INTRODUCTION

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BLOOD MONOCYTES AND TISSUE macrophages are recognized as an early line of defense against infectious microorganisms. They phagocytize microbes and other foreign particulate matter, and they digest them with lytic enzymes and reactive oxygen species. These cells also release inflammatory cytokines such as interleukin (IL)-1 that promote both innate and adaptive immune responses (5). IL-1 and other cytokines orchestrate the "acute phase response" in which physiological effectors, via generation of fever and hepatic production of plasma proteins, produce an internal environment hostile to the invaders. Furthermore, IL-1 and other cytokines induce heat shock proteins and antioxidants in host cells that protect them against current and future stresses (16). Several environmental challenges (radiation, oxidative stress, and thermal injury) can induce IL-1 (5). These observations lead to the question: is the release of IL-1 a generalized response to any environmental stress?

The present study was intended to test the hypothesis that the stresses of high altitude would induce IL-1 secretion. However, the secretion of IL-1 can be affected by physical exertion, stress-induced hormones (4), and other systemic factors that may change on ascent to high altitudes or enclosure of a human subject in a pressure chamber (11). The present study sought to focus on the direct effect of altitude on IL-1-secreting cells and to separate the influence of reduced pressure from the influence of reduced oxygen.

	METHODS

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Six male subjects were recruited (22-26 yr of age) who were moderately active and did not smoke. All subjects were requested not to participate in physical activities or drink alcohol for 24 h before blood samples were drawn. The subjects read and signed informed consent forms. All procedures were approved by The Pennsylvania State University Office for Regulatory Compliance.

With the use of a standard sterile technique, 30 ml of blood were drawn into heparinized tubes, and the mononuclear cells were immediately isolated using a Ficoll-hypaque density gradient (Histopaque, Sigma, St. Louis, MO). Cells were resuspended at a final concentration of 2.5 × 10⁶ cells/ml in RPMI 1640 media (Sigma) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 mM HEPES (all from Sigma). Cells were cultured in two separate 24-well plates (Corning 3526, Corning). Each well received 0.5 ml cell suspension and 10 µl heat-inactivated autologous plasma. Control wells received an additional 0.5 ml of supplemented RPMI, whereas stimulated wells received an additional 0.5 ml of supplemented RPMI containing lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma), yielding a final LPS concentration of 1 ng/ml. Plates were randomly assigned to control or test environment and maintained there for 8 h. In one series of experiments, cytochalasin B (Sigma) was added to the media (1 mM final concentration), and associated control cultures were treated with vehicle (0.1% DMSO in supplemented RPMI). At the end of the environmental exposure, the supernatants were aspirated and frozen at 70°C.

IL-1 concentrations in the supernatants were measured in duplicate with an ELISA kit (Cistron Biotechnology, Pine Brook, NJ). IL-1 receptor antagonist (IL-1RA) and soluble receptor type II concentrations were assayed using sandwich ELISAs constructed with commerially available reagents [antibodies and cytokine standards: R&D Systems, Minneapolis, MN; streptavidin-conjugated horseradish peroxidase: Pierce, Rockford, IL; and 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid): Sigma] as previously described in detail (4). Absorbance at 405 nm was measured with a Labsystems Multiskan MCC/340 plate reader (Needham Heights, MA).

Three test environments and one standard control environment were employed (see Table 1). The control incubator was maintained at ambient pressure with a normal air-5% CO₂ environment and a 37°C temperature. For the test environments, another incubator was placed in a room-sized hypo-/hyperbaric chamber (Environmental Tectonics, Southampton, PA). All environmental gases for this incubator were mixed in a 350-l chain-compensated gasometer (Collins, Braintree, MA) located outside of the chamber and pumped into the incubator at a flow rate of 200 ml/min. The gases were premixed before every experiment and checked using an Applied Electrochemistry oxygen analyzer (model S-3A; Sunnyvale, CA) and a Beckman carbon dioxide analyzer (model 864; Schiller Park, IL) (see Table 1). The barometric pressure was controlled by the chamber at 350 Torr (~20,000-ft altitude) for the hypobaric condition.

Preliminary experiments were performed to verify that changes in the incubator environment caused by the different test conditions did not alter the temperature and pH of the media. A 24-well culture plate fitted with a miniature thermister (YSI Instruments, Yellow Springs, OH) and a Lazar miniature pH probe (model PHR-146) was used. All data were collected by computer (MAC IIcx) and logged using a LabView (National Instruments) program at 1-min intervals. No significant variations in temperature or pH (relative to ambient control) were observed under any of the experimental conditions.

