INTRODUCTION

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IT HAS BEEN DOCUMENTED THAT spaceflight affects the functional integrity of the immune system. The observed immune dysfunction in microgravity is involved in both cell-mediated and humoral immunity (15). These changes may be the result of exposure to the spaceflight environment. Elements of such an environment include microgravity, lack of load bearing, stress, forces of acceleration, and radiation. However, the mechanisms for spaceflight-induced changes in immune responses remain unknown. There have been limited opportunities for in-flight experiments because of technical difficulties and the lack of laboratory space in space to carry out the required experiments (14). However, the anti-orthostatic hindlimb suspension (AOS) of rodents is a ground-based model for simulation of some effects of microgravity. Studies that used this model have shown complex contradictory observations relative to organ-specific immunological changes. Chapes et al. (4) have summarized the specific physiological and immunological changes induced by AOS and indicated a correlation with physiological and immunological changes induced by flight. This AOS position simulates the cephalad fluid and organ shift; a negative balance of water, nitrogen, and potassium; and increased metabolic turnover observed in astronauts during spaceflight. Overall results of such AOS models have shown a decrease in immunity.

Fuchs and Medvedev (6) elucidated countermeasures for ameliorating in-flight immune dysfunction. To ameliorate the immune dysfunction in space, they suggest two options: one is to control and regulate the neurohormonal system, and the other is the use of immunomodulator agents acting on the immune system itself. However, the suggested methods of neurohormonal regulation by using agents that act on the nervous system may have deleterious effects on systems besides the immune system.

Most studies have focused on the immune dysfunction in spaceflight; no studies to our knowledge have been done on the use of the immunomodulation by nutrients to improve the immune system during spaceflight. Our laboratory has previously demonstrated that dietary sources of nucleosides and nucleotides are important and conditional for the maintenance and enhancement of cellular immunity in unit gravity (1, 2, 5, 11, 12, 20-22). The relationship of nucleotides in immune functions is becoming increasingly evident. Dietary nucleotides have been shown to enhance in vivo and ex vivo cell-mediated immunity, augment the immune response to cardiac allograft survival (21), enhance lymphocyte proliferation and interleukin (IL)-2 production (2, 20), and improve host resistance to systemic infection by Staphylococcus aureus and Candida albicans (1, 5). Dietary nucleotides also increase the delayed-type hypersensitivity (DTH) response to chemicals, bacterial antigens, and xenoantigens (12, 22). However, exogenous nucleotides and nucleosides had not been considered as required nutrients and hence have not been studied in detail. It was generally assumed that living organisms, including humans, could synthesize adequate amounts of the compounds required for normal growth and development. On the other hand, it is now known that tissues such as intestinal mucosa, bone marrow hematopoietic cells, lymphocytes, and the brain have limited capacity for the de novo synthesis and depend on the supply by the salvage pathway (23). Our hypothesis is that, under certain stress conditions, such as sepsis, trauma, and extremely unusual environments including spaceflight, the endogenous supply of these compounds may not be adequate for optimal functions, especially the immune system, and dietary supply may be of particular significance.

With the present knowledge of the interrelationship of nutrition and immunity, particularly the immunotrophic effects of nutritional nucleotides, this study was carried out to assess the effect of dietary nucleotides on immune response in microgravity by using the AOS model.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Six- to eight-week old female-specific, pathogen-free BALB/c and C57BL/6 mice were obtained from Harlan Sprague Dawley (Indianapolis, IL). Animals were kept in a constant temperature (25 ± 2°C) and humidity (50-70%) room with a 12-h light period from 0800 to 2000. The Animal Care and Facilities Use Committee at The University of Texas, which complies with National Institutes of Health animal care standards, approved the animal protocol. The mice were allowed to adapt to our laboratory environment for 1 wk before the onset of the experiment, during which period they were maintained on a commercial rodent diet.

Housings and diet. After the period of acclimatization, the mice were randomized into three housing groups: group housing (more than two mice per cage), isolation (one mouse per cage), and tail suspension (single AOS mouse per cage); in addition, each housing group was divided into four dietary groups. Group housing is a normal environment, which provides normal communal behavior and living. Isolation housing (one mouse per cage) is used as control for the isolation factor of the AOS group. Isolation is known to exhibit certain effects of stress in the animal, such as stress hormonal changes, and thus differs from the actual AOS effects. Animals in all groups had free access to food and water ad libitum. The mice were maintained on diets custom-made by Purina Test Diets (Richmond, IN) for the duration of the experiment. The diets were isocaloric and isonitrogenous. They are described as follows: 1) control chow diet (CD), consisting of 21% protein (this diet contains ~0.25% of purines and pyrimidines); 2) RNA-supplemented CD (CDR) (0.25% purified yeast RNA is added to CD; the quantity of nucleotides added was based on the CD total nucleotide content and levels determined in our previous observations, see Refs. 12, 21); 3) adenine-supplemented CD (0.06% wt/wt) (CDA); and 4) uracil-supplemented CD (0.06% wt/wt) (CDU).

