Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys.

During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.

Spaceflight alters immune cell function and distribution.

Experiments were carried out aboard COSMOS 2044 to determine the effects of spaceflight on immunologically important cell function and distribution. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. In one experiment, rat bone marrow cells were examined in Moscow, for their response to recombinant murine granulocyte/monocyte colony-stimulating factor (GM-CSF). In another experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States for analysis on a flow cytometer. Bone marrow cells from flown and suspended rats showed a decreased response to granulocyte/monocyte colony-stimulating factor compared with bone marrow cells from control rats. Of the spleen cell subpopulations examined from flown rats, only those cells expressing markers for suppressor-cytotoxic T- and helper T-cells showed an increased percentage of stained cells. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleukin-2 receptor-bearing pan T- and helper T-cells in the lymphocytic population. Cell populations from rats suspended antiorthostatically did not follow the same pattern of distribution of leukocytes as cell populations for flown rats. The results from COSMOS 2044 are similar, but not identical, to earlier results from COSMOS 1887 and confirm that spaceflight can have profound effects on immune system components and activities.

Effect of spaceflight on natural killer cell activity.

The effects of spaceflight on immune cell function were determined in rats flown on COSMOS 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for 51Cr-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

Methyldopa: effects on the murine immune system.

Mice were treated for 7 weeks with doses of methyldopa somewhat exceeding those given to man, and mixed immunotoxic effects were observed. Daily subcutaneous injections of 5 mg (in 0.1 ml) methyldopa or saline (0.1 ml) did not generally alter body weights, except on day 19, when the methyldopa-treated mice weighed significantly less. During treatment, all mice were immunized twice with sheep red blood cells (SRBC) and bled four times. Anti-SRBC titers were not affected by methyldopa treatment, but leukocyte counts were dramatically decreased, and hematocrits to a lesser degree. Although in methyldopa-treated mice spleen and kidneys were increased in size, liver, lung, heart, and thymus size was not affected. These results are discussed in the context of other studies on the mode of action of methyldopa in eliciting an autoimmune anemia in man treated therapeutically with this drug.

Effect of ethanol on the immune system in mice.

Mice were injected with ethanol (2.5 g/kg body wt., i.p.) 1-3 times daily for 17 days or 3 times daily for 49 days. The primary and secondary antibody titers to sheep red blood cells were determined. In addition, microhematocrits, white blood cell counts, white blood cell differential counts, and organ-to-body-weight ratios were determined. Despite continuous large doses of ethanol, the ethanol-treated mice responded as well as saline-treated controls in all parameters tested. The results of this study are discussed in the context of other studies on the effects of ethanol on the immune system.

Dimethyl sulfoxide: lack of suppression of the humoral immune response in mice.

Mice were injected intraperitoneally (i.p.) with 100% dimethyl sulfoxide (DMSO) or sterile saline (controls) for 36 days. The mice received 0.05 ml daily for one week, 0.025 ml every other day for the second week (because the DMSO-treated mice appeared weak), and 0.05 ml daily for 3 more weeks. All mice were immunized twice with sheep red blood cells (days 13 and 24), and bled twice by caudal incision (days 20 and 29). Hematocrits were significantly decreased (P less than or equal to 0.002) but still within the normal range. The primary and secondary antibody response to sheep red blood cells (SRBC), leukocyte counts, body weight, and the size of the heart, lungs, spleen, thymus, and kidneys were not affected. DMSO treatment resulted in significant liver enlargement (P = 0.02). It is concluded that this dose of DMSO is not deleterious to the humoral immune response in mice responding to a new antigen.

Effect of simulated weightlessness on the immune system in rats.

Rats suspended in a model system designed to simulate many aspects of weightlessness were immunized with sheep red blood cells. Parameters measured on these and control rats included titers of anti-sheep red blood cell antibodies, serum immunoglobulin levels, spleen and thymus weights, hematocrits, and leukocyte differential counts on peripheral blood. No significant differences were found between test and weight-bearing, harnessed controls; however, the thymuses of animals in both these groups were significantly smaller than untreated cage controls. The lack of an effect of simulated weightlessness on the immune system is an interesting result, and its significance is discussed.

Inhibited interferon-gamma but normal interleukin-3 production from rats flown on the space shuttle.

Rats were flown on Space Shuttle SL-3 for 1 week. When spleen cells were removed from these rats and challenged with concanavalin-A, interferon-gamma production was severely inhibited, while interleukin-3 production was unaffected compared to ground-based control rats. These data indicate that there is a defect in interferon-gamma production in rats that have been exposed to spaceflight. This defect could contribute to, and be one reason for, immunosuppression observed after spaceflight.

A suspension model for hypokinetic/hypodynamic and antiorthostatic responses in the mouse.

Hypokinetic/hypodynamic and antiorthostatic responses to weightlessness and bedrest were simulated in mice using a suspension technique. Animals were suspended for 1 or 2 weeks in an antiorthostatic posture and positioned to permit freedom of movement but eliminate load bearing by the hindlimbs. Suspended mice exhibited reduced food and water intakes and rapid 10% decrease in body weight to a level which was maintained for the remainder of the suspension period. Diuresis was evident in suspended mice, but the natriuresis and kaliuresis previously observed in the suspended rat were not evident. Differential hindlimb muscle atrophy (soleus greater than plantaris = gastrocnemius greater than EDL) and increased excretion of urea and ammonia were also noted in suspended mice. Postsuspension recovery studies indicated that the recovery process was highly effective. These results document specific responses to hypokinesia/hypodynamia and antiorthostasis in the mouse and demonstrate similarities in the responses of mice and rats to suspension. These studies expand the utility of the suspension model and suggest that the mouse may be useful in future studies simulating both weightlessness and bedrest.

