1 Department of Transfusions Medicine, University of Tuebingen
2 Center for Bioinformatics Tuebingen, Department of Information and Cognitive Sciences, University of Tuebingen
3 Department of Sports Medicine, Medical Clinic, University of Tuebingen
4 Central Institute for Mental Health, Department of Psychopharmacology, Mannheim, Germany
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ABSTRACT |
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thioredoxin; CD81; oxidative; marathon
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INTRODUCTION |
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In leukocytes, several different systems are activated or regulated after intensive, exhausting endurance exercise. A variety of genes are known to be involved in this process. These comprise the heat shock proteins as well as the cytokines (10, 15, 55).
After prolonged, exhaustive endurance exercise, an increase in different cytokines, like TNF-, IL-1, IL-6, IL-8, and IL-1 receptor antagonist (IL-1ra), were observed in plasma or urine of athletes, with levels falling back to normal on the following day (20, 43, 45, 58, 62). Presently, IL-6, which also has major metabolic functions for the working muscle, is considered to be the major indicator for physical strain in sports medicine (45, 52, 57). Considerably elevated levels of cortisol and adrenaline were suggested to be at least partly responsible for these changes (21, 48). On the other hand, after endurance exercise, ex vivo cytokine production by peripheral blood mononuclear cells was shown to be significantly decreased, probably as a result of counterregulation (63).
Exercise also leads to increased generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) (41). Besides their damaging potential, ROS play a physiological role in cellular signaling and in promoting lymphocyte apoptosis (21, 35).
To date, there is no proven knowledge about the molecular mechanisms by which exercise exerts its effects on the immune system. In humans, the most convenient source available to perform measurements of stress parameters on the cellular and molecular level is peripheral blood. cDNA arrays have evolved into widely used tools for comprehensive analysis of gene expression.
Using spotted cDNA chips, we decided to examine blood samples of one-half marathon runners before and after a run to look for changes in gene expression patterns. The aim, using microarrays, was to find hitherto unknown target genes involved in the reaction of the immune system to exercise. It is hoped that this may finally lead to an improved understanding of physiological and metabolic regulatory pathways that are influenced by endurance exercise.
The genes represented on the cDNA microarray were selected on the basis of up-to-date knowledge about their role in inflammation, apoptosis, stress response, and related pathways. It is also known that exhaustive exercise induces major transit changes in subpopulations of the peripheral blood.
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METHODS |
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We compared the gene expression pattern of each athlete before and after exhaustive exercise.
Each subject gave written informed consent before participation in the study. The experimental protocols were approved by the Institute's Human Ethics Committee according to the principles set forth in the Declaration of Helsinki of the World Medical Association.
Preparation of Glass cDNA Microarrays
cDNA microarrays were produced in the Center for Mental Health in Mannheim, Germany. Human clones were purchased from the Ressourcen Zentrum für Genomforschung Library (RZPD) in Berlin, Germany. The final library contained 277 different genes (5001,500 bp). Insert DNAs of all clones were amplified, each in a 50-µl PCR solution [10x PCR buffer (Qiagen), MgCl2 (Qiagen), Taq polymerase (Qiagen), RZPD vector primers] in 384-well microtiter plates. The plates were incubated for 3 min at 94°C, before 35 cycles of denaturation at 94°C for 75 s, annealing at 59.5°C for 90 s, and elongation at 72°C for 90 s were performed, followed by a final elongation step at 72°C for 10 min. The quality of the PCR products was routinely checked on a 1.5% agarose gel stained with ethidium bromide.
Each PCR sample was concentrated, and the pellet was taken up in 15 µl of 3x SSC plus 1.5 M betaine (Sigma, Munich, Germany) spotting solution. Stratagene's SpotReport-10 Array Validation System included human ß-actin PCR product (3'-sense and 5'-sense) (Stratagene), poly(dA) oligonucleotide (Stratagene), human COT-1 (Stratagene) and cDNA from Universal RNA (Stratagene), 3x SSC buffer, and 1.5 M betaine spotting solution, spotted as positive and negative controls together with the RZPD gene library, in triplicate, on QMT amino slides (75 x 25 mm) (Quantifoil, Jena, Germany). cDNAs were spotted in triplicate with a MicroGrid I spotter (BioRobotics, Cambridge, UK) supplied with 16 solid microspotting pins (BioRobotics). The slides were UV cross-linked with 250 mJ/cm2 and incubated at 80°C for 2 h.
