Peripheral Blood Lymphocytes: A Model for MonitoringPhysiological Adaptation to High Altitude

Maria A. Mariggio,1,4 Stefano Falone,2 Caterina Morabito,1,4 Simone Guarnieri,1,4

Alessandro Mirabilio,2 Raffaele Pilla,1,4 Tonino Bucciarelli,3 Vittore Verratti,1 and Fernanda Amicarelli 2

Abstract

Mariggio, Maria A., Stefano Falone, Caterina Morabito, Simone Guarnieri, Alessandro Mirabilio, Raffaele Pilla,Tonino Bucciarelli, Vittore Verratti, and Fernanda Amicarelli. Peripheral blood lymphocytes: a model formonitoring physiological adaptation to high altitude. High Alt. Med. Biol. 10:333–342, 2010.—Depending on theabsolute altitude and the duration of exposure, a high altitude environment induces various cellular effects thatare strictly related to changes in oxidative balance. In this study, we used in vitro isolated peripheral bloodlymphocytes as biosensors to test the effect of hypobaric hypoxia on seven climbers by measuring the functionalactivity of these cells. Our data revealed that a 21-day exposure to high altitude (5000 m) (1) increased intra-cellular Ca2þ concentration, (2) caused a significant decrease in mitochondrial membrane potential, and (3)despite possible transient increases in intracellular levels of reactive oxygen species, did not significantly changethe antioxidant and/or oxidative damage-related status in lymphocytes and serum, assessed by measuringTrolox-equivalent antioxidant capacity, glutathione peroxidase activity, vitamin levels, and oxidatively modifiedproteins and lipids. Overall, these results suggest that high altitude might cause an impairment in adaptiveantioxidant responses. This, in turn, could increase the risk of oxidative-stress-induced cellular damage. Inaddition, this study corroborates the use of peripheral blood lymphocytes as an easily handled model formonitoring adaptive response to environmental challenge.

Key Words: lymphocytes; oxidative stress; intracellular calcium; hypobaric hypoxia; altitude adaptation

Introduction

Reactive oxygen species (ROS) are radical and non-radical oxygen-centered molecules or ions formed by the

incomplete one-electron reduction of oxygen. ROS are knownto play crucial roles in the pathogenesis of several diseases,such as cardiovascular disorders, neurodegeneration, andcancer (Radak et al., 2008b). Despite the increasing number ofphysiological processes that require ROS for the appropriateactivation of signaling pathways, ROS-based molecularevents remain among the most important factors involved inconditions related to oxidative stress (Brookes et al., 2002;Droge, 2002; Balaban et al., 2005).

Paradoxically, exposure to a hypoxic environment isknown to increase ROS concentration in various cells andtissues as a result of the activity of complex III of the mito-

chondrial respiratory chain (Guzy and Schumacker, 2006;Murphy, 2009). Many researchers have reported importantredox imbalances after prolonged mountain sojourns (Sinhaet al., 2009). Some studies have indicated possible pathogenicroles of oxidatively modified molecules generated in responseto hypoxia (Behn et al., 2007). In fact, an overproduction ofROS can lead to the oxidation of essential biomolecules, in-cluding lipids, proteins, and nucleic acids, and may be asso-ciated with cellular dysfunction, thus promoting variousbiological responses, such as inflammation and apoptosis(Ames et al., 1993; Breen and Murphy, 1995; Droge andSchipper, 2007; Karihtala and Soini, 2007).

Intense physical activity is also known to promote ROSoverproduction, mainly through oxygen overconsumption,ischemia–reperfusion-induced activation of xanthine oxidase,neutrophil-mediated superoxide release resulting from tissue

1Dipartimento di Scienze Mediche di Base ed Applicate, Universita Gabriele d’Annunzio di Chieti-Pescara, Chieti, Italy.2Dipartimento di Biologia di Base ed Applicata, Universita dell’Aquila, L’Aquila, Italy.3Dipartimento di Scienze Biomediche, Universita Gabriele d’Annunzio, Chieti-Pescara, Chieti, Italy.4Center of Excellence on Aging, Gabriele d’Annunzio University Foundation, Chieti, Italy.

HIGH ALTITUDE MEDICINE & BIOLOGYVolume 11, Number 4, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089/ham.2009.1097

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damage, and inflammatory processes (Fisher-Wellman andBloomer, 2009). Therefore, high-energy-demanding mountainexpeditions may aggravate oxidative damage that could re-sult from exposure to low oxygen tension, thus exacerbatingthe risk of developing oxidative-stress-induced dysfunctionand pathologies. On the other hand, exercise-induced increasesin the generation of ROS also trigger adaptation mechanismsthat may decrease the incidence of ROS-associated diseases(Radak et al., 2001).

