Nitric oxide (NO·) is an important effector molecule that accounts for a variety of functions such as vascular regulation, neurotransmission, and control of tissue oxygen utilization (see review 14). NO· is also involved in immune regulation and is of importance in host defense due to its antimicrobiological properties (7). Furthermore, NO· acts as a radical in the development of cell and tissue injury, and it has been suggested that an overproduction of NO· plays an important role in the pathogenesis of several inflammatory diseases (17,28). NO· is synthesized from L-arginine by a family of nitric oxide synthases, of which the inducible form (iNOS) is mainly expressed in immunocompetent cells such as leukocytes. Evidence exists that the expression of iNOS in human leukocytes is induced during several inflammatory processes whereby cytokines are considered to act as potent stimulators (17).
Heavy physical exercise has been shown to induce a cellular stress response including activation of inflammatory cells (23,29) and stimulation of the cytokine release (27). Exercise-induced cytokine release causes an increase in plasma concentrations or urinary excretion of IFN-γ, TNF-α, and IL-1β (21), which are known to stimulate expression of iNOS (19). With regard to the interaction between NO· and cytokines, and looking at the several processes that can be induced by NO· in immunocompetent cells, it seems of interest whether vigorous physical exercise is capable of inducing the expression of iNOS in human leukocytes.
In the present investigation, we studied the influence of two different types of running exercise on the expression of mRNA for iNOS in human leukocytes. Therefore, our exercise protocols consisted of a prolonged intensive endurance run mainly dependent on aerobic capacity and a more intensive treadmill protocol until exhaustion with an augmented demand of anaerobic metabolism. After the treadmill protocol, we additionally visualized the cytoplasmic expression of iNOS protein product separately in lympho-, mono-, and granulocytes by using flow cytometry.
MATERIALS AND METHODS
Subjects and Experimental Design
The subjects were nonsmokers with normal dietary habits, who did not take any medication or vitamin supplements. The protocols of the studies were approved by our institute’s ethics committee and conformed to the policy statement of the American College of Sports Medicine for research on human subjects. All subjects gave informed consent to participate in the investigation.
Ten endurance-trained male subjects (age 32.3 ± 3.3 yr, weight 65.5 ± 1.3 kg; height 174.5 ± 3.0 cm) competed in an official half marathon run (21.1 km). The subjects performed regular endurance training with a running volume averaging 55.7 ± 5.5 km·wk−1. No intensive or prolonged training sessions were performed within the last 4 d before the race.
Eight healthy male subjects (age 25.0 ± 2.2 yr, weight 70.6 ± 1.7 kg; height 180.5 ± 3.4 cm), who did not perform any kind of specific sports conditioning and devoted less than 3 h·wk−1 to recreational and occupational physical activities, participated in study 2. The exercise protocol consisted first of a graded exercise test on the treadmill (Saturn, HP COSMOS, Traunstein, Germany), which was absolved until exhaustion. Initial speed was 6 km·h−1 with an increment of 2 km·h−1 every 3 min. The incline of the treadmill was kept constant at 1%. Capillary blood for lactate measurement was obtained from the earlobe after every stage. The running speed at the individual anaerobic threshold (IAT) was assessed according to Dickhuth et al. (9). After a resting period of 15 min, the subjects performed a continuous run (CR) on the treadmill until exhaustion at a running velocity of 110% of the IAT.
Venous blood samples were taken in a sitting position using EDTA as an anticoagulant at rest, 0, 3, 24, and 48 h after the end of CR and HM. Whole blood aliquots for flow cytometry, RT/PCR, and determination of complete blood cell counts were kept at room temperature and the procedures of analysis were started within 1 h after collection. Ten mL of whole blood were centrifuged immediately after sampling and plasma aliquots were stored at −70°C until further analysis. Capillary blood for lactate measurements was obtained from the earlobe before and 0 and 5 min after the runs.
Complete blood cell counts including hemoglobin, hematocrit, and differential leukocyte counts were performed by an automated Coulter Counter (Coulter Juniors, Coulter Electronics, Miami, FL). 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, Mannheim, Germany). Furthermore, plasma samples were analyzed for myeloperoxidase (MPO) and interleukin-8 (IL-8), which was performed by an enzyme-linked immunoassay method (Myeloperoxidase ELISA Kit, Calbiochem, Germany; IL-8: Genzyme, Duoset, Cambridge, MA). Additionally, tumor necrosis factor-α (TNF-α) was determined after the HM also by using an ELISA assay (R&D Systems, Minneapolis, MN). Post-exercise values of plasma parameters were corrected for changes in plasma volume according to Dill and Costill (10).
