INTRODUCTION

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INTERLEUKIN (IL)-6 is an important cytokine involved in a number of biological processes, including the synthesis of acute-phase proteins (1, 2, 12) and the regulation of the immune and/or inflammatory reaction to various stressors (3, 5, 8, 20). Thus circulating IL-6 is elevated during times when homeostasis is disrupted (sepsis, shock, surgery). Recent evidence has clearly shown that high-intensity exercise is a stressor that also elicits an increase in plasma IL-6 (10, 13, 21-24, 30). The mechanisms responsible for the IL-6 response to acute exercise and/or stress remain unknown.

Hypoxia is another stressor that alters homeostasis (subsequent to a reduction in arterial oxygen saturation), as indicated by a variety of nervous and endocrine responses (15-18). Over time, as one becomes acclimatized to hypoxia (or high-altitude exposure), the extent and severity of the perturbation in homeostasis subside. The IL-6 response to both acute and chronic hypoxia is not known nor are any potential mechanisms that may be involved.

It has been demonstrated that the catecholamines, which are elevated during stressful conditions, play a role in the production of and increase in plasma IL-6 levels (9, 25, 26, 31). These studies implicate the -adrenergic pathway for IL-6 activation, because -adrenergic agonists can mimic the effects of the catecholamines, whereas -adrenergic antagonists block the IL-6 response (26, 31). Very little is known with regard to the -adrenergic contribution to the IL-6 response during periods of stress. Both plasma and urinary norepinephrine levels are elevated during hypoxia (15-18). Because norepinephrine has a strong affinity for the -adrenergic receptors, this represents a potential mechanism that may contribute to the IL-6 response under such conditions. Thus it was the purpose of this study to 1) examine the IL-6 response over time during high-altitude exposure (4,300 m for 12 days) both at rest and during exercise and 2) examine the influence of -adrenergic blockade (prazosin) on these responses. It was hypothesized that the perturbation in homeostasis associated with high-altitude exposure would increase circulating IL-6 levels and that this would be exacerbated by the added stress of exercise under hypoxic conditions. Finally, -adrenergic blockade would blunt these responses.

	METHODS

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Subjects. Sixteen healthy, eumenorrheic women (age 23.2 ± 1.4 yr, weight 68.7 ± 4.5 kg) volunteered to participate in the study. All subjects were nonsmoking, sea-level residents who were recreationally active. None was taking oral contraceptives at the time of testing. All testing procedures received approval from the University of Colorado Health Sciences Center. Sea-level tests were performed at the Veterans Affairs Medical Center in Palo Alto, CA. Altitude tests were performed at the summit of Pikes Peak, CO (4,300 m), where subjects resided for 12 consecutive days. Throughout the duration of the study, subjects' diets and physical activity habits were closely monitored to avoid any weight fluctuations.

Sea-level testing. Subjects were randomly assigned to receive either an -adrenergic blockade drug (blocked; n = 8; prazosin, 3 mg/day), or a placebo (n = 8) in a double-blinded design. Administration of the drugs began 3 days before initial testing at the Veterans Affairs Medical Center in Palo Alto, CA (752 mmHg), and subjects remained on the drugs for the duration of the sea-level testing. The degree of -adrenergic blockade was determined by the dose response of systolic blood pressure to incremental increases in phenylephrine (phenylephrine challenge test), an -adrenergic agonist, on the third and ninth day of the testing protocol at sea level and on day 9 at high altitude.

Altitude testing. The altitude portion of the study was conducted at the Maher Memorial Research Facility on the summit of Pikes Peak, CO (4,300 m, 462 mmHg), ~8 wk after sea-level testing. Administration of the -adrenergic blockade or placebo began 3 days before arrival at altitude. Within 4 h of arrival (day 1) and again on day 12, subjects performed a 50-min submaximal exercise bout at the same absolute intensity that elicited 50% of sea-level O_2 max. A O_2 max test was performed on day 8 at altitude.

Blood and urine collection. Blood samples were collected at rest on days 1 and 6 at sea level and days 1, 3, 6, 9, and 12 at altitude via venipuncture. Blood samples during exercise were collected from the antecubital vein via an indwelling catheter at 15, 30, and 40 min. Twenty-four-hour urine samples were collected continuously during the testing for catecholamine determination: days 1-12 at sea level and days 1-12 at altitude. Urine samples were treated with 5 mM reduced glutathione to control for catechol oxidation.

