production by lipopoly- saccharide (LPS)–stimulated peritoneal macrophages.5 IL-1� also plays an important role in providing a second signal in the acti- vation of lymphocytes, thus raising the question of whether the decreased proliferative response of PBMC from cosmonauts and astronauts could be at least partly due to reduced synthesis of this cytokine.

One of the most important factors in driving in vitro T-cell proliferation is IL-2, which is produced by the activated T cell itself and upregulates the expression of the high-affinity form of its receptor, IL-2R. Thus, defects in IL-2 synthesis or IL-2R induction could underlie the suppressed response of PBMC harvested after space flight to in vitro stimulation with mitogens. In addition, IL-2 stimulates NK cell growth, and deficient IL-2 production could contribute to the reduced NK cell numbers observed in cosmonauts and astronauts. A major stimulus for NK activity is interferon-� (IFN-�), and a reduction in the production of this cytokine could underlie the decreased NK cell activity of cosmonauts and astronauts.

Interleukin-1

Unstimulated and phorbol myristate acetate (PMA)–induced IL-1� production in PBMC of seven cosmonauts after flights of 22-, 151-, or 166-d duration was higher postflight than preflight, but this increase did not reach statistical significance.19 No consistent changes in intracellular PMA plus ionomycin-induced IL-1� pro- duction were found in PBMCs from 14 of 20 Space Shuttle astronauts after 10- to 18-d missions.1 Inexplicably, the remaining subjects, representing the entire crew of one Space Shuttle flight, showed a significant increase in intracellular IL-1�. Only a limited amount of information on the Shuttle missions was provided in this paper; hence, it is difficult to determine whether differences in mission length, workload, or other stresses might have contributed

to the striking difference in IL-1� responses between the three shuttle crews.

Tumor Necrosis Factor-�

There was a two-fold reduction in PMA- and PMA plus phytohe- magglutinin (PHA)–stimulated TNF-� activity in PBMC of a French cosmonaut obtained on L�1 after a 21-d space flight compared with preflight and 7-d postflight values.15 After stimu- lation with PHA alone, however, TNF-� activity was unchanged.

Interleukin-2

Rather conflicting results have been reported concerning changes in IL-2 production and activity after space flight. In PBMCs stimulated with PHA with or without PMA, IL-2 biological activ- ity, measured by the induction of proliferation in the IL-2– dependent CTLL-2 cell line, was reduced in 12 of 13 cosmonauts after prolonged space flights (65 to 366 d).13 There appeared to be a trend for the suppression to become more pronounced with increasing duration of the space flight.12 In contrast, when IL-2 production was measured by enzyme-linked immunosorbent as- say, there were no significant differences between postflight and preflight samples, except that there was a substantial increase in IL-2 production in two of eight cosmonauts. Higher postflight as opposed to preflight synthesis of immunoreactive IL-2 has also been reported in five cosmonauts from flights lasting 26, 151, or 166 d, although this increase was not statistically significant due to the small sample size.19 In two of these cosmonauts (from the 166-d mission), the percentage of cells expressing IL-2R after PHA stimulation was substantially below the normal range. This was not observed in the other five cosmonauts studied. Taken together, these results suggest that IL-2 production is either not affected or actually stimulated by space flight, but that its activity is inhibited by low IL-2R expression or some other as-yet un- known factor.

This hypothesis does not appear to be supported by more recent findings. T cells (CD3�) isolated from Shuttle astronauts on R�0 and stimulated with PMA and ionomycin produced significantly less intracellular IL-2 than T cells obtained on L-10.1 The reduc- tion in IL-2 synthesis was significant in CD4� and CD8� T cells; whereas CD4� cells had recovered their ability to produce this cytokine on R�10, CD8� cells still exhibited suppressed IL-2 levels.

Interferons

In PBMCs isolated from two cosmonauts on R�1 after a 7-d flight, a significant reduction in Newcastle disease virus-induced IFN-� bioactivity was observed.20 In contrast, analyses of PBMCs from a larger number of cosmonauts indicated that IFN-� activity was either unchanged or increased.12,14 In the same analyses, decreased IFN-� activity was found to be typical after short-term flights, whereas reduced and increased IFN-� productions were seen with about equal frequency after flights of long duration.12,14 Intracel- lular IFN-� production by CD3� T cells isolated from seven astronauts on R�0 was similar to that observed on L-10 and R�3 but reduced in CD4� T cells from 12 astronauts.21 Individual data plots indicated that IFN-� production varied widely between sub- jects, some showing increases, some decreases, and others no change.

