Clarence Sams

Immune system dysregulation following short- vs long-duration spaceflight.

INTRODUCTION Immune system dysregulation has been demonstrated to occur during and immediately following spaceflight. If found to persist during lengthy flights, this phenomenon could be a serious health risk to crewmembers participating in lunar or Mars missions. METHODS A comprehensive postflight immune assessment was performed on 17 short-duration Space Shuttle crewmembers and 8 long-duration International Space Station (ISS) crewmembers. Testing consisted of peripheral leukocyte subset analysis, early T cell activation potential, and intracellular/secreted cytokine profiles. RESULTS For Shuttle crewmembers, the distribution of the peripheral leukocyte subsets was found to be altered postflight. Early T cell activation was elevated postflight; however, the percentage of T cell subsets capable of being stimulated to produce IL-2 and IFN gamma was decreased. The ratio of secreted IFN gamma:IL-10 following T cell stimulation declined after landing, indicating a Th2 shift. For the ISS crewmembers, some alterations in peripheral leukocyte distribution were also detected after landing. In contrast to Shuttle crewmembers, the ISS crewmembers demonstrated a statistically significant reduction in early T cell activation potential immediately postflight. The percentage of T cells capable of producing IL-2 was reduced, but IFN gamma percentages were unchanged. A reduction in the secreted IFN gamma:IL-10 ratio (Th2 shift) was also observed postflight in the ISS crewmembers. CONCLUSION These data indicate that consistent peripheral phenotype changes and altered cytokine production profiles occur following spaceflight of both short and long duration; however, functional immune dysregulation may vary related to mission duration. In addition, a detectable Th2 cytokine shift appears to be associated with spaceflight.

Leukocyte subsets and neutrophil function after short-term spaceflight.

Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short‐term spaceflight (8–15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5‐fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10‐fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC‐1 was significantly decreased while L‐selectin was significantly increased. These functional alterations may be of clinical significance on long‐duration space missions. J. Leukoc. Biol. 65: 179–186; 1999.

Altered cytokine production by specific human peripheral blood cell subsets immediately following space flight

In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the result of either microgravity exposure or the physiologic stresses of landing and readaptation to unit gravity. Future studies, including in-flight analysis or sampling, will be necessary to determine the cause of these alterations.

T cell activation responses are differentially regulated during clinorotation and in spaceflight

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground‐based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti‐CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL‐2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL‐1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR‐mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC‐associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead‐Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead‐Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.—Hashemi, B. B., Penkala, J. E., Vens, C., Huls, H., Cubbage, M., Sams, C. F. T cell activation responses are differentially regulated during clinorotation and in spaceflight. FASEB J. 13, 2071–2082 (1999)

The role of psychoneuroendocrine factors on spaceflight-induced immunological alterations

This paper summarizes previous in‐flight infections and novel conditions of spaceflight that may suppress immune function. Granulocytosis, monocytosis, and lymphopenia are routinely observed following short duration orbital flights. Subtle changes within the monocyte and T cell populations can also be noted by flow cytometric analysis. The similarity between the immunological changes observed after spaceflight and other diverse environmental stressors suggest that most of these alterations may be neuroendocrine‐mediated. Available data support the hypothesis that spaceflight and other environmental stressors modulate normal immune regulation via stress hormones, other than exclusively glucocorticoids. It will be essential to simultaneously collect in‐flight endocrine, immunologic, and infectious illness data to determine the clinical significance of these results. Additional research that delineates the neuroendocrine mechanisms of stress‐induced changes in normal immune regulation will allow clinicians in the future to initiate prophylactic immunomodulator therapy to restore immune competence altered by the stress of long‐duration spaceflight and therefore reduce morbidity from infectious illness, autoimmune disease, or malignancy.

Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.

The risk of renal stone formation during and after long duration space flight

Background: The formation of a renal stone during space flight may have serious negative effects on the health of the crewmember and the success of the mission. Urinary biochemical factors and the influence of dietary factors associated with renal stone development were assessed during long duration Mir Space Station missions. Methods: Twenty-four-hour urine samples were collected prior to, during and following long duration space flight. The relative urinary supersaturation of calcium oxalate, calcium phosphate (brushite), sodium urate, struvite and uric acid were determined. Results: Changes in the urinary biochemistry of crewmembers during long duration spaceflight demonstrated increases in the supersaturation of the stone-forming salts. In-flight hypercalciuria was evident in a number of individual crewmembers and 24-hour dietary fluid intake and urine volume were significantly lower. During flight, there was a significant increase in brushite supersaturation. Conclusions: These data suggest acute effects of space flight and postflight changes in the urinary biochemistry favoring increased crystallization in the urine. The effects of dietary intake, especially fluid intake, may have a significant impact on the potential for renal stone formation. Efforts are now underway to assess the efficacy of a countermeasure to mitigate the increased risk.

Immune system dysregulation during spaceflight: clinical risk for exploration-class missions.