The cytoplasmic expression of iNOS in lympho-, mono-, and granulocytes was assessed by flow cytometry. Five mL of EDTA-blood were layered above 5 mL of Lymphoflot (Biotest, Dreieich, Germany) and settled for 60 min by gravity without centrifugation. The overlay was removed and the cell concentration adjusted with PBS to 1 × 107 cells·mL−1; 100 μL of the suspension was used for the flow-cytometric analysis.
The leukocytes were analyzed by indirect immunofluorescence using the iNOS specific antibody (iNOS, rabbit polyclonal antiserum, StressGen Biotechnologies, Victoria, Canada); 1 × 106 cells were first fixed at room temperature in a solution containing formaldehyde, then permeabilized according to the manufacturer’s instructions (Fix & Perm kit, An der Grub, Vienna, Austria) and at the same time incubated with the primary iNOS specific antibody for 15 min. After washing the labeled cells twice and incubating in the presence of the second FITC-conjugated goat anti-rabbit F(ab′)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 second antibody (Dianova, Hamburg, Germany) only. Fluorescence histograms were area-corrected to 10,000 cells. Data are presented as percent positive cells (%) and mean fluorescence channel (mfc). The values were corrected for background fluorescence with the negative controls. The lympho-, mono-, and granulocyte populations were differentiated by size and granularity in the scattergram and gated.
Isolation of RNA and Reverse Transcriptase/Polymerase Chain Reaction (RT/PCR)
Cytoplasmic RNA for RT/PCR analysis was isolated from whole blood samples using the RNeasy-blood kit (Quiagen, Hilden, Germany); 400 ng RNA were reverse transcribed (10 min 20°C, 15 min 42°C, 5 min 99°C, 5 min 5°C). β-actin was amplified under the following conditions: 3 min 95°C, 1 min 95°C, 1 min 55°C, and 1 min 72°C in the thermal cycler (MJ Research, PTC200, Waltham, MA) using the specific primer according to (33). iNOS-cDNA was amplified under different conditions (4 min 94°C, 1 min 94°C, 1 min 66°C, and 1 min 66°C). Sense and antisense primers for iNOS were used according to Amin et al. (1). Reverse transcription and subsequent amplification by the polymerase chain reaction (PCR) were performed utilizing a GeneAmp RNA PCR kit (Perkin Elmer, Wilton, CT). The RT master mix consisted of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 1 mM each of deoxyribonucleoside triphoshates (dNTPs), 1 U·μL−1 RNase inhibitor, 2.5 U·μL−1 reverse transcriptase, and 2.5 μL of Oligo d(T). The final RT-reaction volume was 20 μL. The PCR master mix contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.25 mM MgCl2, 200 μM each of dNTPs, 2.5 U·100 μL−1 AmpliTaq DNA polymerase, and 0.15 μM of each primer. A final 25 μL of PCR reaction solution contained 5-μL RT product (cDNA) and 20-μL PCR master mix. For each primer pair, control experiments were performed to determine the range of cycles in which a given amount of cDNA would be amplified in a linear fashion: iNOS 30 cycles and β-actin 27 cycles. Additionally, we performed a dilution assay to assess the proper input RNA concentration. The resulting amplified products for iNOS (258 bp) were confirmed by sequence analysis (SEQLAB, Goettingen, Germany). Semiquantitative analyses of photographs (Fig. 1) of ethidium bromide stained DNA-gels (2% agarose) were performed by the Lumi-Imager-System (Boehringer, Mannheim, Germany). The data generated were normalized to transcript levels for the constitutively expressed β-actin gene.
All statistical analyses and descriptive methods were computed using the statistical software package JMP (SAS Institute Inc., Cary, NC) for Macintosh computer. Data were expressed as means ± standard error (SE). Calculated differences between post-exercise and resting values showed normal distribution as revealed by the Shapiro-Wilk W-test and were tested for significance using the Wilcoxon signed ranks test. Differences between HM and CR were evaluated by the nonparametric test of Mann-Whitney. A P-value of <0.05 was regarded as significant.