IL-6 determination. Plasma IL-6 concentration was determined by an ELISA kit using 96-well plates (Quantikine High Sensitivity; R&D Systems, Minneapolis, MN).

Catecholamine measurements. Urinary catecholamine levels were determined by means of HPLC (model 1330 pump and model 1340 electrochemical detector, Bio-Rad) with electrochemical detection as previously described (15). Dihydroxybenzylamine (Sigma Chemical) was used as the internal standard. Catecholamines were absorbed onto acid-washed alumina with 1.5 M Tris buffer at pH 8.6 in 2% EDTA. The alumina was then washed 2× with 3 ml of distilled water. The catecholamines were extracted with 100 µl of 0.1 N perchloric acid with 10 min of shaking and final centrifugation at 12,000 g. One hundred milliliters of eluant were then injected into the HPLC column (reverse phase, Bio-Sil ODS-5S, Bio-Rad), and eluted with mobile phase (6.8 g sodium acetate-anhydrous, 1.0 g sodium heptane sulfonate, 60 ml acetonitrile, and 1.0 g Na₂EDTA in 1 liter, pH adjusted to 4.8). The flow rate was set at 1.1 ml/min at 2,000 psi at 0.65 V. The chromatogram was integrated on a Shimadzu integration system (model C-R3A).

Ovarian hormones. Plasma samples were obtained periodically throughout the study to monitor menstrual cycle phase. Ovarian hormones were determined by using DPC Coat-a-Count radioimunnoassay kits.

Statistics. All values reported are means ± SE. Differences across all testing conditions were determined by a repeated-measures two-way analysis of variance with significance set at P < 0.05. Tukey post hoc comparisons were used to identify significant differences among means. Pearson product-moment correlations were used to assess the relationship between urinary norepinephrine excretion rates with that of plasma IL-6 levels.

	RESULTS

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Phenylephrine challenge. The dose of phenylephrine required to raise systolic blood pressure 20 mmHg above baseline was designated as the PD₂₀, which was the end point used to document the degree of -adrenergic blockade. At sea level, PD₂₀ was 1.47 ± 0.73 µg · kg¹ · min¹ before administration of prazosin and increased to 9.45 ± 2.22 µg · kg¹ · min¹ at day 3 (P < 0.05), indicating a significant degree of -adrenergic blockade. PD₂₀ on day 9 at sea level was 6.87 ± 2.99 µg · kg¹ · min¹ and was 15.05 µg · kg¹ · min¹ on day 9 at 4,300 m, indicating no decrement in the degree of blockade due to drug tolerance or altitude exposure. Thus a high degree of -adrenergic blockade was maintained throughout the study at both sea level and high altitude.

Menstrual cycle phase. No differences were found between the luteal and follicular phase for both IL-6 levels as well as urinary catecholamines; consequently, values have been grouped.

Plasma IL-6. At sea level, no differences in plasma IL-6 levels were found at rest between groups (1.5 ± 0.2 and 1.2 ± 0.3 pg/ml plasma for placebo and blocked groups, respectively; Fig. 1). On acute ascent to altitude, IL-6 levels increased significantly in both groups compared with sea-level values (57 and 84% for placebo and blocked groups, respectively), but this response did not differ between the two groups. After 12 days acclimatization, IL-6 levels remained elevated for placebo subjects; however, they returned to sea-level values in the blocked group. As a result, from day 3 to day 12 at altitude, plasma IL-6 levels were significantly lower for the blocked group compared with the placebo group (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Resting interleukin-6 (IL-6) levels taken on days 1 and 6 while at sea level and on days 1, 3, 6, 9, and 12 while at high altitude (4,300 m). Blocked, -adrenergic blockade. Values are means ± SE. * Significantly different from placebo group, P < 0.05.  Significantly different from sea-level values, P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   IL-6 levels taken before and during 50 min of submaximal exercise (50% maximal oxygen consumption) while at sea level (A), on day 1 of high-altitude exposure (B), and on day 12 of high-altitude exposure (C). Values are means ± SE. *Significantly different from placebo group, P < 0.05.  Significantly different from resting values, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Comparison of the IL-6 response to exercise across all 3 conditions studied (sea level, day 1 at high altitude, and day 12 at high altitude) for both placebo (A) and blocked (B) groups. Values are means ± SE. * Significantly different from sea level, P < 0.05.  Significantly different from sea level and day 12, P < 0.05.