As reported by several authors, a close mutual relationshipexists between increased ROS production and intracellularCa2þ concentration ([Ca2þ]i). In fact, modification of cyto-plasmic Ca2þ levels could be one of the most importantmechanisms by which ROS exert their multiple actions in cells(Camello-Almaraz et al., 2006; Feissner et al., 2009). SeveralCa2þ transport systems are modulated by oxidation. For ex-ample, the activity of inositol 1,4,5-trisphosphate and ryano-dine receptors, the main Ca2þ channels of intracellular stores,depends on oxidation status, which thus regulates sponta-neous and stimulus-induced intracellular Ca2þ oscillations(Davidson and Duchen, 2006). Mitochondria, the main ROSproducers, are also known to control intracellular Ca2þ sig-nals through Ca2þ uptake and release during cytosolic Ca2þ

mobilization (Nicholls, 2005). Furthermore, there is evidencethat Ca2þ-mobilizing stimuli generate mitochondrial ROS,which, in turn, could facilitate intracellular Ca2þ signaling(Camello et al., 2000; Xi et al., 2005).

Extensive research has been dedicated to the discovery ofunique and easily available biological models for integratedstudies concerning physiological and pathological conditionsin relation to whole-body exposure to environmental stress-ors. Defining the role of environmental changes in the controland alteration of the oxidative balance is limited by two mainfactors: (1) our understanding of the persistence of environ-mentally induced oxidative alterations and (2) the availabilityof a cellular system easily obtained from a single source, yetcapable of providing valuable information on the status of theentire body. The invasiveness of the procedure required toobtain adequate answers to the questions raised is an addi-tional factor that needs to be taken into account.

As previously reported, peripheral lymphocytes can be areliable model for studying the pathophysiology of oxidative-stress-mediated Ca2þ homeostasis alterations, which inducecell dysfunction and lead to organic pathogenic states (Beliaet al., 2009). In addition, the hematopoietic system is highlysensitive to environmental factors, and the blood system isknown to be directly involved in the development of toleranceto hypoxic conditions (Chouker et al., 2005; Shtemberg et al.,2007; Brooks et al., 2009). Furthermore, because circulatinglymphocytes are readily accessible and retain their originalphenotype during in vitro culture, they offer important ad-vantages for cellular and molecular studies (Nagaeva et al.,2002).

For all these reasons, we used lymphocytes, which are a‘‘time-persistent’’ system (Balakrishnan and Rao, 1999; Gre-goire et al., 2006) capable of reflecting the condition of thewhole organism, as a model to analyze the effects of highaltitude exposure. In a longitudinal study, we analyzed oxi-dative status-related parameters in lymphocyte samples col-lected prior to, soon after, and 6 months after a high altitudeexpedition; we also assessed cell Ca2þ balance and mito-chondrial function, because these two mechanisms are strictlyrelated to oxidative status.

Our overall goal was to determine if high altitude inducesoxidative stress and dysfunction by utilizing peripheral bloodlymphocytes as biosensors of the whole-body response and asa model for adaptation of the organism to environmentalmodifications.

Materials and Methods

Subjects and the expedition

Seven healthy, male, nonsmoking climbers were enrolled inthe study; their characteristics are summarized in Fig. 1. Thehealth conditions of climbers were assessed at the time of eachblood sampling. Written consent was obtained from eachparticipant, and the study was designed in accordance withthe recommendations of the Declaration of Helsinki and ap-proved by the Ethics Committee of the Gabriele d’AnnunzioUniversity of Chieti-Pescara, Italy.

The climbers took part in the INTERAMNIA 8000–MANASLU 2008 expedition that started from Rome (sealevel: SL) on September 8, 2008, and ended in the same city onOctober 20, 2008. The subjects had not experienced high alti-tude conditions within a period of at least 6 months prior tothis expedition. The expedition timetable and altitude profileare shown in Fig. 2.

After landing in Kathmandu, Nepal (*1300 m above SL),on September 9, 2008, climbers began the ascent phase onSeptember 11, gradually reaching the Manaslu Base Camp(5000 m above SL) on September 21. During this 10-day pe-riod, climbers covered approximately 25 km/day and aver-aged about 7 h of trekking each day.