Isolation, Amplification, and Labeling of RNA
Blood was drawn in PAXgene blood RNA tubes (Qiagen). RNA was isolated using the PAXgene blood RNA kit (Qiagen,) including a DNase I treatment, according to the manufacturer's recommendations. The quality of the RNA was checked with Agilent's Lab-on-a-Chip total RNA nanobiosizing assay (Agilent Technologies, Palo Alto, CA). Four micrograms of total RNA from each sample was spiked with Stratagene's SpotReport-10 Array Validation System RNA spikes (amounts per spike varying from 0.0025 to 2.5 ng) and reverse transcribed. Amplification was performed exactly according to the Eberwine procedure (61) using Ambion's MessageAmp kit (Ambion, Austin, TX). After in vitro transcription, the indirect labeling method with aminoallyl was used with equal amounts of all samples. The protocol follows exactly the Institute for Genomic Research (TIGR) protocol from Jeremy Hasseman called "Aminoallyl labeling of RNA for microarrays" (http://pga.tigr.org/sop/M004.pdf), using cyanine-3 (Cy3) and Cy5 ester (Amersham Biosciences, Amersham, UK). For each labeled sample, absorbance was measured at 260 nm, for Cy3 at 550 nm, and for Cy5 at 650 nm, respectively.
Hybridization of Glass cDNA Microarrays
After labeling, the samples were vacuum dried for 3 h. During this time, the cDNA arrays were treated with a prehybridization step according to the Quantifoil prehybridization protocol A (http://www.quantifoil.com), followed by a denaturing step, for arrays spotted with PCR probes [1 x 3 min in boiling distilled water (dH2O; 95100°C)]. The vacuum-dried Cy3- and Cy5-labeled samples were resuspended together in 10 µl of 10 mM EDTA, incubated at 95°C for 5 min, and followed by incubation at room temperature for another 5 min. Prewarmed Ambion's Hybridization Buffer No. 1 (35 µl) was added to the samples and was incubated for 10 s at 68°C before given on the microarray. Slides were hybridized in hybridization chambers (TeleChem International, Sunnyvale, CA). Hybridization was done at 55°C for 1416 h in a water bath. After hybridization, each slide was washed with 2x SSC + 0.2% SDS at 30°C for 10 min, 2x SSC at room temperature for 10 min, and 0.2 x SSC at room temperature for 10 min. Slides were dried immediately with N2.
To compare the gene expression pattern of each athlete before and after exhausting exercise, we hybridized each slide with samples from t0 vs. t1 and from t0 vs. t2. A dye swap was done with each investigation.
Scanning, Feature Extraction, and Analysis
Slides were scanned in a microarray scanner (Genetix Limited, Hampshire, UK).
Photomultiplier tube voltage was always set to 100% for both red and green channels. The two resulting green and red images were overlaid using ImaGene 5 (BioDiscovery).
Signal and Feature Extraction
As a first step of feature extraction, all spots that were flagged as poor by the ImaGene 5 software (due to signal foreground or background contamination, shape irregularity, or poor spot quality) were excluded.
The modus of the pixel distribution for the signal foreground and background of each spot was selected as a robust estimator of the raw signal values of both channels (see Fig. 1).
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Differentially Expressed Genes
Due to the direct design of the experiment, only log ratios M that lie statistically significantly above or below 0 mark correspond to differentially expressed genes. To investigate this, a one-sample t-test procedure against the mean 0 was used (13). The problem of multiple testing was addressed by controlling the family-wise error rate (FWER) and the false discovery rate (FDR) (13, 17). A gene was called differentially expressed if its Bonferroni-corrected (7) P value was <0.05 (5%). In addition, other correction methods like Benjamini and Hochberg (2), Benjamini and Yekutieli (3), Holm (22), and Sidak (54) were also applied and the results compared. The data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI)'s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE2532. A subset of interesting genes was selected for a further evaluation by real-time PCR.
Software
All parts of the feature extraction and analysis procedures were computationally done inside R 1.8.1 (50) with a mixture of Bioconductor structures (18) and self-written functions.
Quantitative real-time PCR.