Urinary catecholamines. At sea level, 24-h urinary norepinephrine excretion rates did not differ over time during the 12 days of collection. However, urinary norepinephrine excretion rates were significantly greater for blocked vs. placebo subjects while at sea level (47.7 ± 3.6 vs. 24.8 ± 2.1 µg/day for blocked and placebo, respectively). After arrival at 4,300 m, norepinephrine excretion rates increased immediately for both groups (Fig. 4A). Urinary norepinephrine excretion continued to increase steadily during subsequent days at altitude, peaking at days 4-6. Thereafter, excretion rates remained constant at this elevated level for the duration of the altitude residence. Similar to sea-level findings, norepinephrine excretion rates at altitude were significantly greater for blocked vs. placebo subjects.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Twenty-four-hour urinary excretion rates for both norepinephrine (A) and epinephrine (B) during the 12 days of residence at high altitude. Values are means ± SE. *Significantly different from placebo group, P < 0.05  Significantly different from sea-level values, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The main findings of the present study are 1) both acute and chronic hypoxia are environmental stressors that elicit an increase in resting plasma IL-6 levels, 2) the IL-6 response to a single bout of exercise is exacerbated under hypoxic conditions, and 3) -adrenergic blockade with prazosin significantly reduced this response during both short-term moderate-intensity exercise and hypoxia, suggesting an -adrenergic component to IL-6 production and mobilization under these conditions.

Numerous studies have consistently demonstrated that a single bout of exercise results in an elevation in circulating IL-6 (10, 13, 21-24, 30). In fact, it appears that, of the cytokines, the IL-6 increase is the most reliable and marked in response to an exercise-induced stimulus (22, 27, 30). The IL-6 response is also intensity dependent, because the greater the exercise intensity (indicated by running speed, lactate levels, circulating catecholamines), the more dramatic are the increases in plasma IL-6 levels (22). It was originally suggested that the increases in IL-6 levels were related to the extent of muscle inflammation and damage inflicted during exercise (4, 5, 7, 13, 29). The IL-6 response to exercise may signify local inflammation resulting from damaged or injured muscle primarily caused by eccentric-type contractions. However, recent studies have shown that, in apparently undamaged and/or uninjured muscles (no markers of damage such as an increase in serum creatine kinase and myoglobin concentrations), IL-6 can be dramatically elevated during exercise. These studies, employing either concentric exercise or eccentric training, have suggested that factors unrelated to muscle damage, such as endogenous catecholamines or endothelial shear stress are possible mechanisms contributing to the increase in IL-6 associated with exercise (28).

Regardless, the present study demonstrates that the environmental stress of altitude exposure alone is sufficient to cause an elevation in circulating IL-6 even under resting conditions (independent of exercise stress). As shown in Fig. 1, resting IL-6 levels increased immediately on arrival to altitude and, for placebo subjects, remained elevated throughout the duration of stay at 4,300 m. To our knowledge, this is the first study to demonstrate that both acute and chronic altitude exposure results in elevated resting IL-6 levels in humans. In cultured endothelial cells from mice acutely exposed to hypoxia, expression of IL-6 was dramatically increased (33). Similar results were reported in cultured rat neonatal cardiac myocytes after 4 h of hypoxia (32). In the one previous study examining the influence of acute hypoxia in humans, serum IL-6 levels were found to be significantly increased, whereas other proinflammatory cytokines remained unchanged (14). Our results are consistent with and extend those findings indicating that this elevation in IL-6 persists while subjects are becoming acclimatized to high-altitude exposure.

Although the physiological significance of the IL-6 response to hypoxia remains unknown, a number of possibilities have been suggested. It is generally believed that the function of IL-6 during acute hypoxia is not as a mediator of inflammation or acute-phase protein response because serum values of IL-1, IL-1ra, tumor necrosis factor-, and C-reactive protein remain unchanged (14). One possible explanation relates to the ability of IL-6 to promote angiogenesis (19). IL-6 expression is elevated in tissues that undergo active angiogenesis and may play a role via the induction of vascular endothelial growth factor (VEGF; Ref. 6). Treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA that is comparable to the documented induction of VEGF mRNA by hypoxia (6). The benefit of forming new blood vessels during extended periods of hypoxia (and reduced arterial oxygen saturation) are obvious and have been reported in both animal and human studies. Additionally, IL-6 can modulate production of erythropoietin because the addition of IL-6 to hypoxic human hepatoma cells resulted in a dose-dependent stimulation of hypoxia-induced erythropoietin production by as much as 81% (11). The associated increases in red blood cell number and oxygen-carrying capacity are well-documented markers of adaptation to high altitude.

