Effects of dietary oxidized fish oil and deficiency of anti-oxidants on the immune response of turbot, Scophthalmus maximus

Free Radical Biology & Medicine, Vol. 26, Nos. 1/2, pp. 184 –192, 1999 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(98)00192-0

Original Contribution EXPRESSION OF THE ANTIOXIDANT STRESS PROTEIN HEME OXYGENASE-1 (HO-1) IN HUMAN LEUKOCYTES Acute and Adaptational Responses to Endurance Exercise ANDREAS MICHAEL NIESS,* FRANK PASSEK,† INGRID LORENZ,‡ ELISABETH MARION SCHNEIDER,§ HANS-HERMANN DICKHUTH,* HINNAK NORTHOFF,† and ELVIRA FEHRENBACH† *Medical Clinic und Policlinic, Department of Sports Medicine, University of Tuebingen, Tuebingen, Germany, †Department of Transfusion Medicine, University of Tuebingen, Tuebingen, Germany, ‡Immunology Laboratory, Institute of Hemostasis and Transfusion Medicine, University of Duesseldorf, Dusseldorf, Germany, and §Experimental Anaesthesiology, University of Ulm, Ulm, Germany (Received 30 March 1998; Revised 6 July 1998; Accepted 6 July 1998)

Abstract—Inducible heme oxygenase (HO-1) is an antioxidant stress protein, that is mainly induced by reactive oxygen species (ROS), cytokines and hyperthermia. By using flow cytometry the present investigation demonstrated a rise in the cytoplasmic expression of HO-1 in lympho- (L), mono- (M) and granulocytes (G) of 9 endurance-trained male subjects after a half marathon run. The expression was more pronounced in M (median: 98.3% HO-1 positive cells/4.31 mfc) and G (94.8%/1.93 mfc) than in L (80.1%/ 1.51 mfc) when measured 3 h post-exercise. Additionally the exercise protocol caused a rise in the plasma levels of myeloperoxidase, TNFa and interleukin-8 (IL-8), indicating an inflammatory response. We could detect a correlation between IL-8 and HO-1, directly after exercise, that was apparent in G (r 5 0.67, p , .05) and L (r 5 0.80, p , .05), but did not reach significance in M (r 5 0.65, p 5 0.06). An additional detection of HO-1 at rest in 12 untrained subjects showed a higher baseline expression of HO-1 compared to the athletes. The regulatory pathways leading to an increased expression of HO-1 after endurance exercise are not completely clear, but a causal involvement of a cytokine-mediated generation of ROS must be discussed. We supposed that the down-regulation of the baseline expression of HO-1 in athletes reflects an adaptional mechanism to regular exercise training. © 1998 Elsevier Science Inc. Keywords—Exercise, Heme oxygenase, Oxidative stress, Leukocytes, Myeloperoxidase, Cytokines, IL-8, TNFa, Flow cytometry, Free radical

INTRODUCTION

kocytes themselves and are assumed to be induced in part by oxidative stress. On the other hand ROS are involved in modulation of gene expression by activating transcription factors, a mechanism that may also signal adaptive responses to exercise [9] and augment stress tolerance. Nutritive antioxidants as well as antioxidant enzymatic systems, including superoxide dismutase, catalase and glutathione peroxidase, play an important role in lowering the prooxidant state of the cell and therefore are also responsible for the maintenance of cell viability and function in leukocytes. Tolerance to oxidative stress is also provided by stress proteins such as the inducible heme oxygenase (HO-1) [10,11], a 32 kD enzyme, that expression is up regulated during exposure to oxidants, UV-A irradiation and a series of agents including cyto-

Heavy physical exercise has been shown to induce an acute response of the immune system, including an activation of inflammatory cells [1,2]. Immunological reactions lead to an augmented generation of reactive oxygen species (ROS) by leukocytes, a mechanism that is partly mediated by cytokines such as plasma interleukin-8 (IL-8) and tumor necrosis factor a (TNFa) [3,4,5]. Exercise-induced changes such as depressed proliferation of lymphocytes [6], impaired neutrophil function [7] or DNA damage [8] have been shown to occur in leuAddress correspondence to: Andreas M. Niess, Medizinische Klinik und Poliklinik, Abteilung Sportmedizin, Universita¨t Tu¨bingen, Ho¨lderlinstr. 11, D-72074 Tu¨bingen, Germany; Fax: 10049-7071-295162; E-Mail: [email protected]. 184