Effects of spaceflight on levels and activity of immune cells.

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of spaceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

Effect of treatment of mice with sera containing gamma interferon on the course of infection with Salmonella typhimurium strain LT-2.

Swiss/Webster mice pre-treated with gamma interferon preparations and then infected with Salmonella typhimurium showed significantly increased survival when compared to mock interferon-treated or control mice. Nude mice similarly pre-treated with gamma interferon showed similar increased survival after S. typhimurium infections. If gamma interferon treatment were delayed until the commencement of the S. typhimurium infection, no effect on mortality was observed. The increased survival of the gamma interferon-treated mice did not appear to be due to increased antibody production, more rapid clearing of bacteria from the circulation, or enhanced uptake of bacteria by phagocytic cells of the spleen, liver, or lungs. The number of viable bacteria isolated from the spleens and livers of interferon-treated mice was significantly lower over time than in organs of mock interferon-treated or untreated mice, suggesting the gamma interferon treatment may have resulted in more efficient killing of bacteria by phagocytic cells.

In vitro production and cellular origin of murine type II interferon.

Antigen-specific type II interferon was produced in vitro by harvesting supernatants of spleen cell cultures from Swiss-Webster mice sensitized with Mycobacterium bovis strain BCG and challenged with old tuberculin. Treatment of C3H mouse spleen cell cultures with appropriate anti-Ia, anti-IgG, anti-Thy-1 or anti-Ly-2,3 sera resulted in a significant decrease in production of type II interferon. Removal of nylon wool adherent cells or cells with histamine receptors by column chromatography similarly caused reduced production of type II interferon. Recombination of spleen cell cultures treated with anti-Ia and anti-Thy-1 sera or of cells treated with anti-IgG and anti-Thy-1 resulted in restored production of type II interferon. Interferon production was also restored by combination of cells passed through histamine columns with anti-Ia treated cells, or those passed through nylon wool columns with anti-Thy-1 treated cells. Anti-Ly-1 serum treatment had no effect on interferon production. Removal of plastic-adherent cells or cells that had phagocytosed carbonyl iron also decreased interferon production, suggesting that macrophages were also involved in type II interferon production. Recombination of non-adherent spleen cells with anti-Ia and anti-Thy-1 sera treated spleen cells, however, did not restore interferon production, suggesting that other cells in addition to macrophages are depleted by the adherence procedure. These findings indicate that type II interferon is produced by suppressor or cytotoxic (Ly-2,3+) T lymphocytes in co-operation with one or two additional cell types: (i) B lymphocytes, and (ii) macrophages.

Current research on regenerative systems.

Multiple studies directed toward the development of a regenerative life support system have shown that easily synthesized organic compounds and microbiological materials are potentially capable of being used as foods for long-duration space missions. Animal feeding studies have supported these views. The organic compounds presently believed to offer the greatest potential are glycerol, simple glycerol derivatives such as triacetin, and formose sugars. Laboratory studies indicate that glycerol can be synthesized from formaldehyde which in turn is obtained by the direct catalytic oxidation of methane, a by-product of the Sabatier reaction used in spacecraft atmosphere control system. Formose sugars are derived from the self-condensation of formaldehyde. Mixtures of glycerol and triacetin have been shown to be suitable as a major component of diets fed to weanling rats for prolonged periods. These compounds do not exist as stereoisomers and therefore offer advantages over the formose sugars. Hydrogenomonas eutropha is the microbiological system under investigation. An automated system for the continuous autotrophic production of Hydrogenomonas bacteria is in operation, and the nutritional requirements for growth in the system using urea as a nitrogen source are being studied. Nutritional evaluation of Hydrogenomonas bacteria has shown they are capable of supplying the total protein requirement of growing rats for prolonged periods. The potential and problems of these regenerative systems and the prospects for the accomplishment of a totally regenerative food system will be discussed.

Protection of chicken embryos by viridans streptococci against the lethal effect of Staphylococcus aureus.

Chicken embryos were used to investigate the mechanism by which viridans streptococci inhibit the growth of pathogenic staphylococci. Ten-day-old embryonated eggs were infected allantoically. At a concentration of 1.8 x 10(2) colony-forming units (CFU) of viridans streptococci, the percentage of fatalities was less than 10%. There was 80% fatality with 8 x 10(1) CFU of Staphylococcus aureus strain 502A and 100% when a 100-fold increase in concentration was used. An inoculum size of 10(2) to 10(3) CFU of viridans streptococci was chosen to protect the embryos against the lethal effect of strain 502A when challenged 24 h later. The survival after challenging at 4 days was 93% in protected eggs and 37% in unprotected eggs. Chicken embryos receiving heat-killed viridans and challenged with strain 502A when examined after 4 days did not demonstrate a protective effect. This protection of embryonated eggs could not be transferred by administration of sterile filtrate of allantoic fluid in which protecting strain was grown. The experimental infection of embryonated eggs has demonstrated that prior allantoic infection with viridans streptococci affords significant protection against subsequent challenge with virulent staphylococci.