The finding that -adrenergic blockade significantly reduced the IL-6 response to both short-term moderate intensity exercise and hypoxia is noteworthy. It is known that the catecholamines can act as a potent stimulator for IL-6 production and release into plasma; however, this effect has generally been thought to result from activation of the -adrenergic pathways (9, 25, 26, 31). Infusion of epinephrine has been reported to induce a dose-dependent increase in plasma IL-6 concentrations in rat. Importantly, this epinephrine-induced increase in plasma IL-6 was blocked by the -adrenergic receptor antagonist propranolol (9). A separate study in rats found similar results with propranolol but also demonstrated that isoprenalin, a ₂-adrenergic agonist, also elicited very high levels of plasma IL-6, indicating that the release of IL-6 can be mediated via the ₂-adrenergic receptors (31). However, very little is known with regard to the -adrenergic influence on IL-6 induction, particularly during periods of stress, in humans.

In our study, resting IL-6 levels increased significantly in both groups on immediate exposure to high altitude (Fig. 1). However, whereas resting IL-6 levels remained elevated throughout the 12-day duration at altitude for the placebo group, IL-6 levels were significantly lower for the blocked group from day 3 through day 12, returning to sea-level values. Thus the presence of -adrenergic blockade clearly influenced the IL-6 response to the chronic stress of hypoxia. This relationship was also apparent for the IL-6 response during exercise. Both at sea level as well as on day 12 at altitude, the blocked group demonstrated dramatic reductions in plasma IL-6 levels in response to the exercise stimulus (Fig. 2, A and C). With acute exposure to altitude (day 1), no differences were observed between groups either at rest or during the submaximal exercise bout (Fig. 2B). This can be explained by the large and significant increase in the adrenal epinephrine response to acute hypoxia. Epinephrine (both plasma and urinary) increases immediately on exposure to high altitude (15-18). Furthermore, the plasma epinephrine response during exercise is exacerbated with acute hypoxia, resulting in large increases compared with sea-level values (15-18). Thus this strong mediator of the -adrenergic mechanism for IL-6 release (as mentioned above) is present in both groups with acute exposure, thus accounting for the similar increase in IL-6 levels. Under the presence of high levels of epinephrine, the lack of an effect of -adrenergic blockade is understandable. However, as shown in Fig. 4B, after day 3, this systemic epinephrine response rapidly returns to sea-level values, removing this potent -adrenergic stimulus, allowing -adrenergic blockade to effectively reduce IL-6 levels. It must be noted that factors unrelated to -adrenergic stimulation could also participate in the IL-6 response to acute hypoxia.

Thereafter, IL-6 levels remained elevated throughout the duration at altitude (placebo group only) in a similar pattern to that found for the increase in urinary norepinephrine excretion. Urinary norepinephrine excretion (a marker of overall sympathetic nerve activity) continued to increase steadily during days at altitude, peaking at days 4-6. Because norepinephrine has a strong affinity for the -adrenergic receptors, this is a potential mechanism likely to contribute to the continued elevation in plasma IL-6 levels overtime at altitude, particularly in the face of declining -adrenergic stimulation. This is supported by a significant correlation (P = 0.004) between resting IL-6 and urinary norepinephrine excretion rates for placebo subjects over the course of time while at altitude (Fig. 5). One other study has reported a similar relationship between peak plasma norepinephrine and IL-6 levels during high-intensity treadmill running in humans (24).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Relationship between resting IL-6 levels and 24-h urinary norepinephrine excretion rates, for placebo group only, during the 12 days of residence at high altitude.

Further support for an -adrenergic contribution to the IL-6 response is found when examining resting IL-6 values in subjects treated with prazosin. Once the -adrenergic stimulation (epinephrine) had subsided, IL-6 levels returned quickly to sea-level values for the -adrenergic-blocked subjects, differing significantly from that found for the placebo group. Thus the presence of -adrenergic blockade completely eliminated the persistent elevation in the IL-6 response observed in placebo subjects over time at altitude.

In summary, we have demonstrated that hypoxia, as with other forms of stress, induced marked elevations in plasma IL-6 levels that was attenuated by -adrenergic blockade. We believe this to be the first study to demonstrate, in humans, a significant -adrenergic contribution to the IL-6 response under the types of stressful conditions studied (short-term moderate-intensity exercise, hypoxia). Further research is required to more fully examine the mechanisms responsible for the IL-6 response to stress, its source(s) as well as the functional significance of this cytokine under these conditions. Finally, future studies are necessary to determine whether these responses found in women are similar for men.