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kines, hormones, heme and heavy metals [12,13]. The important role of HO-1 in the antioxidant defense arises from an induction of ferritin synthesis, diminishing the cellular pool of free iron [14], and also from enhancement of bilirubin, that is known to be a potent antioxidant [15]. The effect of physical exercise on the expression of HO-1 has not been previously investigated in humans. Recently Essig et al., [16] demonstrated increased mRNA levels of HO-1 in rat muscle following repeated contractions. However, to date, no investigation has focused on whether an exercise-induced expression of this stress protein occurs in immunocompetent cells. It has been shown that acute endurance exercise induces various cellular defense systems, including antioxidant enzymes [17]. Furthermore regular training improves exercise tolerance, that is partly provided by a chronic adaptation of antioxidant enzymes [18-21]. Recognizing that strenuous physical exercise leads to an immunological response that is associated with augmented generation of ROS, we questioned whether heavy endurance exercise is capable of increasing the expression of the cytoplasmic HO-1 in leukocytes. Furthermore we intended to verify whether the regulation of the basal cytoplasmic HO-1 expression exhibits adaptation due to regular endurance training. In the present study we used a competitive half marathon run as an endurance exercise stress model. Blood samples of moderately-trained athletes were drawn at rest and at three time points after the run. The cytoplasmic expression of HO-1 was analyzed separately in mono-, lympho- and granulocytes by an indirect immunofluorescence method using flow cytometry. The cytokine response and the activation of neutrophils were assessed by measuring IL-8, TNFa and myeloperoxidase (MPO). Additionally cytoplasmic HO-1 was determined in a control group of untrained men at rest to examine the influence of regular endurance training on the baseline expression. MATERIALS AND METHODS

Subjects Ten endurance-trained (weight 64.4 6 3.7 kg; height 175.0 6 3.3 cm) and 12 inactive healthy male subjects (weight 75.9 6 3.2 kg; height 176.7 6 2.8 cm) gave informed consent to participate in the study. The trained individuals were engaged in specific endurance training and reported a regular running volume of 4.33 6 1.7 h z wk21 over a minimum of 2 years. The untrained individuals did not perform any kind of sports conditioning and devoted less than 2 h z wk21 to recreational and occupational physical activity. All subjects were non-

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smokers with normal dietary habits and did not take any medication or vitamin supplements. Experimental design The protocol of the study was approved by our institute’s Ethics Committee, and conformed to the guidelines of the Helsinki Conference for research on human subjects. The trained volunteers participated in an official half marathon competition (21.1 km, running time 89.9 6 10.6 min), that started at 10:00 AM. No intensive or prolonged training sessions were absolved within the last four days before the race. Venous blood samples were taken in a sitting position using EDTA as an anticoagulant at rest (09:00 AM), 0 (11:30-12:00 AM), 3 (2:30-3:00 PM) and 24 h (09:00 AM) after the half marathon. One of the volunteers did not finish the half marathon due to a knee injury, his post-exercise values were excluded from the study. Additionally blood was collected from 12 untrained subjects at rest (09:00 AM). Whole blood aliquots for flow cytometry and determination of complete blood cell counts were kept at room temperature and the analytical procedures were started within 1 h after collection. 10 mL of whole blood was centrifuged immediately after sampling and plasma aliquots were stored at 270°C until further analysis of creatine kinase (CK), myeloperoxidase (MPO), tumor necrosis factor a (TNFa) and interleukin-8 (IL-8). Capillary blood for lactate measurement was obtained from the earlobe before and directly after the run. Analytical methods Complete blood cell counts including hemoglobin, hematocrit and differential leukocyte counts were performed by an automated Coulter Counter (Cell Dyn 3500, Abbott, Delkenheim, Germany). The lactate concentrations of hemolyzed capillary blood were measured electrochemically using a lactate analyzer (EBIO, Eppendorf, Germany). Plasma CK activity was determined in our clinical laboratory routine (Hitachi 717, Boehringer, Germany). For determination of the plasma MPO concentrations we used an enzyme-linked immunoassay method (Myeloperoxidase ELISA Kit, Calbiochem, Germany) with a detection limit of 1.5 ng z mL21. Plasma levels of TNFa (R&D Systems, Minneapolis, USA), detection limit 1.0 pg z mL21, and IL-8 (Genzyme, Duoset, Cambridge, USA), detection limit 1.5 pg z mL21, were also assessed by ELISA. All post-exercise values of plasma parameters were adjusted for changes in plasma volume according to Dill and Costill [22]. The cytoplasmic expression of HO-1 in lympho-, mono- and granulocytes was assessed by flow cytometry.