Is There Enhanced Risk of Disease in Astronauts Because of Immune Suppression?

During the early phases of space flight, a substantial number of astronauts was affected by infectious diseases during the 3 wk before launch and during and after flight.22 After the Apollo 13

Nutrition Volume 18, Number 10, 2002 891Microgravity and Immunity

mission, the Health Stabilization Program was introduced and continues to be practiced in a somewhat modified form. This program limits crew contact and exposure before each mission by restricting crew to their quarters and allowing only medically screened and badged personnel access to them. This has substan- tially reduced the numbers of overt infections. Such measures, however, do not rule out that the suppressed immune status ob- served after space flights could have more subtle and possibly delayed clinical consequences. It was recently reported that Epstein-Barr virus (EBV), a herpesvirus found in a vast majority of adults and persisting in a latent form throughout the lifetime of the infected person, was reactivated in eight of 23 male and three of five female astronauts.23 These astronauts had participated in Space Shuttle missions of 9 to 16 d in duration. They showed some of the typical immune changes associated with space flight, i.e., leukocytosis due mostly to pronounced neutrophilia without sig- nificant changes in lymphocyte or monocyte populations. A highly significant increase in geometric mean antibody titers against EBV viral capsid antigen had been observed on L-10 compared with that at the annual medical examination, suggesting that the reactivation was stress induced. A follow-up study found that, in the 11 astronauts who had serologic evidence of EBV reactivation, epi- nephrine and norepinephrine were increased by 220% and 200%, respectively, versus 1% and 1% in the non-reactivating group.24

Reactivation of EBV has also been reported during periods of academic stress for medical students during examinations25 or the various stresses associated with Antarctic expeditions.26,27

Possible Factors Involved in the Changes in Immune Parameters During and After Space Flight

It has been proposed that microgravity is not a factor, or only a minor one, in the effects of space flight on various immune parameters.8 Instead, many investigators have suggested that the physical and psychological stresses of space flight account for a vast majority, if not all, of the changes observed in immune parameters during and after space flight. Table II lists many of the factors potentially contributing to this stress. There certainly are many similarities between the effects of stress and space flight on immune functions. The level of stress is likely to vary between missions, and it can be markedly influenced by individual mission characteristics (e.g., schedule changes in launch and landing dates, duration, workload, type of work, number of extravehicular activ- ities, etc.). In addition, subject variability in susceptibility and response to various types of stress are likely to play a major role in affecting immune function.

Unfortunately, much of the published literature contains little information on mission characteristics (other than length) and essentially no information on any of the stressors, including those most likely to affect immune parameters, such as nutrition (much less, details on macro- and micronutrients), energy balance, dis- turbances in sleep and diurnal rhythms, amount of exercise, etc. In addition, although some data on stress hormone levels during and immediately after space flights are available, such data are rarely provided in the same studies in which immune parameters are characterized. Correlations between the two have only occasion- ally been attempted.3,24

Hormones

Before, during, and after space flights, particularly in the periods surrounding launch and landing, a large variety of hormonal changes has been observed in cosmonauts and astronauts.1,28,29 It is not uncommon for changes in immune4,16 and hormonal4,24,30

parameters to occur even before launch, quite likely due to the intensive mission preparation and the excitement about soon en- tering space. Large increases in plasma cortisol concentrations were found in six of 21 male and three of six female astronauts on

L-10 compared with values from their annual medical examina- tions, whereas others had greater than 30% decreases in plasma cortisol at the same time point.24 Similarly, an increase in serum cortisol levels occurred in two cosmonauts on L-15 versus L-60.30

