Antiorthostatic suspension stimulates profiles of macrophage activation in mice.

The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice.

Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

Influence of spaceflight on the production of interleukin-3 and interleukin-6 by rat spleen and thymus cells.

Six adult male Sprague-Dawley rats were flown on the 7-day US space shuttle mission STS-54. After flight, the spleen and thymus from each animal were assayed for the capacity to secrete the cytokines interleukin-3 (IL-3) and IL-6. Spleen and thymus cells (5 x 10(6)/ml) were incubated for 48 h in the presence of 5 micrograms/ml of concanavalin A or 2 micrograms/ml of bacterial lipopolysaccharide to stimulate the production of IL-3 and IL-6. IL-3 activity was measured using the IL-3/colony-stimulating-factor-dependent cell line 32D. IL-6 activity was measured using the IL-6-dependent cell line 7TD1. Spleen and thymus cells harvested from flown rats secreted significantly higher titers of biologically active IL-3 compared with ground control rats. Spaceflight significantly enhanced IL-6 production by thymus, but not spleen, cells. The results of this study demonstrate that spaceflight can enhance the production of certain cytokines by cells of the immune system.

Effects of hypergravity on immunologic function.

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rat spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

Inhibition of murine splenic T lymphocyte proliferation by 2-deoxy-D-glucose-induced metabolic stress.

Female Swiss-Webster mice were injected with the glucose analogue 2-deoxy-D-glucose (2-DG), which when administered to rodents induces acute periods of metabolic stress. A single or multiple injections of 2-DG invoked a stress response, as evidenced by increases in serum corticosterone levels. The influence of this metabolic stressor on the blastogenic potential of splenic T lymphocytes was then examined. It was found that one, two, or three injections of 2-DG resulted in depressed T cell proliferative responses, with an attenuation of the effect occurring by the fifth injection. The 2-DG-induced inhibition of T cell proliferation was not attributable to 2-DG-induced cytolysis, as in vitro incubation of naive T cells with varying concentrations of 2-DG did not result in a reduction in cell number or viability, and flow cytometric analysis demonstrated that percentages of CD3, CD4, and CD8 splenic T cells were not altered as a result of 2-DG-induced stress. Incubating naive T cells in varying concentrations of 2-DG resulted in a dose-dependent inhibition of T cell blastogenic potential. Following in vivo exposure to 2-DG, T cell proliferation did not return to normal levels until 3 days after the cessation of 2-DG injections. Administering the beta-adrenergic receptor antagonist propranolol did not reverse the inhibited lymphoproliferation in 2-DG-treated mice. The inhibition in T cell proliferation was not observed, however, in mice that had been adrenalectomized or hypophysectomized and injected with 2-DG.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of suspension on the oxidative burst by rat neutrophils.

The influence of spaceflight on the oxidative burst of neutrophils is not known. The present study was designed to evaluate the influence of antiorthostatic suspension, a ground-based modeling system designed to simulate certain aspects of weightlessness that occur after spaceflight, on the capacity of rat neutrophils to express the oxidative burst, an important host defense mechanism against microbial pathogens. Rats were suspended in whole body harnesses in the antiorthostatic orientation for a 3- or 7-day period. Control rats were suspended orthostatically or allowed to remain in vivarium cages without the attachment of any suspension materials. After suspension, peripheral blood was harvested and neutrophils were isolated by density gradient centrifugation. The enriched neutrophil preparations were stimulated with N-formyl-methionyl-leucine-phenylalanine and phorbol myristic acid to induce the oxidative burst. It was found that neutrophils isolated from suspended animals released the same levels of superoxide anion as did vivarium control animals that were not suspended, indicating that whole body suspension did not alter this aspect of rat neutrophil function.

The metabolic stressor 2-deoxy-D-glucose (2-DG) enhances LPS-stimulated cytokine production in mice.

The present study was designed to evaluate the influence of metabolic stress on murine cytokine production in vivo. Female Swiss-Webster mice were exposed to a single or multiple injections of the metabolic stressor 2-deoxy-D-glucose (2-DG; 500 mg/kg body wt) once every 48 h and then were injected intravenously with 10 micrograms of bacterial lipopolysaccharide to stimulate macrophage-derived cytokine production. Plasma samples were harvested 2 h later and were assayed for interleukin (IL)-1, IL-3, and IL-6 activities using a panel of standardized bioassays. It was found that one, two, or three injections of 2-DG enhanced the production of all three cytokines. An attenuation of the enhancing effect was observed following the fourth and fifth injections. These results demonstrate that the metabolic stressor 2-DG can alter immune cell effector functions, including the enhancement of normal patterns of cytokine production in vivo.

Increased glucose and 2-deoxy-D-glucose uptake in skeletal muscle of suspended rats.