Climbers remained at the base camp for 21 days, duringwhich symptoms of acute mountain sickness were insignifi-cant and decreased after a few days; no climber needed anymedication. Throughout the expedition, balanced food intakeand ad libitum fluid ingestion were allowed. Antioxidantvitamins were not administered before or during the expe-dition. When the meteorological conditions were favorable,the climbers reached Camp 1 (5900 m above SL) and Camp 2(6400 m above SL), covering approximately 8 to 10 km/day inan average 6 h/day of trekking. During the period at the basecamp, the climbers busied themselves attending to their per-

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334 MARIGGIO ET AL.

sonal needs, rearranging tents, and maintaining the camp.Nepalese Sherpa packers and base-camp personnel supportedthe expedition; however, no data were collected from theseNepalese citizens.

On October 19, the subjects reached Kathmandu and leftthe following day. After landing in Rome on October 20,climbers were transferred to Chieti, Italy, for health evalua-tion. Since the logistics and the equipment of the expeditioncould not ensure safe, controlled, and standardized time-course blood withdrawals, serum isolation, and sample stor-age, each subject underwent blood withdrawal before, soonafter (on October 20, once arrived in Italy), and 6 months afterthe expedition (follow-up).

Materials

All media, sera, antibiotics, and culture solutions werepurchased from Gibco BRL (Paisley, Scotland, UK). All sterileculture plastics were obtained from Falcon (Plymouth, UK).All other reagents were analytical grade.

Isolation of Serum

Sera were isolated from peripheral blood samples, drawnfrom each subject before and after the expedition, using Ter-umo test tubes (Venosafe Serum Gel VF-106SAS, TerumoEurope NV Laboratory Systems, Rome, Italy). Blood sampleswere centrifuged at 3500 rpm for 15 min. Each serum samplewas collected and frozen at �808C prior to biochemicalmeasurements.

Isolation of Human Lymphocytes

Peripheral venous blood samples from climbers were col-lected in sodium-heparinized vacutainers. Peripheral bloodlymphocytes were separated under sterile conditions on aFicoll-Histopaque 1077 (SIGMA, Milan, Italy) gradient usingthe Boyum method (Boyum, 1976). Aliquots of heparinizedwhole blood, diluted with an equal volume of Dulbecco’sphosphate-buffered saline (1:1), were gently applied to anequal volume of Ficoll-Paque PLUS in centrifuge tubes.Samples were centrifuged at 400�g for 30 min, and the re-sultant interface (buffy coat) was carefully aspirated from the

gradient and washed twice in Dulbecco’s phosphate-bufferedsaline by centrifugation at 200�g for 10 min. The cell pelletwas resuspended in RPMI 1640 medium supplemented with10% (v/v) fetal bovine serum (FBS), 2% (w/v) l-glutamine,and 1% (w/v) penicillin/streptomycin. Monocytes were re-moved from the mononuclear fraction by adherence to Petridishes during overnight incubation at 378C. Purified lym-phocytes were resuspended in complete RPMI 1640 mediumat a density of 1 to 2�106 cells/mL and used in experimentalanalyses within 2 days after isolation. Cell viability was de-termined by Trypan Blue dye exclusion.

Markers of Oxidative Stress

Serum

Total antioxidant status. Because of interactions amongantioxidants and difficulties in measuring each antioxidantcomponent separately, methods were developed to assessthe total antioxidant status of serum or plasma. For thispurpose, we used the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity(TEAC) assay, which is a widely used kit-based commercialmethod. This assay is based on suppression of the absorbanceof radical cations of 2,20-azino-di-(3-ethylbenzthiazoline sul-phonate) (ABTS) by antioxidants in the test sample whenABTS is incubated with a peroxidase (metmyoglobin) andhydrogen peroxide (H2O2) (Rice-Evans and Miller, 1994).Assays were performed as described by the manufacturerof the kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly,10-mL aliquots of serum was added in duplicate to 10mL ofmetmyoglobin and 150mL of a chromogen solution, and re-actions were initiated by the addition of 40mL of H2O2. Reac-tion mixtures were incubated for 3 min at room temperatureand read using a Victor3 microplate reader (PerkinElmer,Waltham, MA, USA). TEAC values in samples were deter-mined by reference to a linear calibration curve prepared usingpure Trolox-containing reactions (range: 0–0.33 mmol/L).