Samples obtained during space flight for measurements of plasma or urinary cortisol have produced highly variable results. During the Skylab missions (28, 59, and 84 d), plasma cortisol concentrations were generally increased over preflight values in the nine crewmen, but this change only rarely reached statistical significance.31 In contrast, plasma adrenocorticoptropin (ACTH) levels were depressed, at times significantly. Urinary cortisol ex- cretion was reportedly elevated in a Shuttle astronaut on the first day of space flight and again, to a lesser extent, preceding the landing.32 During the remainder of the flight, cortisol excretion was similar to the preflight values. A similar pattern was reported in four astronauts during the 9.5-d SLS-1 Shuttle mission.18 The mean plasma cortisol concentration in four astronauts on a dual- shift mission of unspecified length was similar to L-2 levels and within the reference range.29 During the Euromir 95 mission, the serum cortisol levels of a cosmonaut fluctuated considerably, with the lowest concentration (below baseline levels) observed on FD 125.30 Data obtained from six cosmonauts and astronauts between FD 88 and FD 186 in orbit on the Mir space station showed an increase in urinary cortisol excretion compared with preflight values in two of the subjects and a decrease in the four others33; one of those four subjects had very high concentrations in all three preflight samples.

On R�0 after the Skylab missions, there was an increase in plasma cortisol, but it did not achieve statistical significance.31 In contrast, in 20 cosmonauts returning from orbital flights of 4 to 14 d, the rise in the mean blood concentration of cortisol was significant.34 More recently, serum and urinary cortisol and serum ACTH were increased in astronauts (between 12 and 17) imme- diately after Space Shuttle flights of 8 to 10 d in duration.1

However, only the changes in urinary cortisol, reached statistical significance. This agrees with other studies showing no significant changes in plasma cortisol levels on R�0,2,4,23 whereas urinary cortisol excretion is generally higher on R�0,2,4,23 but not always significantly higher.18,36 A return to preflight levels is commonly

TABLE II.

LIST OF STRESSORS ENCOUNTERED BY ASTRONAUTS AND COSMONAUTS DURING SPACE FLIGHTS AND STAYS ON THE

SPACE STATION

Physical Launch and landing gravitational forces Vibration Noise Microgravity Radiation

Low, but higher than on Earth; may become a factor during prolonged space flights or stays on space stations

Increased microbial load Malnutrition

some of it related to space motion sickness Disturbances of circadian rhythms and sleep

Psychological Anxiety

Related to the danger of the missions, the hostile environment, the inability to return to Earth even if severe illness/trauma occurs

Intense work load Isolation

From family, friends, normal social settings Confinement Difficulties of a small group living together closely for long periods

892 Borchers et al. Nutrition Volume 18, Number 10, 2002

observed within a few days, although, in at least one case, plasma cortisol was still significantly elevated on R�7.34

Taken together, these data suggest that take-off and landing are associated with considerable stress, whereas the level of stress during the time in space is highly variable and greatly dependent on individual mission characteristics.

Nutrition

Astronauts and cosmonauts generally lose weight during space flights.17,35,37–39 It was long assumed that this reduction in body mass was attributable to the absolute fluid loss early during expo- sure to microgravity due to exaggerated diuresis and natriuresis resulting from the upward fluid shift (reviewed by Drummer et al.40). In several recent studies, however, such exaggerated diure- sis and natriuresis was not observed at the beginning of flights; instead, water and sodium were retained during that period. Al- most daily body mass measurements aboard the Mir space station confirmed these findings and indicated that reduced energy intake, rather than fluid loss, was a major contributor to the loss of body mass.40

Decreases in energy intake during space flights have consis- tently been reported, with reductions of up to one-third compared with preflight intake not being uncommon.33,35,37,41 Until recently, energy expenditure on space flights had not been determined, and calculations for energy requirements were based on the assumption that they would be similar to those on Earth during moderate activity. During some recent Shuttle missions, energy expenditure during space flight was measured with doubly labeled water,35,41

as were isotope dilution and body fat measurements by dual- energy x-ray absorptiometry.35 Although the mean energy require- ment for the astronauts was similar to that for moderate activity on Earth in both studies, there was no correlation between calculated and measured values.35 Despite fairly constant intake levels after the first day, energy intake was consistently below the require- ment, which resulted in, at times severe, negative energy balance. Frequently, the most pronounced reduction in energy intake occurs during the first 1 or 2 d of space flight due to space motion sickness,17,35 although sharply lower intake was observed in the absence of overt symptoms of space motion sickness.17 These results strongly suggest that much of the weight loss observed on numerous missions is due to insufficient energy intake, with mus- cle atrophy and minor fluid losses contributing.