AOS of mice. Animals were anti-orthostatic suspended by using modified metabolic cages. The method is similar to the Wronski and Morey-Holton tail suspension cage described by Chapes et al. (4). Briefly, mice were tail suspended at an angle of 20-25° so that their hindlimbs were skeletally unloaded. Cables connected to the tails of mice were attached to a low-resistance pulley system above the cages, which allows the animals to move in a complete 360° area within the enclosure. Mice were weighed before suspension and during experimental periods.

Popliteal lymph node assay (in vivo proliferation). This assay is based on the method described by Twist and Barnes (19) and later modified for host vs. graft response assay by Van Buren et al. (21). Four- to six-week-old BALB/c (H2^d) and C57BL/6 (H2^b) mice were killed by cervical dislocation. The spleens were removed aseptically and placed in separate sterile petri dishes containing RPMI-1640 medium containing penicillin (100 U/ml) and streptomycin (100 mg/ml) (GIBCO, Grand Island, NY) at 4°C. Single-cell suspensions from the donor spleens (allogeneic C57BL/6 and syngeneic BALB/c) were obtained by teasing tissues through sterile 50-mesh stainless steel screens. All cell suspensions were then treated with 0.1 M Tris · HCl, pH 7.2, containing 0.8% ammonium chloride (Sigma Chemical) to lyse the red blood cells and were centrifuged at 200 g for 10 min at 4°C. Cell pellets were washed three times with RPMI-1640 and resuspended in the medium. The cell suspension was irradiated (3000R), and cell concentration was adjusted appropriately. Spleens from 10 animals were used for allogeneic and syngeneic donor spleen cell preparations.

Splenocyte proliferation assay (ex vivo proliferation). These assays measured the degree of lymphoproliferative response to mitogenic stimulation. After 7 days of experimental diets and housing, the mice were killed, and their spleens were removed. Single-cell suspensions from spleens were prepared as described earlier. The splenocytes were resuspended in complete RPMI (RPMI-1640 medium containing 20 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum). Cell viability was determined by 0.4% trypan blue (GIBCO) dye exclusion method. Stock cells were suspended at 1 × 10⁶ cells/ml.

Splenocyte cytokine release. Splenocytes (1 × 10⁶ cells/ml) in complete RPMI-1640 were cultured in polypropylene tubes (Becton Dickinson Labware, Franklin Lakes, NJ) with 1:25 diluted stock PHA. At the end of the incubation period, supernatants were collected and stored at 40°C before assaying for IL-1, IL-2, IL-3, interferon (INF)-, and tumor necrosis factor (TNF)- levels. The cytokine production was quantified in triplicate with commercial mouse ELISA kits (IL-1, IL-2, INF-, and TNF-: Endogen, Woburn, MA; IL-3: R&D Systems, Minneapolis, MN), by using the manufacturer's instructions.

Corticosterone assay. Immediately before each animal was killed, blood was collected from the orbital sinus cavity with a Pasteur pipette. The serum was collected and stored at 40°C before corticosterone levels were measured. The concentration of serum corticosterone was assayed by competitive inhibition radioimmunoassay (ICN Biomedical, Shelton, CT) by the Texas Veterinary Medical Diagnostic Laboratory of Texas A&M University, College Station, TX.

Statistical analysis. The data presented are the means of experiments ± SE. Statistical analyses were performed with Statview software program (version 4.0; Abacus Concepts, Berkeley, CA). Duncan's multiple-range test and ANOVA were used to determine significant differences among means at P < 0.05.

	RESULTS

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After 7 days of tail suspension, there were no differences in body weights in any of the treatment groups (Table 1). The overall food intakes in all treatment groups were similar (data not shown). Spleen weights of AOS mice were significantly lower in control CD and CDA groups compared with those from group and isolation housing conditions. In CDR and CDU groups, splenic weights did not change in AOS (Table 1). Splenic weight from AOS animals in CDU was significantly higher than it was in those from the control CD group (P < 0.05).