Thiobarbituric acid-reactive substances. The measure-ment of thiobarbituric acid-reactive substances (TBARS) is awell-established method for detecting lipid peroxidation(Yagi, 1998). We used TBARS Assay Kit, which allows rapid

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HIGH ALTITUDE AND LYMPHOCYTE RESPONSES 335

photometric detection at 532 nm of the thiobarbituric acid–malondialdehyde (TBA-MDA) adduct, as described by themanufacturer (cat. 10009055; Cayman Chemical). In brief,100 mL of serum was added in duplicate to 100mL of sodiumdodecyl sulfate (SDS) and 4 mL of color reagent. Reactionmixtures were then incubated for 1 h in boiling water andcentrifuged at 1600�g for 10 min at 48C. After warming for5 min at 258C, samples were read on a Lambda25 spectro-photometer (PerkinElmer). Values for samples were calcu-lated from a linear calibration curve prepared using pureMDA-containing samples (range: 0–50 mmol/L).

Protein carbonyl content. A colorimetric assay (ProteinCarbonyl Assay Kit, cat. 10005020, Cayman Chemical) wasused to evaluate oxidized proteins (Levine et al., 1994), asdescribed by the manufacturer. In brief, each serum samplewas treated in duplicate with 2,4-dinitrophenylhydrazine(DNPH) dissolved in 2.5 mol/L HCl. The formation of a Schiffbase between protein carbonyls and DNPH produced thecorresponding hydrazones, which could be isolated froma 20% trichloroacetic acid and ethanol–ethyl acetate (1:1) so-lution by centrifugation at 10,000�g for 10 min at 48C.Hydrazone-containing pellets were redissolved in guanidinehydrochloride and read at 370 nm using a Victor3 microplatereader (PerkinElmer), as described by the manufacturer. Theabsorbance of samples treated with 2.5 mol/L HCl was sub-tracted from that of DNPH-treated samples, and the correctedvalues thus obtained were used to determine the concentra-tion of protein carbonyls (e¼ 22,000 (mol/L)�1cm�1). Valueswere then normalized to the total protein concentration in thefinal pellet (OD280) to take into account protein loss during thewashing steps, as suggested in the kit manual.

Vitamin determinations. A high-performance liquid chro-matographic (HPLC) method was used to measure vitaminsA and E and b-carotene levels in serum using wavelength-programmed, ultraviolet–visible, absorbance detection. Afterde-proteinization with ethanol, a 200-mL aliquot of plasmafrom each subject, containing tocopherol acetate as an internalstandard, was extracted with butanol–ethyl acetate. Sodiumsulfate was added for dehydration. Analytes of extracted sam-ples were found to be stable for at least 4 days. A 10-mL aliquotof each organic extract was used for HPLC analysis. The mo-bile phase was methanol:butanol:water (89.5:5:5.5, v/v) andthe flow rate was 1.5 mL/min. Analytes of interest were wellseparated from other plasma constituents within 22 min at458C. The lowest detection limits of vitamins A and E andb-carotene were 0.02, 0.5, and 0.1mg/mL, respectively. The re-covery and reproducibility of the method were *90%. Themethod is sensitive, specific, and appropriate for epidemiolog-ical studies and routine determination of vitamin deficiency(Lee et al., 1992).

Isolated lymphocytes

Determination of H2O2 production. H2O2 generation inlymphocytes from climbers was assayed before and after the21-day period in the high altitude environment using a col-orimetric method involving the oxidation of iodide in thepresence of ammonium molybdate, with photometric analy-sis of the resulting blue starch–iodine complex at 570 nm(M’Bemba-Meka et al., 2005). Briefly, human blood lympho-cytes were treated with 38.5 mmol/L HCl, 80 mmol/L po-

tassium iodide, 80 mmol/L ammonium molybdate in H2SO4,and 0.38% (w/v) starch. Twenty minutes after adding po-tassium iodide, sample absorbance was measured at 570 nmusing a SpectraMax 190 microplate reader (Molecular De-vices, Sunnyvale, CA, USA). The H2O2 concentration wasestimated using a standard curve. Results are expressed asmilligrams H2O2 per 15�104 cells. For each experimentalcondition (pre- and postexpedition and follow-up) and foreach sample derived from any subject, at least five wells wereanalyzed.

Determination of mitochondrial potential. Mitochondrialmembrane potentials were determined using JC-1 (5,50,6,60-tetrachloro-1,10,3,30 tetraethylbenzimidazolylcarbocyanineiodide/chloride; Molecular Probes, Eugene, OR, USA), acationic carbocyaninic dye that accumulates in mitochondria.When the transmembrane potential is high, as in normal cells,JC-1 forms dimers ( J-aggregates) that emit red fluorescence;when the potential is low, an index of oxidative stress, the dyeforms monomers that emit green fluorescence, and there isa concomitant decrease in red fluorescence. The red:greenfluorescence ratio depends on the mitochondrial membranepotential and not on other factors, such as mitochondrial size,shape, or density, that might influence single-componentfluorescence signals (Molecular Probes).