Interestingly, there have been reports of some astronauts main- taining adequate energy intake.38,41,42 All of these crew members were told to maintain a predefined and controlled dietary intake for the purpose of metabolic studies, and they essentially did. These observations suggest that, with conscious effort on the part of the cosmonauts and astronauts, it is possible for them to meet their energy requirements. This may, however, not be the case if they exercise intensively, as done by Russian cosmonauts.17 Without conscious effort to maintain preflight energy intake levels, there appears to be a stabilization of energy intake at a lower level, suggestive of a rapid metabolic adaptation.

In addition to, and in part because of, the reduction in overall energy intake, intake of individual macro- and micronutrients also appears to be compromised in cosmonauts and astronauts. Rela- tively little has been published about the nutrient composition of space food,43 but there are indications that diets freely chosen from the available space food items are deficient in protein, calcium, and fluid,37,39 although inconsistent results have been reported about nitrogen intake and balance.17,33,42

In addition to nutrient intake, nutrient absorption and metabo- lism appear to be altered during space flight. Fractional calcium absorption by the stable strontium test was markedly reduced in flight compared with pre- and postflight values in one cosmonaut during a 20-d mission.44 A tracer kinetics study involving two cosmonauts and one astronaut of a 115-d flight to the Mir space

station also showed a decrease of up to 50% in fractional calcium absorption.39 Excretion was increased by up to 50%.

It is well known that energy and individual nutrient intake can have profound effects on immune function.45–48 Many of the immune function alterations described in cosmonauts/astronauts to date have also been reported in states of nutrient deficiencies. For example, decreased proliferative responses to mitogens can be associated with vitamin B (pyridoxine, B12, or biotin), vitamin E, copper, or selenium deficiency. Depressed DTH responses can result from vitamin B6 or B12, vitamin C, or iron deficiency. NK activity can be reduced after nucleic acid restriction. Protein and individual amino acid deficiencies can have profound effects on a variety of immune functions. Improving the nutrition of cosmo- nauts and astronauts may, therefore, prove to be an appropriate countermeasure to some of the immunological disturbances ob- served during and after spaceflight. It will, however, be necessary to gather detailed information on the absorption and metabolism of many micronutrients before specific nutritional recommendations can be made. Given the considerable interactions between the immune system and bone remodeling, such nutritional counter- measures could also prove beneficial in reducing the massive bone loss occurring during long-term space flights.

Models of Space Flight

Because of the limited number of space flights and crew on each space flight, several space agencies have made attempts to develop suitable Earth models for the parameters—except microgravity— experienced during space flight. A variety of such models have been proposed, including head-down tilt bed rest (HTBR),50–53 various types of physical and psychological stress,54 isolation and confinement during Antarctic expeditions and other settings,26,27,55–57 sleep deprivation,58 and acute and chronic physical exercise.59 It is noteworthy, however, that researchers who studied both cosmonauts/ astronauts as well as subjects from such models generally agree that none of the existing models completely reproduce the changes ob- served during and after space missions. One of the difficulties in creating such models is, of course, that it is not always clear what changes exactly the model should reproduce. While some of the alterations in immune cell distribution and function are observed in almost all astronauts/cosmonauts, many other changes are seen only in some, while changes in the opposite direction occur in others.

Two of these models will be discussed in some detail. 1) Antarctic expeditions, because NASA itself has recognized their many similarities to space flight and has funded human research programs in the Antarctic;27 and 2) HTBR, because it is the most popular model for many aspects of spaceflight.