Selected cDNAs were quantified by real-time PCR on a LightCycler instrument (Roche, Mannheim, Germany). For PCR-assisted amplification, the LightCycler Fast Start DNA Master SYBR Green I-kit (Roche) was used according to the manufacturer's instructions. Final concentrations of primers and magnesium were optimized for each set of primers (for primer sequences, see Table 2).
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Relative quantification of gene expression levels was done by running an external standard curve together with the samples of interest. Standards were prepared by serially diluting a post-PCR amplicon of the respective templates. The linear range was determined for each PCR run by blotting the crossing point (CP) value calculated by the LightCycler software against the dilution factor of these standards, thus allowing the quantitative comparison of the samples. For samples within the linear range of the standard curve, the respective value was calculated using regression analysis. The ratio of specific sample to ß-actin values allowed the relative quantification of gene expression.
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RESULTS |
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DISCUSSION |
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Using an "inflammation-centered" cDNA chip, we intended to find evidence and new indications to further clarify the area. We did not expect to find all exercise-induced alterations of immunological parameters that are described in the literature to come out as significant changes in our experiments, since these changes may occur at different times or even outside peripheral blood (e.g., IL-6). Despite applying very stringent statistics for microarray analysis to avoid false positives, we found interesting candidate genes that showed significant regulation. Using real-time PCR, we confirmed most of the candidate genes. These and some further genes of special interest shall be discussed in detail below, trying to find out possible relevance in the given setting.
The present data reveal up- and downregulated transcripts in peripheral blood cells of one-half marathon runners immediately after the run compared with preexercise levels. Most of these significantly changed mRNAs encode proteins hitherto unknown to be involved in exercise physiology. Nevertheless, these transcripts neatly fit into the context of exercise stress, including oxidative stress and protective proteins [thioredoxin (TRX), GSTM3, IL-1ra], cellular contact mechanisms (CD81, adhesion proteins), signal transduction, and cellular protection [mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2), TRX]. Some of these genes were also shown to be significantly changed in peripheral blood mononuclear cells (PBMC) by Connolly et al. (10) using Affymetrix arrays.
Gene Expression Changes in Concordance with Cellular Shifts
Being aware of the exercise-induced changes of cell counts that may influence mRNA expression levels of whole blood cells, as measured here, we start by discussing those genes in which changes in the microarray-analyzed gene expression were closely paralleled by relevant cellular shifts. This concerns mainly genes for cell surface markers that are differently expressed in individual cell populations of peripheral blood. Sonna et al. (55) already stated that cell type-specific microarray-detected gene expression changes after heat injury may reflect cellular shifts rather than changes within a cell.
In our study it pertains to L-selectin, which was significantly changed, but also to some additional genes like CD1c, CD2, CD3e, CD14, and CD19, which show a strong trend but did not reach significance (see Table 4).
L-Selectin (CD62L) mRNA was upregulated significantly in this study. This pattern corresponds to the situation known from the literature (30), where it was shown by flow cytometric analysis that, during recovery after exercise, the expression of CD62L on granulocytes was significantly increased. However, in this study a 3.4-fold increase in gene expression may well be attributable to the increase in granulocyte numbers (4.3-fold). CD62L is strongly expressed on granulocytes.
CD1c, CD2, CD3e, and CD19 are markers of lymphocytes. All of these were downregulated in nearly the same range as the downshift of lymphocytes, which was 45% in this study. In contrast to this, CD14 as a monocyte marker, which was upregulated by
88%, nicely reflected changes in numbers of monocytes, which were increased by 69% after exercise.
The expression of CD244, another surface molecule, may also have been altered due to cellular shifts. CD244 is the NK cell receptor 2B4 and is expressed as coreceptor on human NK cells, on macrophages, and on a subset of CD8+ T cells (36). It is known that numbers of NK cells in peripheral blood increase immediately after exhaustive exercise but can show a fast and sharp drop with high individual variation (39). Further expression studies should show whether CD244 data parallel the kinetics of NK cell numbers.
The finding of a highly significant correlation of the fold changes of cell surface markers with the respective fold changes in cell counts underscores the accuracy of the performed methodology, but nevertheless pinpoints the problem to securely separate expression differences from the bias induced by cellular shifts (see Table 6). This is not only a particular problem of this study (10, 55) but may also be true for microarray studies of solid tissue, for instance, for the frequently performed comparison of tumor with benign tissue, where differences in the presence of immune competent cells may severely bias expression analysis.