In study 1, mean running time in the half marathon (HM) was 90.5 ± 11.0 min and maximal lactate values were measured directly after finishing (5.04 ± 0.74 mmol·L−1). In study 2, the subjects achieved a maximal running velocity of 15.8 ± 0.7 km·h−1 in the incremental exercise test (duration 17.7 ± 1.1 min). Running time in the following CR averaged 11.3 ± 1.3 min, mean running velocity during CR was 12.8 ± 0.6 km·h−1. Maximal lactate values were 9.31 ± 1.04 mmol·L−1 after the incremental exercise test and 8.85 ± 0.86 mmol·L−1 after CR, respectively.
Plasma CK rose after HM and CR, but peak values 24 h post-exercise were significantly higher in HM than CR (Table 1). White blood cell counts are also summarized in Table 1. Neutrophils showed an increase, which peaked 3 h after CR and HM and were more pronounced after HM. A decrease of lymphocyte counts below resting values could only be observed 3 h after the HM.
Plasma MPO increased significantly directly after HM and CR and reached baseline levels 24 h post-exercise (Fig. 1). Significant changes of plasma IL-8 could only be detected after HM (Fig. 2). Before HM, plasma TNF-α was undetectable, but 0 and 3 h post-exercise we were able to determine a rise above the detection limit of 1.0 pg·mL−1 in 6 of 10 subjects.
RT/PCR revealed an increase of iNOS mRNA expression in leukocytes directly after the HM (Fig. 3), which was apparent in 8 of 10 subjects (Fig. 4). Corresponding mean values peaked directly after exercise (Table 2). In contrast to HM, significant changes of iNOS mRNA could not be detected after CR (Table 2).
Cytoplasmic expression of the iNOS protein in lympho-, mono-, and granulocytes in study 2, as measured by flow cytometry, are presented in percent iNOS-positive cells and mean fluorescence intensity (Fig. 5). The exercise protocol induced a clear increase of iNOS-positive lymphocytes which reached significance 3 h after the CR and was still apparent 48 h post-exercise. Most of the monocytes were already iNOS-positive at rest, and only minor changes could be detected after exercise. Compared with lymphocytes, a higher percentage of iNOS-positive granulocytes was found at rest and after CR. Mean fluorescence channel for iNOS in lymphocytes exhibited an increase only 3 h post-exercise. By contrast, the expression of iNOS in monocytes was enhanced up to 48 h after exercise in both mono- and granulocytes, whereby the extent was lower in granulocytes.
The present investigation yields indications of an increased expression of the inducible nitric oxide synthase (iNOS) in circulating leukocytes in response to vigorous running exercise. After a half marathon run (HM), we observed in 8 of 10 endurance trained subjects an up-regulation of the iNOS transcript that peaked directly and 3 h post-exercise. By contrast, our measurements failed to detect any significant changes of leukocyte iNOS mRNA after the graded exercise test and following continuous run (CR) above the lactate threshold until exhaustion. Nevertheless, this type of exercise caused an increased expression of cytoplasmic iNOS protein product as measured by indirect immunofluorescence 3 h post-exercise and this induction was still apparent 48 h after cessation of running.
Our flow cytometric results revealed differences in the protein expression of iNOS between lympho-, mono-, and granulocytes at rest as well as after exercise. A more pronounced baseline expression was assessed in mono- and granulocytes. We observed that most of the lymphocytes became iNOS positive after exercise. But as reflected by the mean fluorescence channel (mfc), the amount of iNOS protein expressed was less compared to the other two leukocyte populations investigated in the present study. Within the human immune system, iNOS can be expressed in monocytes/macrophages, neutrophils, eosinophils, T-lymphocytes, and mesangial cells (3,20,32,34). Although its induction was rather low in lymphocytes, our results show a stimulation of iNOS protein expression by exercise throughout all leukocyte populations investigated.
The regulatory pathways leading to an exercise-induced expression of iNOS in human leukocytes are not completely clear. Numerous factors, activators as well as inhibitors, have been identified to modulate the expression of iNOS (), whereby cytokines act as potent regulators. In vitro, a stimulation of iNOS in human cells can be achieved successfully by a combination of the cytokines IFN-γ, TNF-α, and IL-1β (19). Furthermore, thermal stress (19) and low concentrations of NO· itself (30) are both known to exert stimulating effects on the expression of iNOS, whereas our own results could not reveal an effect of in-vitro heat shock on iNOS mRNA in peripheral leukocytes of untrained subjects (Fehrenbach et al., unpublished data).