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Table 1. Differential Leukocyte Counts at Rest, 0, 3, 24 and 48 h After the Half Marathon (n 5 9) presented as Median (Minimum–Maximum) Values Rest 21

Neutrophils (10 z L ) Lymphocytes (109 z L21) Monocytes (109 z L21) 9

a

3.2 (2.0–4.6) 2.2 (1.6–3.0) 0.8 (0.2–0.9)

10 h

13 h a

10.1 (5.0–15.6) 2.2 (1.01–4.2) 0.7 (0.4–1.5)

124 h a

13.3 (8.9–15.4) 1.0 (0.7–1.4)a 1.3 (0.9–1.3)a

4.6 (2.3–6.1) 2.4 (1.4–3.3) 0.9 (0.6–1.4)

148 h a

3.7 (2.7–8.5) 2.7 (1.7–3.1) 0.4 (0.3–0.6)

Denotes significant changes compared to resting values ( p , .05).

Five mL of EDTA-blood were layered above 5 mL of Lymphoflot (Biotest, Dreieich, Germany) and settled down for 60 min by gravity without centrifugation. The overlay was removed and the cell concentration adjusted with PBS to 1 3 107 cells z mL21. One hundred mL of the suspension was used for the flow-cytometric analysis. The leukocytes were analyzed by indirect immunofluorescence using the HO-1 specific antibody OSA-100 (Heme oxygenase-1, rabbit polyclonal antiserum, StressGen Biotechnologies, Canada). Cells 1 3 106 were first fixed at room temperature in a solution containing formaldehyde, then permeabilised according to the manufacturer’s instructions (Fix & Perm kit, An der Grub, Vienna, Austria) and at the same time incubated with the primary HO-1 specific antibody for 15 min. After washing the labeled cells twice and incubating in the presence of the second fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit F(ab9)2 IgG antibody (Dianova, Hamburg, Germany) for 20 min, the cells were analyzed using the flow cytometer EPICS-XL-MLC (Coulter, Krefeld, Germany). Negative controls were performed by using normal

rabbit serum (DAKO, Glostrup, Denmark) and the secondary antibody (Dianova, Hamburg, Germany). For routine phenotyping by two-color-immunofluorescence staining the cells were labeled with the following FITC/ PE-conjugated antibodies: CD45/CD14 and CD3/ CD16-56 (Becton-Dickinson, Germany). The lympho-, mono- and granulocyte populations were differentiated by size and granularity in the forward vs. side-scattergram and gated. Dead cells were excluded by electronic gating and fluorescence histograms were area-corrected to 10,000 cells. Data are presented as percent positive cells (%) and mean fluorescence channel (mfc), corrected for background fluorescence with the negative controls (Fig. 2). Statistics All statistical analyses and descriptive methods were computed by the statistical software package JMP (SAS Institute Inc., Cary, USA) for Macintosh computer. Data were expressed as median and minimum/maximum values. The presentation of the data was performed using a

Fig. 1. Plasma levels of interleukin-8 (IL-8) and myeloperoxidase (MPO) at rest, 0, 3, 24 and 48 h after the half marathon (n 5 9). 1 Denotes significant changes compared to resting values (p , .05).