Figure 1 shows results of the in vivo lymphocyte proliferation (PLN assay) in four dietary groups; results are expressed as SI with the following formula: weight of allogeneic-stimulated PLN/weight of synogeneic-stimulated PLN. In the control CD group, SIs of AOS mice were significantly lower compared with those from group and isolation housing conditions (P < 0.05). Responses were similar in the group and isolation housing conditions. In other supplemental diet groups, there were no differences among the housing conditions. The SI from AOS animals in the CDU group was significantly higher than that from the control CD group (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of dietary nucleotides and housing on in vivo popliteal lymph node proliferative response in mice. CD, chow diet; CDR, RNA-supplemented CD; CDA, adenine-supplemented CD; CDU, uracil-supplemented CD. Values are means ± SE of stimulation index (SI) from 2 experiments; n = 6 per group. * Statistically significant from group housing within same dietary group, P < 0.05.  Statistically significant from tail suspension in CD, P < 0.05.

Figure 2 shows the ex vivo blastogenesis response of splenocytes from the housing and diet groups that were stimulated in the presence of PHA (Fig. 2A), Con A (Fig. 2B), and LPS (Fig. 2C). The data are expressed as SI calculated by the following formula: mitogen-stimulated counts per minute/unstimulated counts per minute. SIs stimulated by PHA and Con A were significantly decreased in the AOS animals, whereas isolation housing did not show significant change compared with control group housing. RNA and uracil supplementation restored the blastogenesis response that was originally decreased in AOS (P < 0.05). SI stimulated by LPS was not changed among the housing and diet groups.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of dietary nucleotides and various housings on in vitro splenocyte proliferative response in mice. Splenocytes were stimulated with phytohemagglutinin (A), concanavalin A (B), or lipopolysaccharide (C) for 48 h. Experimental mice were kept in various housings and diets for 7 days. Values are means ± SE of SI from 3 experiments; n = 9 per group. * Statistically significant from tail suspension in CD, P < 0.05.  Statistically significant from tail suspension in CD and CDA, P < 0.05.

Various cytokine levels were tested in supernatants collected from PHA-stimulated splenocytes, and results are shown in Table 2. PHA-stimulated production of cytokines IL-2 and INF- from the control CD group was significantly decreased in isolation and AOS conditions. The values in tail-suspended CDU groups were significantly higher than those in the control CD group (P < 0.05). Although IL-1 and TNF- levels tended to be lower in isolation and AOS mice compared with control group housing mice, there were no significant differences. IL-3 was not changed in any of the housing or dietary groups.

Serum corticosterone levels from experimental animals are shown in Fig. 3. Corticosterone levels in AOS animals of the control CD group were significantly higher (P < 0.05) than those of group and isolation housing conditions. In other dietary groups, serum corticosterone levels were not changed among the group and isolation housing conditions. In CDR and CDU, corticosterone levels of AOS mice were significantly decreased compared with those of the control CD group.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of dietary nucleotides and housing on serum corticosterone levels. Experimental mice were kept in various housings and diets for 7 days. Values are means ± SE of corticosterone levels; n = 6 per group. * Statistically significant from group housing within same tail-suspended animal group, P < 0.05.  Statistically significant from tail suspension in CD, P < 0.05.  Statistically significant from tail-suspended animals in CDA, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To demonstrate that our hypothesis, i.e., under microgravity condition, the endogenous supply of nucleotides may not be adequate for optimal functions of cellular immunity and dietary supply, may be of particular significance, the effect of dietary nucleotides on the immune response in the microgravity analog in ground-based AOS mice was carried out in the present study. The results of our study have demonstrated that the mice fed both CDR and CDU enhanced in vivo and ex vivo lymphocyte cell proliferation and IL-2 and INF- production and decreased the stress level, indicated by corticosterone levels, compared with mice fed control CD. The diets used in the present study were isonitrogenous and isocaloric, and the only difference is the source of nitrogen from supplemental nucleotides, which significantly reversed the immune suppression caused by microgravity.

Nutrients may be considered conditionally essential when the endogenous supply is shown to be inadequate for normal function. However, their lack does not lead to clinical deficiency syndrome. The body may have its own biochemical pathways to synthesize the conditionally essential compounds, but regulatory and/or developmental factors may hinder the full expression of this capacity, particularly during periods of relative deficiency, as may be the case in certain stress conditions such as sepsis, trauma, and extremely unusual environments including spaceflight. In such circumstances, exogenous dietary supplementation or supply may be necessary to optimize function, thereby sparing the organism the cost of their synthesis.