In the past 30 years, a great many investigations have been conducted to determine the effects of spaceflight on human immunity. Probably more than in any other space-life sciences discipline, many qualifiers must be considered related to the published evidence. These include whether studies involved actual spaceflight or ground analogs of flight, whether animal or humans were the subjects, differing vehicles and mission durations, which of the immune cell subpopulations were monitored, and which types of functional assays were used. These qualifiers aside, the current review by Gueguinou et al. [1] in this issue does an admirable job of summarizing the current knowledge base. The authors thoroughly describe the factors that will affect humans during prolonged, explorationclass space missions, the expected dysregulation of immune parameters based on the current evidence, and potential clinical risks. When considering the evidence to date, it cannot yet be firmly concluded that a clinical risk related to immune dysregulation actually exists for exploration-class spaceflight. This is primarily due to a lack of data about human immunity during long-duration spaceflight. Post-flight data have the potential to be influenced by landing and readaptation stress. Shortduration, in-flight data may not reflect immune status, as it equilibrates during long-duration spaceflight. For example, Pierson et al. [2, 3] have demonstrated reactivation of latent herpesviruses during short-duration flight, and frequency and magnitude of reactivation are much higher than observed terrestrially in analog conditions. It is currently unknown if this reactivation persists for the duration of a 6-month ISS mission or is transiently related to launch stress and early adaptation to the flight environment. Data derived from ground-based analogs, although valuable, must be interpreted with caution. Analogs, despite high fidelity with certain aspects of flight, often fail to replicate other aspects. Obviously, factors such as radiation and microgravity cannot be ground-replicated for human subjects. The closest ground analogs for immune dysregulation are likely those that replicate remote deployment, mission stress, environmental hazards, prolonged isolation, and disrupted circadian rhythms. Immune dysregulation has been reported during bed rest, closed chamber confinement, undersea missions (NASA Extreme Environment Mission Operations), and deployment to the Arctic and Antarctic [4–9]. Given the extreme isolation conditions experienced during Antarctic winter-over, this analog may be the closest on Earth for flight-associated immune changes. Without question, all of the published evidence has contributed greatly to our knowledge base about expectations for immune changes, potential mechanisms, monitoring strategies, and likely countermeasures. However, firm conclusions about clinical risk for humans minimally require in-flight human data collected during long-duration spaceflight. In a series of recent internal and external science program reviews associated with the advent of the Constellation Program, NASA has defined specific potential risks for lunar and Mars missions. These risks and other supporting documents are listed in the Program Requirements Document (PRD) for the NASA Human Research Program and are publicly available (http://humanresearch.jsc.nasa.gov/elements/smo/ nra.asp). The potential for adverse health events related to prolonged immune dysregulation is one of the listed risks; however, a specific magnitude and direction for immune dysregulation are not identified. Instead, the potential for immune hyperactivity resulting in risks such as hypersensitivities or autoimmunity is discussed, as is the potential for immune hypoactivity with corresponding risks for infectious diseases and persistent viral reactivation. Thus, multiple possible adverse health events are listed. For each adverse event, ample terrestrial data exist to correlate dysregulated immunity with clinical disease [10, 11]. Studies by Glaser and others [12, 13] have thoroughly connected physiological stress to changes in specific immune parameters associated with spaceflight [14].

Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells.

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-gamma) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS+CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

Plasma Cytokine Concentrations Indicate That In Vivo Hormonal Regulation of Immunity Is Altered During Long-Duration Spaceflight

Aspects of immune system dysregulation associated with long-duration spaceflight have yet to be fully characterized and may represent a clinical risk to crewmembers during deep space missions. Plasma cytokine concentration may serve as an indicator of in vivo physiological changes or immune system mobilization. The plasma concentrations of 22 cytokines were monitored in 28 astronauts during long-duration spaceflight onboard the International Space Station. Blood samples were collected 3 times before flight, 3-5 times during flight (depending on mission duration), at landing, and 30 days after landing. Analysis was performed by bead array immunoassay. With few exceptions, minimal detectable mean plasma concentrations were observed at baseline (launch minus 180) for innate inflammatory cytokines or adaptive regulatory cytokines; however, interleukin (IL)-1ra and several chemokines and growth factors were constitutively present. An increase in the plasma concentration, tumor necrosis factor-α (TNFα), IL-8, IL-1ra, thrombopoietin (Tpo), vascular endothelial growth factor (VEGF), C-C motif chemokine ligand 2 (CCL2), chemokine ligand 4/macrophage inhibitory protein 1b (CCL4), and C-X-C motif chemokine 5/epithelial neutrophil-activating protein 78 (CXCL5) was observed associated with spaceflight. No significant alterations were observed during or following spaceflight for the inflammatory or adaptive/T-regulatory cytokines: IL-1α, IL-1β, IL-2, interferon-gamma (IFN-γ), IL-17, IL-4, IL-5, IL-10, G-CSF, GM-CSF, FGF basic, CCL3, or CCL5. This pattern of cytokine dysregulation suggests multiple physiological adaptations persist during flight, including inflammation, leukocyte recruitment, angiogenesis, and thrombocyte regulation.