Exercise-induced changes of HO-1

Fig. 2. Flow cytometric histograms (intracellular indirect immunofluorescence) of HO-1 positive lympho-, mono- and granulocytes at rest, 0, 3 and 24 h after the half marathon (Subject 9). The histograms depict HO-1 expression (right peak) of the cells vs. their negative controls (left peak).

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Fig. 3. Expression of HO-1 in lympho-, mono- and granulocytes at rest, 0, 3 and 24 h after the half marathon (n 5 9). The results are expressed in percent HO-1 positive cells. 1Denotes significant changes compared to resting values (p , .05).

quantile box-plot visualizing also the single values. Comparisons of repeated measurements in the trained subjects were tested for significance by the Wilcoxon signed ranks test. Differences of the resting values between the trained and untrained group were evaluated by the non-parametric test of Mann–Whitney. Pearson product moment correlations were calculated for plasma IL-8 vs. HO-1. A p-value of p , .05 was regarded as significant. RESULTS

Lactate values were significantly elevated (median 5.2 mmol z L1, range 2.8-7.8 mmol z L21) directly after the half marathon. The changes in the differential leukocyte counts are presented in Table 1. Neutrophil and monocyte counts rose significantly 3 h after the end of the half marathon, whereas lymphocyte counts exhibited a decrease 3 h post-exercise. Figure 1 depicts the significant increase in plasma myeloperoxidase (MPO) directly after exercise. Plasma CK activity rose from 51 (31-101) U z L21 at rest to maximum values of 244 (76-788) U z L21 24 h after exercise. Plasma TNFa was undetectable at rest but directly and 3 h after the half marathon we could find a rise above the detection limit of 1 pg z mL21 in 5 (1.5, 1.1-3.2 pg z mL21) of 9 subjects. Plasma IL-8 (Fig. 1) increased directly after exercise (p , .05), reaching baseline values in most of the subjects 3 h later. Analysis by flow cytometry revealed a significant increase of HO-1 positive leukocytes 0, 3 and 24 h after the half marathon. Figure 2 presents the flow cytometric histograms of HO-1 positive cells of Subject 9 at rest and three time points after the half marathon. Post-exercise percentage of HO-1 positive cells was more pronounced

in mono- and granulocytes than in lymphocytes (Fig. 3), whereas the relative increase directly after exercise was higher in lymphocytes. The number of HO-1 positive cells at rest was higher in mono- compared to granulocytes. HO-1 positive lymphocytes did not reach 2%, indicating that a baseline-expression of HO-1 was nearly undetectable in the lymphocytes of the trained subjects. The mean fluorescence channel (mfc) of HO-1, the second flow-cytometric parameter we used in the present study, mainly confirmed these results, but the values of the lymphocyte mfc declined already 3 h after the half marathon (Fig. 4 and 5). Significant correlations were revealed between the plasma concentrations of IL-8 and the cytoplasmic expression of HO-1 in lympho- and granulocytes directly after exercise, although this relation did not reach significance in monocytes (Table 2 and Fig. 6). The comparison of the baseline levels between athletes and untrained subjects revealed a significantly

Fig. 4. Expression of HO-1 in lympho- and granulocytes at rest, 0, 3 and 24 h after the half marathon (n 5 9). The results are expressed in mean fluorescence channel (mfc). 1Denotes significant changes compared to resting values (p , .05).

Exercise-induced changes of HO-1

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Table 3. Correlation Between Plasma IL-8 and the Expression of HO-1 in Lympho- (L), Mono- (M) and Granulocytes (G) Directly after the Half Marathon (n 5 9)

IL-8 vs. HO-1 (mfc) HO-1 (%) a

Fig. 5. Expression of HO-1 in monocytes at rest, 0, 3 and 24 h after the half marathon (n 5 9). The results are expressed in mean fluorescence channel (mfc). 1Denotes significant changes compared to resting values (p , .05).

lower percentage of HO-1 positive mono-, granulo- and lymphocytes in the active individuals (Fig. 7). No difference between the groups could be detected for the lymphocyte mfc of HO-1 at rest (Table 3). DISCUSSION