It is well established that heavy physical exercise induces an immune response that includes activation of immunocompetent cells (13,29), stimulation of the cytokine release (27), and also augments leukocyte synthesis of stress proteins (12,26). As reported in earlier studies, exercise-induced rises in plasma concentrations or urinary excretion of IFN-γ, TNF-α, and IL-1β confirm that heavy physical stress is thoroughly capable of increasing the release of inflammatory cytokines (21). The activation of neutrophils due to sepsis is associated with an augmented expression of iNOS especially in this leukocyte population (31). A growing body of evidence indicates that IL-8 mediates neutrophil activation (2,15), a scenario that is also paralleled by a rise in plasma MPO (5). After the HM, our studies revealed a marked neutrophilia and lymphopenia 3 h post-exercise, an increase of plasma CK, IL-8, MPO, and in 6 of 10 subjects a detectable rise of TNF-α. These findings clearly indicate a pronounced inflammatory response that may exert stimulating effects on the expression of leukocyte iNOS. Although a clear relation between the behavior of TNF-α as well as IL-8 in plasma and iNOS was not found in our study, we assume that cytokines are causally involved in exercise-induced changes of iNOS expression in leukocytes. After CR, an exercise protocol of shorter duration, the increase of neutrophils, plasma MPO and CK was lower and neither lymphopenia nor a rise in plasma IL-8 could be observed. This points to a lower extent of overall stress induced by CR and may be the reason for our failing to detect post-exercise changes of iNOS mRNA.
The functional significance of an induction of iNOS expression in leukocytes by exercise has to be focused on the resulting generation of NO·. Increased urine levels of nitrate as an indicator of NO· generation have been shown to occur after a triathlon race (4). Besides other sources such as endothelium and neuronal cells, this may be influenced in part by an increased expression of iNOS in immunocompetent cells.
NO· is an important effector molecule in neuronal communication and vascular regulation. In addition, it plays an important role in immune modulation and contributes to host defense by its antimicrobical properties (7). On the other hand NO·, produced in excess, is known to act as a cell-damaging agent that is involved in various disease processes such as septic shock, diabetes, neurodegeneration, and chronic inflammation (). The protective as well as damaging role of NO· associated functions is represented for example by findings that knockout mice, lacking iNOS, are more susceptible to infections (33) than the wild type, but also exhibit a lower fall of systemic blood pressure during septic shock (22).
The biological significance of an exercise-induced generation of NO· by immunocompetent cells must be discussed in context with other features of the immune response to exercise. Changes such as depressed proliferation of lymphocytes (11,24), partial suppression of the oxidative burst in neutrophils (13,18) and DNA strand breaks in leukocytes (16) have been described to occur as effects of prolonged physical exercise and are assumed to be induced in part by oxidative stress (25). With regard to the various functions of NO·, it has to be discussed whether an induction of iNOS in leukocytes, as observed in the present investigation, exerts immunomodulating effects. NO· is capable of inhibiting cell proliferation (14) via depression of mitochondrial energy metabolism and hence ATP synthesis. NO· and related products are known to block NADPH oxidase (6) and therefore reduce the capacity of the oxidative burst in neutrophils. Physiological levels of NO· are able to result in DNA damage (8). This may result from direct damage of DNA and/or from inhibition of DNA repair enzymes (35) by NO· and peroxynitrite which is formed from the interaction between NO· and superoxide. Considered as a whole, the variety of iNOS-associated mechanisms underline the need for complex control mechanisms.
In summary, we could show that heavy running exercise is capable of inducing the expression of iNOS in human leukocytes. Baseline and post-exercise expression of the iNOS protein is more pronounced in mono- and granulocytes and exhibits a delayed and prolonged time course. Our finding of an increased expression of iNOS in immunocompetent cells may contribute to an exercise-induced rise of endogenous NO· production and reflects a systemic inflammatory response to heavy exercise. The immunoregulatory as well as the possible cell damaging role of an enhanced expression of iNOS after exercise warrants further research.
This investigation was supported by a grant (VF 0407/01/21/97) from the Bundesinstitut für Sportwissenschaft (Cologne, Germany). We wish to express our appreciation to Mrs. M. Faigle for the ELISA measurements of MPO, IL-8, and TNF-α. We would also like to thank the volunteers who participated in the study.
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