The cytokine data were analyzed on a Macintosh G3 computer using StatView software (SAS, Cary, NC). Differences between control and experimental conditions (in cells from the same donor) were analyzed by paired t-tests (Figs. 1, 3, and 4). Pressure-related vs. oxygen-related differences were tested by two-way, repeated-measures ANOVA using experimental values normalized as percentages of control values (see Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Influence of hypobaric hypoxia (A), hypobaric normoxia (B), and normobaric hypoxia (C) on interleukin (IL)-1 secretion from lipopolysaccharide (LPS)-stimulated human mononuclear cells.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Data from Fig. 1 expressed as percent change from control to analyze pressure vs. PO2 influences on IL-1 secretion. Two-way, repeated-measures ANOVA indicated a significant pressure effect (P = 0.039) but insignificant oxygen effect (P = 0.184).

	RESULTS

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IL-1 concentrations were below the detection limit of the assay (10 pg/ml) for all unstimulated human mononuclear cell cultures (no LPS) regardless of environmental condition (data not shown). Stimulation with 1 ng/ml LPS resulted in median IL-1 concentrations ranging between 1,350 and 1,750 pg/ml in the three repetitions of the control condition (normobaric normoxia; Fig. 1).

Exposure of LPS-stimulated cells to the hypobaric hypoxia of 20,000 ft caused consistent reductions in IL-1 secretion of >20% for all subjects (P = 0.016; Fig. 1A). When supplemented with oxygen, cells from four of six subjects still responded to hypobaric conditions with >20% decreases in IL-1 secretion (P = 0.059; Fig. 1B). Hypoxia under normobaric conditions had no effect on IL-1 secretion (P = 0.758; Fig. 1C). Two-way, repeated-measures ANOVA indicated that the change in pressure was the significant factor influencing IL-1 secretion (P = 0.039; Fig. 2), and the change in oxygen partial pressure was not a significant influence (P = 0.184). Treatment with cytochalasin B did not block the altitude-induced reductions in IL-1 secretion (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Lack of influence of cytochalasin B (1 mM) on the altitude-induced inhibition of IL-1 secretion.

The biological activity of IL-1 is modulated by a naturally occuring competitive inhibitor, IL-1RA, and by two types of soluble IL-1 receptors (sIL-1RI and sIL-1RII) produced by blood mononuclear cells. In the present study, sufficient culture supernatant remained to allow measurement of sIL-1RII for five subjects and IL-1RA for three subjects. These factors were detectable in both unstimulated and LPS-stimulated conditions. The hypobaric hypoxia of 20,000 ft did not inhibit the secretion of either factor in unstimulated or LPS-stimulated conditions (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Lack of inhibition by altitude on soluble IL-1 receptor type II (sIL-1RII; A) secretion or IL-1 receptor antagonist (IL-1Ra; B) secretion in either unstimulated () or LPS-stimulated () culture conditions.

	DISCUSSION

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In this study, a simulated altitude of 20,000 ft caused a significant reduction in IL-1 secretion. Altitude did not inhibit sIL-1RII or IL-1RA secretion, suggesting that the diminished IL-1 secretion is a specific, regulated response rather than a generalized depression of cellular function. Furthermore, the IL-1 response was associated with reduced total barometric pressure rather than oxygen partial pressure.

Hydrostatic pressure alters the passive permeability characteristics and ATPase activity of cellular membranes (12). Exposure to high centrifugally applied hydrostatic pressure for 1 h caused changes in membrane lipid order and shedding of membrane proteins from human red blood cell ghosts (1). The authors proposed that the changes in membrane lipid order would influence phospholipid signaling and transmembrane transport. In another study, heart tissue from rats exposed to chronic (30 day) hypobaric hypoxia (5,500 m, 380 Torr) exhibited reduced functional activity of G_s, although mRNA expression and protein concentrations for this signaling protein were unchanged (10). The authors speculated that these functional changes may be due to alterations in the association of G_s proteins with the plasma membrane cytoskeleton.