Isolated lymphocytes were loaded with 5 mg/mL JC-1 for15 min at 378C in growth media. At the end of the incubation,the cells were centrifuged, washed, and resuspended in nor-mal external solution [NES: 140 mmol/L NaCl, 2.8 mmol/LKCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, 10 mmol/L glucose,10 mmol/L 4-(2-hydroxyethyl)1-(piperazineethanesulfonicacid (HEPES); pH 7.3] and seeded at 5�103 cells/well inspecial-optics 96-well plates (Corning-Costar, Milan, Italy).Fluorescence was detected on a Gemini Spectramax XS fluo-rescence plate reader (Molecular Devices) using an excitationwavelength of 485 nm and recording the emissions of the JC-1monomer and aggregate at 530 and 590 nm, respectively. Foreach experiment, the aggregate/monomer (red/green) ratioswere plotted. For each experimental condition (pre- andpostexpedition and follow-up) and for each sample derivedfrom any subject, at least five wells were analyzed.

Glutathione peroxidase activity. Glutathione peroxidaseactivity was measured in samples derived from sonicatedlymphocytes suspended in 100 mmol/L phosphate buffer(pH 7) containing 1.5 mmol/L dithiothreitol. Protein concen-trations were determined using Protein Assay Kit (Bio-RadLaboratories Srl, Milan, Italy) (Bradford, 1976), using bovineserum albumin (BSA) as a standard. Enzymatic activity wascalculated according to the method of Paglia and Valentine(1967). The oxidation of nicotinamide adenine dinucleotidephosphate (NADPH) was monitored at 340 nm and 258C. Oneunit of enzymatic activity was defined as the oxidation of1 mmol NADPH/min.

Ca2þ Signaling Analysis

The intracellular Ca2þ content was monitored using theCa2þ-sensitive fluorescent indicator Fura-2-AM (MolecularProbes), and an inverted Olympus microscope connected to ahigh-speed wavelength switcher (Polychrome II, Till Photo-nics, Germany), equipped with a 75-W stabilized xenon lamp(Ushio Inc., Cypress, CA, USA) and a cooled, charge-coupled

336 MARIGGIO ET AL.

device (CCD) camera (C6790 model; Hamamatsu Photonics,Hamamatsu, Japan). Isolated lymphocytes (1�105 cells/mL)were loaded in suspension with 5 mmol/L Fura-2-AM (fura-2-acetoxymethyl ester) for 30 min at 378C in NES, supple-mented with 1% (w/v) BSA. Cells were centrifuged at 400�gfor 10 min and washed twice to remove extracellular dye.Next, cells were resuspended in fresh NES, transferred tospecial-optics 96-well plates (Corning-Costar) coated withpoly-l-lysine, and maintained for 10 min at room temperatureto allow adhesion before image acquisition.

Fura-2-AM-loaded lymphocytes were sequentially and re-petitively excited at 340 and 380 nm; fluorescence imageswere acquired with a CCD camera and stored on an interfacedcomputer. The acquisition time was one image ratio per sec-ond. The image ratio calculations were carried out pixel bypixel on a pair of corresponding 340- and 380-nm image files.The temporal plots (mean value of the fluorescence signal in aselected cellular area) were calculated from the image ratios.[Ca2þ]i in a single cellular field, recorded by a [Ca2þ] cali-bration plot of the 340/380 ratio, was calculated using Cal-cium Calibration Kit for video imaging (Molecular Probes)(Pietrangelo et al., 2002). For each experimental condition(pre- and postexpedition and follow-up) and for each samplederived from any subject, at least five different wells wereanalyzed

Statistical Analysis

Statistical analyses, where indicated, were performed usingPrism 4 for Windows (GraphPad Software Inc., San Diego,CA, USA). The data are presented as means� SEM, as spec-ified in the figure legends. Comparisons between groups weremade using t-tests.

Results

The first results to emerge from the current study wereobtained during isolation of lymphocytes from blood sam-ples. Counts of isolated lymphocytes showed an increasein cell number in samples collected from climbers after the 21-day period of high altitude exposure relative to samplesisolated before the expedition. Lymphocyte cell number re-turned to preexpedition values in samples obtained in thefollow-up (Fig. 3).

Three markers of oxidative stress, total antioxidant status,lipid peroxidation, and protein oxidation, were analyzed insera isolated before and soon after the expedition, as de-scribed in Materials and Methods. These results are summa-rized in Fig. 4, which shows that none of these parameters wassignificantly different between pre- and postexpedition con-ditions, although lipid peroxidation levels measured afterhigh altitude exposure were slightly higher than those mea-sured at sea level.