Antarctic

Antarctic expeditions model many of the stresses encountered during spaceflight, including those stemming from isolation, con- finement, hostile environment, danger, and difficulties of small group living and working together in close quarters for long periods. The immune responses of crewmembers participating in Antarctic expeditions, especially winter expeditions, in many ways resemble those observed in astronauts/cosmonauts. A reduction of PHA-induced T cell proliferation by �50% has been reported during a 9-mo Antarctic research expedition.26 In the same study, DTH responses were significantly reduced during the expedition, but not after return. These responses were measured with the same multitest device that has been used during and after spaceflights. Similar decreases in cell-mediated immune responses were ob- served in other groups of researchers in the Antarctic,57 including members of a summer expedition.56

One of the contributing factors in these functional changes may have been cytokine imbalances. TNF-� production in PHA- stimulated PBMC was reduced by 74% during a winter expedi-

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tion.26 IL-1 secretion declined from the first to the third measure- ment during isolation, but all three time points were higher than the measurement taken 9 mo after return to Australia. No clear trend emerged in the changes in IL-6 synthesis. The amount of IL-2 secreted into the supernatant was dependent on the length of culture and was markedly higher at the time of the third measure- ment in both the 48 h and 72 h culture, but was higher after return in the 24 h culture.

Unlike in astronauts/cosmonauts, no major changes in lympho- cyte subsets were observed in members of either a summer56 or a winter expedition to the Antarctic.26 In the wintering personnel, however, a novel population of CD14� monocytes was detected, characterized by smaller size and lower expression HLA-DR, but increased expression of CD64 (the high-affinity IgG receptor). A population of smaller, less granular monocytes was also detected in some astronauts, together with significant monocytosis.1 This subset of monocytes exhibited reduced expression of the receptors for IGF-1 and insulin; unfortunately, HLA-DR expression was not analyzed.

Head-down Tilt Bed Rest (HTBR, With 6° Head Tilt)

One of the most popular Earth-based models for some of the effects of space flight is HTBR, most commonly with a 6° head tilt.

It reproduces some of the physiological changes observed in space, including the redistribution of body fluids, cardiovascular decon- ditioning, bone demineralization, and muscle atrophy. In terms of the responses to these changes and the mechanisms contributing them, however, HTBR frequently differs from space flight.40,60,61 Furthermore, it seems questionable whether HTBR constitutes an appropriate model for the immune changes occurring during and after space flight, since many of these changes are not reproduced (summarized in Table III).

In vitro Studies With Space-flown Cells and Simulated Microgravity

Although the various physical and psychological stresses of space flight most likely play a major role in the changed immune cell distribution and function observed during and after spaceflight, it cannot be ruled out that microgravity also affects these parameters. In recent years, it has become increasingly evident—both from in vitro experiments conducted in space and from ground simulations of microgravity—that a vast majority of cell types are sensitive to changes in gravity (hyper- and hypogravity) and react to such changes with alterations in cell morphology and function,8 some- times in a matter of a few seconds.62 Some of these changes appear

TABLE III.

COMPARISON OF SPACE FLIGHT AND HEAD-DOWN TILT BEDREST (6 DEGREES) STUDIES

Outcome measure Spaceflight HTBR

Leukocyte count 1 � During and after 120-d HTRB52,85

Lymphocyte count 1 or � � During and after 10-d HTBR,49 42-d HTRB51 and 120-d HTRB52,85

CD4�/CD8� ratio 1 or � 2 120 (decrease was not significant)85

PMN/neutrophils 11 � During and after 10-d HTBR49

1 During 42-d HTRB51

� During and after 120-d HTRB52,85

Monocytes 1, 2 � During and after 10-d HTBR,49 42-d HTRB,51 and 120-d HTRB52,85

NK cells 2 2 After 10-d HTBR49

2 During 42-d HTBR versus 12 d before and 13 d after, but � versus 33 and 47 d after51

2 On d 110 of 120-d HTRB52

1 Continuously throughout 12-d HTRB (still higher after HTRB, but not significantly so)85

Lymphocyte proliferation 2 During and after (but not 1 or 2 d before) 2 Just before, during and after 10-d HTBR49

DTH response 2 � During and after 10-d HTBR49

IL-2 2 When measured by CTLL-2 based bioassay 2 IL-2 production measured by ELISA after 28-d HTBR66

1 When measured by ELISA 1 ELISA51

2 IL-2 positive CD3�, CD4�, and CD8� T cells on R�0, but not on R�31

1 IL-2 secretion, measured by ELISA, 7 d after 120-d HTRB, but 2 IL-2-positive CD8� (but not CD4�) T cells 7 d after 120-d HTRB52