In the present investigation we could demonstrate a rise in the cytoplasmic expression of the antioxidant stress protein HO-1 after a half marathon run, that was significant in lympho-, mono- and granulocytes. Until now, exercise-related changes of the HO-1 expression have only been reported in one study, showing an increase of HO-1 mRNA levels in mouse muscle cells following repeated contractions [16]. Analysis by flow cytometry allows quantification of leukocytes, exhibiting

Cell type

p-value

L M G M

0.80a 0.65 0.67a 0.46

p , .05.

an expression of cytoplasmic HO-1 and the amount of HO-1 expressed in the cells. We could detect a rise in percent HO-1 positive cells as well as an increase in fluorescence intensity in all cell populations directly after exercise and both parameters remained elevated in a comparable manner 3 and 24 h after exercise in monoand granulocytes. By contrast the lymphocyte fluorescence intensity returned to baseline values within 3 h after exercise, although the percentage of HO-1 positive cells remained elevated. This discrepancy may reflect that the expression of HO-1 is induced by exercise in most of the lymphocytes, but the amount of HO-1 expressed is less compared to mono- and granulocytes. ROS have been shown to induce autoxidation and impair the function of leukocytes [11,23]. Other results indicate the occurrence of DNA damage in leukocytes after intensive endurance exercise [24], that is also attributed to oxidative stress. Several lines of data suggest that HO-1 provides protection against cellular oxidative damage [9], but the mechanisms of the protective effects are not completely known. The synthesis of both bilirubin and ferritin [14,25,26] seem to play an important role. Augmented levels of ferritin reduce the pool of intracellular iron and therefore decrease the iron-catalyzed generation of ROS via the Fenton-reaction. Free iron has been shown to increase in muscle tissue of rats after exhaustive treadmill running [27]. Additionally

Table 2. Expression of HO-1 in Lympho- (L), Mono- (M) and Granulocytes (G) at Rest of the Trained (n 5 10) and Untrained Subjects (n 5 12)

HO-1 (mfc)

Cell type

TR

UT

L M G

1.3 (1.3–1.5) 1.6 (1.5–2.0) 1.3 (1.2–1.4)

1.3 (1.2–1.5) 2.5 (1.7–3.1)a 1.5 (1.2–1.8)a

The results are expressed as mean fluorescence intensity (mfc) and presented as median (minimum–maximum) values. a Denotes significant differences between trained (TR) and untrained (UT) subjects ( p , .05).

Fig. 6. Relationship between plasma levels of interleukin-8 (IL-8) and the expression of HO-1 (percent HO-1 positive cells) in lympho- and granulocytes directly after the half marathon.

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Fig. 7. Expression of HO-1 in lympho-, mono- and granulocytes at rest in the trained (TR, n 5 10) and untrained subjects (UT, n 5 12). The results are expressed in percent HO-1 positive cells. #Denotes significant differences between trained and untrained subjects (p , .05).

HO-1 serves as the rate-limiting enzyme in the catabolism of heme to biliverdin, that is further converted to bilirubin. Bilirubin acts as an effective physiological antioxidant [15] and therefore contributes to the antioxidant defense system. Previous research could reveal the important role of HO-1 in the maintenance of the proliferative capacity as well as the survival of leukocytes [11] and it has to be discussed, whether its antioxidant function is also of significance during and after intensive endurance exercise. The exact mechanisms leading to an augmented cytoplasmic expression of HO-1 after exercise are not completely clear. At present influences of the exerciseinduced cytokine release as well as the involvement of ROS have to be discussed. Hyperthermia has been shown to enhance the expression of HO-1 in liver, kidney and heart of rats [28]. However as observed by Okinaga et al. [29], HO-1 is not inducible in human alveolar macrophages by heat shock due to a repressed heat shock element (HSE) in humans. Various investigations [12,13,30] could demonstrate that HO-1 is induced by agents, that are themselves ROS or are capable of generating active intermediates. Studies by Kurata et al. [31], revealed an oxidative activation pathway of the HO gene in monocytes exposed to H2O2. The more pronounced and prolonged expression of HO-1 in mono- and granulocytes compared to lymphocytes at rest and after exercise let us suggest that the expression of this antioxidant stress protein is affected by oxidative stress. Granulocytes generate O2•2, •OH, H2O2 and chlorinated compounds upon activation, a scenario that is known to occur also during intensive or prolonged exercise [7]. TNFa primes neutrophils for oxidant release in response to other stimuli and IL-8 plasma levels have been correlated with the generation of ROS by neutrophils [32-35]. After exercise we could observe TNFa