In the same serum samples, vitamins A, E, lycopene, andb-carotene were analyzed before and soon after the expedi-tion. As shown in Fig. 5, values obtained after the expeditionwere not significantly different from those measured before theexpedition, although the mean values found at the end of highaltitude exposure did trend lower than preexpedition values.

The oxidative machinery was also assessed in lymphocytecell populations by analyzing (1) live-cell H2O2 productionand release, (2) live-cell mitochondrial potential, and (3) glu-tathione peroxidase activity in cell extracts. Figure 6A showsthat release of H2O2 caused by cell oxidative activity wasunchanged in samples collected soon after or 6 months afterthe end of the expedition compared with that in preexpeditionsamples. The enzymatic activity of glutathione peroxidase, amajor antioxidant enzyme, was also unchanged (Fig. 6C).Interestingly, mitochondrial membrane potential was signif-icantly reduced in lymphocytes isolated soon after and 6months after the end of the expedition compared with basal(preexpedition) values (Fig. 6B).

[Ca2þ]i was assayed in the same cell populations by video-imaging analysis of single cells. These assays revealed thatmean basal [Ca2þ]i was significantly higher in cells isolatedsoon after the expedition, but had returned to preexpeditionvalues in cells collected at the 6-month follow-up (Fig. 7).

The effect of the high altitude expedition on lymphocyte[Ca2þ]i homeostasis was also confirmed by monitoringspontaneous intracellular Ca2þ oscillations in the same cell

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HIGH ALTITUDE AND LYMPHOCYTE RESPONSES 337

populations (Fig. 8A). In lymphocytes assayed before the ex-pedition, spontaneous Ca2þ waves were recorded in about26%� 1.6% of cells; this percentage increased to 44%� 2.0%and 38%� 5.0% soon after and 6 months after the end of theexpedition, respectively (Fig. 8B).

Changes in [Ca2þ]i in response to an oxidant stressor(H2O2) were also monitored by video imaging to assay thepossible involvement of intracellular Ca2þ variation in theoxidative stress machinery. Addition of H2O2 (1 mmol/L)induced an increase in [Ca2þ]i in a subpopulation of cells. Inlymphocytes isolated before the expedition, about 30%� 2.7%of cells were H2O2 responsive; this percentage trendedslightly higher (36%� 5.0%) in lymphocytes isolated soonafter the expedition and significantly increased to 46%� 6.0%in cells isolated at the 6-month follow-up (Fig. 9).

Discussion

It is well known that a high altitude environment can affectvarious organ systems, notably the nervous, cardiovascular,and pulmonary systems, and the effects reflect modificationsat the cellular level (Grocott et al., 2007; Hainsworth andDrinkhill, 2007; Hainsworth et al., 2007; Wilson et al., 2009).These effects result from the simultaneous influence of dif-ferent environmental parameters, such as absolute altitude,humidity, temperature, climatic conditions, and duration ofexposure. In addition, the level of physical activity needs to betaken into account. For these reasons, it is often difficult orunrealistic to attempt to directly correlate experimental re-sults with a specific environmental or physical parameter.Accordingly, our goal was to provide a partial picture of thehuman response to such an unfavorable environment.

We used peripheral blood lymphocytes as biosensors of thewhole-body response to high altitude and as a model foradaptation of the organism to environmentally inducedmodifications. Peripheral lymphocytes exhibit a number ofimportant features. From an experimental point of view,primary lymphocytes can be easily and repeatedly collected.In addition, lymphocytes not only participate in inflammationmechanisms or as part of the immune response, but alsoconstitute an important system that contributes to organ ho-meostasis and adaptation to pathological conditions and newenvironments (Walsh and Whitham, 2006; Zhang et al., 2007;Belia et al., 2009).

In addition, blood lymphocytes have also been used asbiosensors of whole-body exposure to hypoxia, radiation, andphysical activity, all challengers characterized by oxidative-stress-related phenomena (Niess et al., 1996; Moller et al.,2001; Lee et al., 2007). Consistent with previous reports(Klokker et al., 1993; Walsh and Whitham, 2006), we found anincrease in lymphocyte number after high altitude exposurethat returned to preexpedition values at the 6-month follow-up. Conversely, some authors found different responsesin blood cell number and activation to hypobaric hypoxia,

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338 MARIGGIO ET AL.

exercise, or both of these challengers and hypothesized thatthe observed effects depend on the cellular subpopulationsand individual sensitivity to these stimuli (Chouker et al.,2005; Shtemberg et al., 2007; Wang and Lin, 2010). It is wellknown that lymphocytosis has resulted from different envi-ronmental conditions, such as variable physical activity lev-els, time exposure, and hypobaric hypoxia, probably owing tothe mobilization of cells from the secondary lymphoid organsin response to stress (Hong et al., 2005).