IL-6 1 IL-6 excretion in urine on FD1 and in some astronauts on R�018

1 During, but not after, 120-d HTRB85

IFN-� � IFN-�–positive CD3� T cells, but 2 IFN-�– positive CD4� T cells on R�0 (no longer significant on R�3)1

1 On d 34 of 42-d HTBR, ELIZA51

1 IFN-� secretion, measured by ELISA, 7 d after 120-d HTRB, but 2 IFN-�–positive CD8�, but not CD4�, T cells 7 d after 120-d HTBR25

Catecholamines � or 1 or 2 during and after flight � Epinephrine, 1 norepinephrine on D65 and after 120-d HTBR85

Cortisol 11 and last FD, 1 or � during the rest of the flight and on R�0

2 Throughout 42-d HTRB51; 2 Morning cortisol only on d 65, evening cortisol steadily increasing throughout the 120-d HTBR85

DTH, delayed-type hypersensitivity; ELISA, enzyme-linked immunosorbent assay; FD, flight day; HTBR, head-down tilt bedrest; IFN, interferon; IL, interleukin; NK, natural killer; PMN, polymorphonuclear leukocyte; R�0, day of re-entry; R�3, 3 d after re-entry; 1, increase; 2 decrease; �, no statistically significant change occurred.

894 Borchers et al. Nutrition Volume 18, Number 10, 2002

to be direct effects of microgravity rather than merely gravity- dependent changes in culture conditions, such as the absence of thermal convection (i.e., the potential of accumulation of secretory and waste products near the cells producing them rather than their dispersion within the culture system).

When interpreting results obtained in space-flown cell cultures, it is helpful to understand some of the constraints imposed by limitations in space, equipment, and available crew-time on such experiments. It is quite common for experiments conducted on the in-flight 1g reference control to produce different results from those observed in ground-based (1g) controls (e.g., [63–66]). Occasionally, this has been explained by the fact that the in-flight centrifuge was not activated until several hours after launch (ex- posing the controls to hours of microgravity)65,66 and/or was shut off for certain periods of time in order to accommodate other experiments.63,65 This explanation is supported by the finding that, when the 1g in-flight centrifuge was run continuously, most of the results were almost identical to those in the ground control.64 In the same experiment, however, the production of IL-1 still differed between the in-flight centrifuge and the ground control. This suggests that other space flight factors, such as acceleration during launch, temperature, vibration, and potentially radiation exposure, differ from those in the ground controls, and that at least some of these factors influence the cell culture results. This underscores the importance of including both in-flight and ground-based 1g con- trols in all space-flown cell culture experiments. It may also explain why results obtained in simulated microgravity are occa- sionally different from those observed in cells cultured during actual spaceflight.67

Since there are severe limits to the number and type of exper- iments that can be conducted during spaceflights, several ground- based simulations of microgravity have been developed. These include the fast-rotating clinostat (FRC), rotating wall vessel (RWV) bioreactor, random positioning machine (RPM), and high- aspect ratio vessel (HARV). To date, most experiments with immune cells have been conducted in the FRC or RWV and appear to give comparable results.

Qualitatively, the results from space-flown cell cultures, as well as cultures exposed to simulated microgravity, are often similar to the effects space flight exerts on functional aspects of immune cells obtained from cosmonauts/astronauts. Given the rather con- tradictory nature of some of the changes reported in astronauts, however, such comparisons are not always productive. This is particularly true for changes in cytokine production.

Cell-cell and Cell-substrate Contacts Under Conditions of Microgravity

As is observed in a majority of cosmonauts/astronauts, PHA- or Concanavalin A (ConA)-stimulated proliferation in actual or sim- ulated microgravity is significantly reduced compared to 1g con- trols (see Table IV). Lack of sedimentation in actual or simulated microgravity may result in reduced cell-cell and/or cell-substratum interactions, which could in turn contribute to the severely reduced proliferative response to mitogens observed under these condi- tions. Light microscopic autoradiography revealed that, although cell aggregates formed in space-flown ConA-stimulated PBMC, they were found with markedly lower frequency.68,69 In contrast, binding of ConA to either PBMC or Jurkat T cells was normal and, although a significant reduction of patching and capping was observed in microgravity, this appeared to be due to a retardation of these processes rather than their suppression.68,70