The influence of 7-d of whole-body suspension (WBS) on insulin stimulated glucose and 2-deoxy-D-glucose uptake in resting skeletal muscle was examined. Differential hindlimb muscle atrophic effects characteristic of this model (soleus > gastrocnemius > EDL) were observed. WBS and control (C) rats underwent hindlimb perfusion following an overnight fast. The rate and magnitude of glucose uptake across the entire hindlimb and within six individual muscles were assessed following the addition of 150, or 1000 microU/ml insulin to a perfusate containing 10 mM glucose and 5 microCi of the non-metabolizable glucose analog 2-deoxy-D-glucose (2-DG). The rate of uptake across the hindlimbs (A-V glucose difference x perfusate flow rate) was not different between C and WBS groups in the absence of exogenous insulin. At insulin concentrations of 150 and 1000 microU/ml the hindlimb musculature of suspended rats exhibited uptakes significantly (p < 0.05) more rapid (increased initial slope of the uptake curve) and of greater magnitude (increased area under uptake curve) than that of C. Overall, no differences in basal (no exogenous insulin) 2-DG uptake were observed between C and WBS muscles. When 150 and 1000 microU/ml of insulin were added to the perfusate, individual muscles from suspended rats exhibited significantly (p < 0.05) greater accumulation of 2-DG than C. These in situ results derived from an intact hindlimb preparation are the first to correlate both total hindlimb and individual muscle glucose uptakes in suspended rats, and suggest an enhanced effect of insulin in skeletal muscle of suspended rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon production by and leukocyte phenotyping of rhesus monkey lymph node and peripheral blood cells.

The ability of peripheral blood leukocytes to produce interferon-gamma (IFN-gamma) and be labeled with monoclonal antibodies against cell-surface markers was determined in this study. Both peripheral blood leukocytes and lymph node cells were able to produce IFN-gamma after challenge with mitogens. The rhesus monkey IFN-gamma was detectable by means of a biological assay but not by means of a radioimmunoassay for human IFN-gamma. Peripheral blood leukocytes and lymph node cells from rhesus monkeys (Macaca mulatta) were treated with fluoresceinated antibodies directed primarily against cell-surface antigens of humans. The degree of binding was determined by means of flow cytometry. Several of the anti-human antibodies did bind to the rhesus monkey peripheral blood leukocytes, as expected. In a novel study, the antibodies bound in a similar fashion to rhesus monkey lymph node cells. Binding of the antibodies was equivalent whether the cells came from inguinal or axillary lymph nodes. Rhesus monkey peripheral blood leukocytes incubated with recombinant human IFN-gamma showed enhanced expression of class II major histocompatibility complex antigens, as detected with anti-HLA-DR antibodies.

Morphometric and metabolic indices of disuse in muscles of hibernating ground squirrels.

1. Morphological, biochemical and metabolic characteristics of hindlimb muscles from summer-active (SA), winter-active (WA) and hibernating (H) golden-mantled ground squirrels (Spermophilus lateralis) were examined to identify alterations resulting from seasonal periods of inactivity. 2. Cross-sectional areas of fibers from the soleus were reduced in both WA and H, although only significantly (P less than 0.05) in WA. Fibers in the EDL exhibited significant reductions in cross-sectional areas in both H and WA groups. Muscle fiber and capillary densities were altered in quantitative agreement with changes in cross-sectional areas. 3. Protein content was reduced 20% (P less than 0.05) in EDL from H and WA groups, but reductions (10%) in the soleus were not statistically significant. RNA content in WA and H groups was significantly decreased in soleus (20%) and EDL (35%) compared with SA, but DNA content was unchanged. 4. In the plantaris, triglyceride content was unchanged, but citrate synthase activity in H (210 +/- 13 mumol min-1 g-1) was significantly greater than in SA (177 +/- 10). In contrast, LDH activity in H was reduced by 25% (P less than 0.05) compared with SA. 5. These results demonstrate atrophic effects associated with seasonal inactivity in hibernating ground squirrels, but suggest the existence of natural mechanisms which limit the response.

Biochemical changes in skeletal muscles of denning bears (Ursus americanus).

1. Biopsies of the extensor hallucis longus (EHL) and gastrocnemius (G) muscles of four captive black bears (Ursus americanus) were obtained prior to denning (PRE), during denning (DEN) and following the Spring arousal (POST). 2. Glycogen, triglyceride and protein concentrations did not differ significantly between the three groups. Likewise, the activity of citrate synthase, a mitochondrial oxidative enzyme, was not significantly different between the three groups. 3. DNA concentrations in DEN samples increased 30% compared to other groups while RNA concentrations were significantly elevated in POST samples. The RNA/DNA ratios were significantly depressed during DEN. 4. These results suggest a degree of muscle atrophy during DEN, with the potential for an increased capacity for muscle protein synthesis following the Spring arousal.