It is possible to assess the functional status of lymphocytesunder different conditions by evaluating cellular oxidativestatus and Ca2þ dynamics, which are important in regulatingthe functions of these cells and their ability to communicatewith other blood cells (Li, 2008; Feissner et al., 2009). A reliableassessment of oxidative stress or damage in biological speci-mens requires a battery of assays that monitor oxidative sta-tus, associated changes in [Ca2þ]i, and the levels of oxidativelymodified macromolecules (Halliwell and Whiteman, 2004;Belia et al., 2009). As extensively reviewed by Hwang andKim (2007), assays of malondialdehyde and protein carbonylcontent are among the most widely used to determine lipidperoxidation and oxidative damage to proteins, respectively.Further, because ROS removal rate is controlled primarily by

Mean value of intracellularbasal Ca2+ levels

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*** *

cell

per

cen

tag

e

A

B

FIG. 8. Spontaneous Ca2þ waves in isolated lymphocytes:(A) representative traces depicting the spontaneous activityin a single cell; (B) summary data showing the percentages ofcells isolated before (Pre: n¼ 268 tested cells from 7 climb-ers), soon after (Post: n¼ 241 tested cells from 7 climbers),and 6 months after (Follow-up: n¼ 129 tested cells from 7climbers) the expedition that displayed Ca2þ waves. Thedata are expressed as means� SEM. *p< 0.05, ***p< 0.001vs. Pre.

Cell response to H2O2

0 3 6 9 120

100

200

300

400

500

600

70A

B

0

1 mMH2O2

min

[Ca2+

]i (

nM

)

H2O2-responsive lymphocytes

Pre Post Follow-up0

20

40

60

80

100

*

cell

per

cen

tag

e

FIG. 9. H2O2-induced changes in [Ca2þ]i in isolated lym-phocytes: (A) representative trace depicting the response of asingle cell to the addition of 1 mmol/L H2O2; (B) summarydata showing the percentages of cells isolated before (Pre:n¼ 193 tested cells from 7 climbers), soon after (Post: n¼ 187tested cells from 7 climbers), and 6 months after (Follow-up:n¼ 203 tested cells from 7 climbers) the expedition showing[Ca2þ]i increases after the addition of 1 mmol/L H2O2 to themedium. The data are expressed as means� SEM. *p< 0.05vs. Pre.

HIGH ALTITUDE AND LYMPHOCYTE RESPONSES 339

a variety of low-molecular-weight antioxidants, and consid-ering that the net antioxidant effect reflects cooperationamong a variety of enzymatic and nonenzymatic compoundswith antioxidative properties, it is important to measure totalantioxidant status in tissues and body fluids (Rice-Evans andMiller, 1994). Our data revealed that serum and lymphocytemarkers that correlate with oxidative stress, tested here, werenot significantly changed after hypobaric hypoxia exposure.These findings seem to contrast with other published resultsthat show the activation of ROS generating systems and theweakening of enzymatic and nonenzymatic antioxidant sys-tems, depending on the degree of altitude and time exposure(Dosek et al., 2007). The high altitude-induced oxidative un-balance can occur in different tissues. Indeed, this alterationmay have relevance for the high altitude-induced microcir-culatory dysfunction, as well as for pulmonary and cerebraledema (Gonzalez and Wood, 2001; Serrano et al., 2003). Themacromolecular damage resulting from the hypobaric hyp-oxia-induced oxidative unbalance appears to be tissue spe-cific. It has been reported that in rat skeletal muscleshypobaric hypoxia may induce lipid peroxidation and an in-crease of carbonyl derivatives, markers of oxidative proteindamage, that conversely appeared reduced in the rat brain(Radak et al., 1994; Radak et al., 1997; Radak et al., 1998). Inthe Operation Everest III study, Joanny et al. (2001) reportedthat the levels of plasma lipid peroxidation were increased by23% at simulated 6000 m and by 79% at simulated 8848 m,thus indicating that high altitude-induced oxidative stressis augmented as the severity of hypoxia increases. Anotherstudy showed that 3-month exposure to high altitude(4500 m) revealed increased lipid peroxidation and decreasedenzymatic and nonenzymatic defense, whereas a 13-monthsojourn induced normalization of redox balance (Vij et al.,2005).