Incubation of PBMC with PHA in teflon culture bags, which reduce cell-substratum interactions, did not significantly affect proliferation compared to cultures in standard T flasks.71 Other

experimental results, however, indicated that a microgravity- associated decrease in cell-substratum interactions might contrib- ute to the suppression of proliferation. Incorporation of PBMC into collagen beads before incubation with PHA in an RWV restored the proliferative response from almost complete suppression to about 50% of that seen in static cultures. Similar results were obtained with PBMCs preincubated with Cytodex beads.64 Com- pared with suspended cells, bead-attached cells also exhibited markedly different cytokine profiles. In particular, IL-2, TNF-�, and IFN-� production was significantly higher than in ground controls but lower in suspended cells.

Binding of cells to collagen is mediated by adhesion molecules, which connect to the cytoskeleton at focal adhesion points. Thus, attachment to collagen could prevent microgravity-induced changes in adhesion molecules or cytoskeletal structure. The ex- pression of adhesion molecules in lymphocytes cultured in simu- lated microgravity (RWV) was unchanged or even slightly upregu- lated compared to static controls.73 Different results on the level of expression of two non-overlapping epitopes on �1-integrin sug- gested, however, that a conformational change might have oc- curred due to simulated microgravity. Short-term exposure of PBMCs or Jurkat cells to microgravity (on sounding rockets or during space flight) resulted in structural changes in various ele- ments of the cytoskeleton.71,74,75 It seems likely, then, that attachment of cells to collagen beads may prevent such alter- ations in the cytoskeleton and subsequent changes in cell shape and function.

It should be noted, however, that in three models of antigen- specific lymphocyte activation, preincubation with collagen beads did not overcome the almost complete inhibition of proliferation associated with simulated microgravity.76 Further, proliferation of purified human T cells incubated in an RWV in the presence of CD2/CD28 and CD3/CD28 (i.e., stimuli that do not require co- stimulatory signals provided by cell-to-cell contact) was com- pletely suppressed compared with static controls.72 These results suggest that alterations in signal transduction rather than absence of cell-to-cell contact is responsible for the defective proliferative response in simulated microgravity.

Protein Kinase C

There are indications that signal transduction pathways, in partic- ular those involving protein kinase C (PKC), are affected by microgravity.77 To date, however, highly variable results have been obtained as to the precise nature and location of these alterations. In one case, it was reported that inhibition of PKC counteracted the space flight–induced TNF-mediated cytotoxicity, suggesting that PKC activity was enhanced by microgravity in this experimental system.78 More commonly, microgravity is associ- ated with a decrease in PKC activity. Some have localized the target of this inhibition upstream of PKC.68,72 Other results have suggested that PKC activity itself, or a downstream event, is affected by microgravity.79

Among the possible explanations for these discrepancies is that different isoforms of PKC are differentially affected by micrograv- ity. The 11 isoformes of PKC known to date are localized to different subcellular compartments, and this localization is thought to regulate their substrate specificity. Upon activation, a redistri- bution of PKC isoformes to different cellular compartments oc- curs.80,81 Cells exposed to microgravity during space flight exhib- ited different redistribution patterns from in-flight and 1g ground controls.65,66 Isoforms of PKC are associated with various ele- ments of the cytoskeleton,80 in particular intermediate filaments and stress fibers in a variety of cell lines, including U937 (a monocytic cell line) and CEM (a human T cell line).82 Changes in cytoskeletal structure have been observed in cells exposed to

Nutrition Volume 18, Number 10, 2002 895Microgravity and Immunity

microgravity, and such changes could contribute to the alterations in PKC distribution patterns and activity.