It can be seen that, in the AOS animals, there was a significant decrease of the in vivo PLN response in the control CD group compared with group or isolation housing environments. This effect of immunosuppression was reversed by supplementation of dietary nucleotides, with uracil being the most and being statistically significant from the control CD group in AOS animals (Fig. 1). Because the PLN responses have been used extensively in assessing in vivo cell-mediated immune states of hosts and as a possible mechanism(s) for reduced T-cell function (11), these results suggest that T-cell function was suppressed in AOS and was restored by nucleotide supplementation.

The results from the ex vivo splenocyte proliferation are similar to the in vivo results. SIs of splenocytes stimulated by PHA and Con A were significantly decreased in AOS animals, whereas, in isolation housing, there were no differences compared with control group housing. RNA and uracil supplementation restored the responses decreased in AOS (Fig. 2). Cytokines related to T-cell proliferation, such as IL-2 and INF-, were also significantly decreased in AOS animals (Table 2). The decrease in T-lymphocyte blastogenesis, IL-2, and INF- production by splenic cells in mice fed control CD in AOS was consistent with the decrease in the PLN response. The present results are consistent with our laboratory's previous observation in unit gravity (2, 20). Previous reports indicate that the addition of dietary nucleotides influences the number and function of T-helper cells and enhances their immunocompetence and that dietary nucleotides increase the expression of IL-2 receptor and Mac-1 cells (12). Consequently, dietary nucleotides decreased allograft survival (21); increased DTH to chemicals, bacterial antigens, and xenoantigens (2, 12, 22); and enhanced lymphocyte proliferation and IL-2 production (2, 20). In addition, nucleotides improved host resistance to systemic infection by Staphylococcus aureus and Candida albicans (1, 5).

T lymphocytes are important components in the induction of cell-mediated immunity, including DTH. The results from in-flight studies of DTH with the use of kits showed significant suppression in one-half of the subjects who spent 3-5 mo in space and on landing (7). In short-duration flights, the majority of the outcomes are from the postflight period analysis and showed decreased cellular response to mitogens, decreased T-cell counts, and somewhat variable leukocyte counts (16). Long-duration studies (1-12 mo), which were performed by the Russians on board the Mir space station, have documented a 50% reduction in lymphocytic response to PHA on the day after the mission, compared with preflight response. Other studies showed decreased mitogen-induced IL-2 production (10). These foci of immune dysfunction in spaceflight are also consistent with the results of modeled microgravity experiments in animals here on Earth.

Taylor and colleagues (17, 18) suggested that stress might be the primary cause of the decreased proliferation in human T cells postflight. Cortisol suppresses the immune response to foreign substances. This hormone decreases the number of circulation T cells, especially the proportion of helper T₄ lymphocytes, as well as their migration to the site of antigenic stimulation and their function. Cortisol also inhibits the production of IL-1 and IL-2, as well as that of INF- and other macrophage and lymphocyte products (3). Because rodents do not have cortisol and do have corticosterone, hydroxylated from cortisol, serum corticosterone levels were measured as the stress hormone in the present study. Serum corticosterone levels of tail-suspended animals in CD were significantly higher (Fig. 3) than those of group and isolation housing animals. Although isolation housing is a recognized stress environment, immune function and corticosterone level did not show any change compared with those for group housing in the given period of time. These results suggest that immune suppression in AOS animals may be influenced only in part by the central nervous system, in addition to other primary microgravity stressors, which differ from other environmental stress factors. However, in the AOS mice, nucleotide-supplemented CDR and CDU groups showed significantly lowered corticosterone levels, resulting in a decreased stress response in AOS mice in induced simulated microgravity. Such a decrease in corticosterone levels may be one of the mechanisms of immune restoration. Interestingly, nucleotides have been implicated in central nervous system function (9, 13). Thus it is possible that RNA and uracil may have suppressed the production of this stress hormone and enhanced certain cytokines that resulted in the significant increase in immune response shown only in the AOS mice.

On the other hand, the in vivo and ex vivo results, decreased proliferative response of murine lymphocyte in AOS and its restoration by nucleotide, were remarkably similar to those observed in the in vitro rotating cell culture system model, a National Aeronautics and Space Administration-developed bioreactor (8). The bioreactor has been shown to be an ideal ground-based model system for examining the effects of microgravity on cells of the immune system without the presence of psychoneuroendocrine factors. These results suggested that the direct effect of nucleotides on cell proliferation may be via a defect in signal transduction mechanisms.

Although these results document that nucleotide supplementation of normal diets maintains and restores cell-mediated immune response in simulated microgravity, more research is needed to understand its mechanisms. Therefore, to our knowledge, this is the first experimental evidence suggesting strongly that a nutritional immunomodulatory countermeasure is beneficial and can safely be applied and evaluated for human use in true microgravity.