In our study, we assayed samples before and soon after theexpedition and did not observe any persistent unbalancedoxidative mechanisms or damaged molecules; on the otherhand, we cannot exclude a possible transient increase in in-tracellular levels of ROS. Our data came from a real highaltitude experience, and not from simulated hypobaric hyp-oxia. In addition, our study is based on a high-mountainexpedition characterized by multiple parameters (such asgradual ascent and descent, moderate physical exercise,severe hypobaric hypoxia, and poor weather conditions) thatcould differently modulate the oxidative status of the climb-ers. The discordance between our results and those obtainedby Joanny and co-workers may result also from the hormeticresponse triggered by high-impact and long-lasting aerobicexercise practiced by the subjects during the expedition.Indeed, it has been previously demonstrated that recurringbouts of nonexhausting physical efforts were able to enhancethe main antioxidative protection systems and the activity ofcellular machinery involved in the repair and detoxificationmolecular processes, as reviewed by Radak and collegues(2008a).

The environmental conditions did, however, influenceboth [Ca2þ]i and mitochondrial functions of lymphocytes.After high altitude exposure, the mitochondrial potentialdecreased; this effect appeared associated with an increasein [Ca2þ]i, consistent with the strict linkage between lev-els of this ion and mitochondrial membrane permeability(Feissner et al., 2009). It is possible that this decrease inmitochondrial function reflects a compensatory response to

oxidative stress. Alternatively, because the oxidative statusof the cells was unchanged after high altitude exposure,this decrease might be independent of the oxidative stressmachinery. Surprisingly, the lymphocyte mitochondrialmembrane potential persisted at lower than preexpeditionvalues during the follow-up period, a time when basal[Ca2þ]i had returned to mean preexpedition values. Inter-estingly, the spontaneous Ca2þ wave activity of lympho-cytes and their sensitivity to H2O2-induced increases in[Ca2þ]i appeared to remain elevated at the 6-month follow-up point. During the 6-month period after the expedition,all subjects were still healthy, did not undergo any phar-macological therapy or treatment, and led a normal dailyworking life. For this reason, we cannot exclude that aposthypoxic reoxygenation process could be involved ininducing these results.

Because lymphocytes have a relatively long life-span, thecells are useful substrates for monitoring the incidence ofcumulative, long-lasting stimuli (Balakrishnan and Rao, 1999;Gregoire et al., 2006). In addition, circulating lymphocytesexperience tissue-specific and organ-function modificationsinduced by the environment. Over the lifetime of peripherallymphocytes, transient responses of some parameters to ex-pedition conditions recovered during the follow-up period.However, the cumulative impact of the environmental stimulicontinued to be evident in certain residual modifications oflymphocyte function, here represented by an increase inspontaneous Ca2þ waves and enhanced cellular responses tothe oxidative stressor H2O2.

Conclusions

The results obtained here reflect the aggregate responses oflymphocytes to the collective conditions that define the 21-day, high altitude expedition, including the effects of hypoxia,hypobaric pressure, temperature, humidity, and physical ac-tivity. In this study, high altitude conditions did not have aspecific effect on global oxidative status as assayed by serumand lymphocyte markers. However, lymphocyte functionsdid show sustained effects of exposure, as revealed by chan-ges in [Ca2þ]i and Ca2þ signaling dynamics, as well as inmitochondrial membrane potential. Collectively, these find-ings suggest that mitochondria are the targets or source of theincreased intracellular Ca2þ recorded in lymphocytes soonafter the expedition. The importance of these observations liesin the fact that mitochondria represent the main metabolicsystem of the cell and serve a significant intracellular Ca2þ

buffering function.

Acknowledgments

We wish to thank the seven climbers who enthusiasticallyparticipated in this study. In addition, the authors wish tothank Prof. G. Fano, director of the Department of Basic andApplied Medical Sciences, for having sponsored the expedi-tion and for helpful discussion of the results presented here.This work was also supported by research funds to Mariggioand Amicarelli.

Disclosures

The authors have no conflicts of interest or financial ties todisclose.

340 MARIGGIO ET AL.

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Address correspondence to:Maria A. Mariggio, PhD

Section of Physiology and Pathology of the Nervous SystemDepartment of Basic and Applied Medical Sciences

Gabriele d’Annunzio University of Chieti-PescaraVia dei Vestini 31, 66013 Chieti, Italy

E-mail: mariggio@unich.it

Received December 18, 2009;accepted in final form June 2, 2010.

342 MARIGGIO ET AL.