REVERSIBILITY OF THE EFFECTS OF MICROGRAVITY

During the STS-76 Shuttle mission, cells were kept at ambient temperature for 77 h before being introduced into the incubator or 1g reference centrifuge.65 The changes in the amount of PKC translocated and the translocation kinetics of in-flight cell cultures exposed to microgravity or to 1g were almost identical and clearly different from those observed in ground controls. This suggested that some of the changes induced by exposure to microgravity are not easily reversed. This was, indeed, observed in cultures of PBMC in simulated microgravity (RWV), where locomotion through collagen was inhibited or even completely suppressed, and transfer to static culture after 6 h on the RWV resulted in some restoration of locomotion, whereas lymphocytes did not recover after 72 h in simulated microgravity.73 In contrast, cells preincu- bated in a random positioning machine for 24, 48, or 72 h and then stimulated with concavalin A for 72 h showed similar mitotic indices as static controls,83 even though proliferation is strongly inhibited in cells incubated with concavalin A during exposure to microgravity (see Table IV). This indicates that the microgravity-

induced changes resulting in suppression of proliferation are quite easily and quickly reversed.

SUMMARY

There are clear indications from the existing data that space flight can have profound effects on the immune system. These data also make evident, however, that there is enormous variability among cosmonauts and astronauts in the nature and extent of these effects. To overcome some of this variability and generate statistically significant results, it is becoming an increasingly common practice to combine immunologic data of crew members from several different missions. Because mission characteristics appear to have a major effect on the measured outcomes, this does not seem to be an acceptable strategy. Unfortunately, these mission characteristics and the adaptations they necessitate in individual crew members (e.g., food intake, sleep patterns, etc.) are seldom described in sufficient detail and are rarely correlated to experimental data. Clearly, further studies are needed with particular emphasis on the immunobiology of long-term space travel on subsequent infectious challenge.

TABLE IV.

SUMMARY AND COMPARISON OF RESULTS FROM CELL CULTURE IN SIMULATED AND ACTUAL MICROGRAVITY*

Outcome measure Cell type Effect in simulated microgravity versus

static culture† Effect in true microgravity versus ground

or in-flight 1g controls

Proliferation‡ PBMC 272,76 263,67,69,89

2 In FRC86

2 In FRC and RPM87,88

Lymphocyte locomotion through collagen PBMC 273 273

TNF-� production B6MP102 1 After LPS90

PBMC 1 �10-fold after PHA or Newcastle disease virus in FRC91

IL-1 production PBMC 1 After PHA72 1 After ConA93

2 After IL-292 2 After ConA67

B6MP102 1 After LPS90

THP-1 2 After PMA79

U937 2 After phorbol ester versus in-flight 1g control 1 versus ground 1g control50

IL-2 production PBMC 2 Completely suppressed after PHA72

and after IL-292 2 Slightly, but not significantly after

ConA67

Jurkat � Anti-CD3 � THP-1 cells, after PMA � ionomycin

Soluble IL-2R PBMC 2 After rIL-292 2 After ConA (� IL1 or IL-2)67,93

IFN-� production PBMC 2 (Complete suppression initially, recovery after 72 h) after PHA72

2 After ConA67,89,93

1 After ConA94

Apoptosis (spontaneous) and sFas/APO-1 Jurkat 175,95

Apoptosis (spontaneous and induced) and sFas/APO-1

PBMC � Spontaneous apoptosis and sFas/ APO-1 2 Radiation-induced apoptosis73,96

* In general, the assays described in this table require incubations of 48 to 72 h, and cells were exposed to microgravity for the entire incubation pe- riod. In some cases,73,75 additional experiments indicated that shorter periods of exposure to microgravity (4 and 6 h, respectively) were sufficient to induce significant changes in apoptosis and locomotion through collagen. † In RWV unless indicated otherwise. ‡ A decrease was observed regardless of what stimulus. B6MP102, murine bone marrow macrophage cell line; ConA, concavalin A; FRC, fast-rotating clinostat; IFN, interferon; IL, interleukin; Jurkat, lym- phoblastoid T-cell line; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; PMA, phorbol myristate ace- tate; rIL, recombinant interleukin; RPM, random positioning machine; RWV, rotating wall vessel; sFas/APO-1, soluble cell death factor; THP-1, human monocyte cell line; TNF, tumor necrosis factor; U937, human promonocytic leukemia cell; 1, increase; 2, decrease; �, no statistically significant change occurred.

896 Borchers et al. Nutrition Volume 18, Number 10, 2002

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