In the present study, normobaric hypoxia had no effect on IL-1 secretion (Fig. 1). These data are consistent with the conclusion of Gerlach et al. (9) and others that the inflammatory response seen in the hypoxia/ischemia of trauma and surgery is predominately a function of reoxygenation. In support of this contention, Matuschak and co-workers (15) inhibited hypoxia/reoxygenation-induced IL-1 production in isolated, perfused liver by treatment with antioxidant compounds, suggesting that the inhibition was mediated by reactive oxygen species generated in the reoxygenation phase. Nonetheless, the present study does not rule out a potential influence of hypoxia in vivo. Alveolar macrophages may exist in oxygen concentrations similar to those used in this study, but circulating monocytes experience ~14% O₂ in arterial blood and ~5% O₂ in venous blood (19). Macrophages have been estimated to exist in an ~5% O₂ environment in the spleen and somewhat lower oxygen tensions in the liver (19). Scannell and associates (21) reported that 9% O₂ for up to 24 h caused no significant change in tumor necrosis factor- secretion by a monocyte cell line, but 1% O₂ was a significant stimulus.

If barometric pressure decreases rapidly, tiny bubbles of gas form in solution. These bubbles also form in the blood of divers who ascend to the surface too rapidly (the "bends" or decompression sickness) and in individuals who ascend too rapidly from sea level to extreme altitudes. Microbubbles have been observed in the blood of astronauts decompressing to altitude (200 Torr or ~38,000 ft) for space-suit operations (18). In the present study, formation of microbubbles in cell cultures and media alone (starting at 18,500 ft) was documented with a video camera attached to an inverted microscope. Microbubble formation, either internal or external to the cell, could be a mechanical stress on the cellular membrane causing the depressed IL-1 secretion. Microbubble interaction with platelets causes aggregation that is dependent on cAMP, but not products of cyclooxygenase or phospholipase C (13, 22).

In the present study, cytochalasin B was added to some cultures to determine if cytoskeletal interactions were involved in the pressure-related inhibition of IL-1 secretion. Low concentrations of cytochalasin B do not disrupt existing microfilaments, but they prevent f-actin polymerization (3). Thus the influence of cytochalasin B will depend on the turnover rate of actin, which is cell-type specific (6). Our experiment was predicated on the report that 1 mM cytochalasin B inhibited volume regulation of murine peritoneal macrophages exposed to hypotonic stress (8). In another study, 5 µM cytochalasin B inhibited this response to hypotonic conditions in Jurkat cells (6). In the present study, cytochalasin B (1 mM) had no effect on the human mononuclear cell response to hypobaric stress, indicating that either microfilament associations are not involved in the response or the turnover rate was slow enough that existing microfilaments were sufficient for a response.

The results of this investigation are diametrically opposed to the original hypothesis. But, in retrospect, the altitude-induced decreases in IL-1 secretion make sense in terms of physiological adaptation. A principal component of altitude acclimatization is expansion of red cell mass, which is mediated by erythropoietin. Frede et al. (7) have reported that intraperitoneal injections of IL-1 inhibited expression of renal erythropoietin mRNA in rats exposed to normobaric hypoxia. In humans, recombinant erythropoietin therapy was less effective in multiple myeloma patients with high circulating IL-1 concentrations than those with low concentrations (17). Furthermore, IL-1-mediated intracellular sequestration of iron interferes with iron delivery to the bone marrow for hemoglobin synthesis (2). Thus inhibition of IL-1 synthesis may facilitate erythropoiesis.

Perspectives

The novel's protagonist Hans Castorp makes this statement not long after arrival at a tuberculosis sanatorium in Davos, Switzerland. In the middle to late 1800s, institutions situated at high altitude such as Davos and Denver (The National Jewish Hospital) were established for consumptives (tuberculosis patients). The purported benefits of residence at altitude have been debated for decades, with cool air temperature, low humidity, increased exposure to solar radiation, cosmic radiation, ozone, and reduced atmospheric pressure (from the standpoint of both hypoxia and the need for deeper inspiration) considered as the critical elements (20). An altitude-induced reduction in bacterial toxin-induced IL-1 secretion, as reported here, may have had the effect of reducing symptoms, because IL-1 is a mediator of fever and cachexia. As Hofrat Behrens, the directing physician of the sanatorium in The Magic Mountain, commented: "Its curious about the metabolism of protein with us up here. Although the process of combustion is heightened, yet the body at the same time puts on flesh" [p. 47 (Ref. 14)].