plasma levels slightly above the detection limit in 5 of 9 subjects, that is in accordance with earlier investigations [36]. A more pronounced exercise-induced increase was detected for IL-8, that exhibited elevated concentrations in all runners, reflecting an augmented inflammatory response. Additionally a rise in plasma MPO was found, indicating a systemic neutrophil activation and degranulation during the half marathon. The correlation between IL-8 and the expression of HO-1 in the granulocytes directly after exercise may confirm our assumption that, at least in granulocytes, a cytokine-mediated generation of ROS is responsible for the induction of HO-1. Several investigations [37,38] could reveal the role of zNO as a mediator of the expression of human HO-1 and therefore an involvement of zNO in the exercise-induced induction of HO-1 must also be taken into account. A more recent investigation confirm the assumption that exhaustive exercise is capable of increasing the expression of iNOS in human leukocytes [39]. Compared to mono- and granulocytes, the capability of lymphocytes to generate relevant amounts of ROS is lower [40]. Having regard to the mfc-values found, the expression of HO-1 in lymphocytes was only elevated directly after exercise and exhibited a correlation to the changes in IL-8. A direct stimulating effect of cytokines on the expression of HO-1 has been described for TNFa in liver cells [41]. It might be expected that such a more direct pathway is responsible for the exercise-related changes in the HO-1 expression in lymphocytes. The second major finding of the present investigation was a different baseline regulation of HO-1 in trained compared to untrained subjects. It is noteworthy that the endurance-trained individuals exhibit a lower expression of HO-1 at rest. This down-regulation of cytoplasmic HO-1 may be due to a lower prooxidant state of the cells in trained

Exercise-induced changes of HO-1

individuals at rest. Adaptation to regular endurance exercise involves a reduced neutrophil oxidative activity as shown by a decreased ability of circulating neutrophils to generate ROS after stimulation with phorbol myristate acetate [42,43]. These adaptional mechanisms may affect the amount of ROS already generated at rest. Furthermore an augmented activity of several antioxidant systems such as SOD, catalase and glutathione peroxidase caused by regular training [18-21], may mediate a downregulation of HO-1. Evidence exists that the cellular glutathione content influences in part the expression of HO-1 [44,45] and feeding of rats with ascorbic acid is capable of suppressing mRNA levels of HO-1 [46]. A recent study could show that complete refractoriness of HO-1 is apparent in human fibroblasts within 48 h after UV-A irradiation [47], a mechanism that may be also responsible for a lower expression of HO-1 in athletes exposed to regular exercise stress. Considered as a whole, we could show that the cytoplasmic expression of HO-1 is induced in human leukocytes after intensive endurance exercise. The pattern of the HO-1 expression, that is more pronounced in monoand granulocytes than in lymphocytes suggests that a cytokine-mediated generation of ROS may induce HO-1 expression in these leukocyte populations. The biological significance of the enhanced expression of HO-1 remains unclear, but effects on the maintenance of viability, function and proliferative capacity in leukocytes during heavy exercise must be discussed. The down-regulation of the baseline expression of HO-1 in trained individuals reflects an adaptional mechanism due to regular endurance training. Further investigations are necessary to gain more insight into the regulatory pathways of the HO-1 expression after acute exercise and during regular training.

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[20] Acknowledgements — This investigation was supported by a grant from the Bundesinstitut fu¨r Sportwissenschaft (VF 0407/01/21/97). We wish to express our appreciation to Mrs. M. Faigle for the ELISA measurements of MPO and TNFa. We would also like to thank the volunteers who participated in the study.

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