Gene Expression Changes Not Attributable to Obvious Cellular Shifts
Cellular communication.
Integrin-x (CD11c) and CD81 mRNA were both downregulated significantly in this study. This cannot be attributed to cellular shifts, since CD11c is strongly or moderately expressed in monocytes, macrophages, NK cells and granulocytes, while it is low in T and B cells (26), and CD81 is expressed on polymorphonuclears (PMN), monocytes, and B and T lymphocytes (59). The lymphocytes are the minor population and therefore can hardly explain the strong 50% overall downregulation of gene expression.
Regular moderate exercise leads to a decreased expression of adhesion proteins, especially ß2-integrins (26). It was suggested that the decreased expression of adhesion molecules immediately after exercise negatively influences adhesion and transmigration of the leukocytes into the vessel wall and the surrounding tissue. Shortly after exercise, this may contribute to a reduced defense against infectious agents (37).
To our knowledge, the downregulation of the CD81 gene expression has not yet been demonstrated in context with exhaustive exercise. The protein encoded by this gene is a member of the transmembrane 4 super family (tetraspanins) involved in signal transduction events in the regulation of cell development, activation, growth, and motility. The tetraspanins are common components of exosomes. The CD81 protein may play an important role in the reaction or adaptation to strenuous exercise. As mentioned above, reduced interaction with integrins may contribute to downregulation of undue proinflammatory stimuli resulting from acute muscular (cellular) stress.
Integrins also play a role in the cell contact area during interaction between T cells and antigen presenting cells (APC), which is termed immunological synapse (IS) (33). During formation of IS, the tetraspanin CD81 is redistributed to the contact area of the cells in an antigen-dependent manner and is involved in the antigen-specific B cell-mediated immune response (33). Furthermore, the presence of CD81 at the IS may play an essential role during TH1/TH2 polarization (11). Downregulated CD81 protein may partly contribute to the attenuated cell-mediated immunity and a change of the TH1/TH2 balance in favor of TH2 after exhaustive exercise, rendering the athlete more susceptible to infections. However, CD81 mRNA and corresponding protein changes in individual leukocyte subpopulations still warrant further analyses.
Signal transduction and cellular protection.
In contrast to the cellular communication genes, signal transduction and cellular protection cannot be clearly related to individual subpopulations of peripheral blood. In this study, MAPKAP-K2, TRX, GSTM3, and IL-1ra were changed and confirmed as significantly altered by real-time PCR.
The finding that the gene expression level of MAPKAP-K2 is significantly upregulated in athletes after exhausting endurance exercise has already been shown for the muscle (65).
MAPKAP-K2 is a downstream substrate in the pathway of the MAPK family. The MAPKAP-K2 protein phosphorylates the small heat shock protein 27 (HSP27, HSPB1) (16, 49, 65). It is also known that MAPKAP-K2 is involved in IL-6 synthesis by stabilizing IL-6 mRNA (34). A recent study showed that MAPKAP-K2 is an immediate early gene that is posttranslationally regulated by the mRNA stabilizing protein HuR, which may explain the early and isolated increase in peripheral blood as reported here (32).
We recently showed that inducible nitric oxide synthase (iNOS), IL-6, and HSP27 are upregulated after exercise at mRNA and protein levels (15, 16, 44). It remains to be shown which role the upregulation of MAPKAP-K2 mRNA as demonstrated in this study may play for the posttranscriptional modification of these and possibly other substrates in exercise. Interestingly, hsp27 mRNA (HSPB1) is 1.8-fold upregulated in our study and is included in our list of differentially expressed genes (see Table 4).
The finding of increased TRX mRNA after the one-half marathon is new but does not come entirely unexpected. The TRX protein is a 12-kDa oxidoreductase enzyme containing a dithiol-disulfide active site and is released from various types of human cells, excluding neutrophils (28), in response to oxidative stress (25). It possesses a variety of biological functions including the ability to modulate transcription factor activity (5, 23). The multifunctional redox-active TRX scavenges free radicals, modulates chemotaxis and suppresses leukocyte infiltration into sites of inflammation, and protects cells against TNF-induced cytotoxicity and general oxidative stress (12, 24). The TRX enzyme was found to be elevated in patients with oxidative stress profiles (29, 47). Similarly, heavy exercise is associated with oxidative stress (1, 31, 41, 42). Therefore, the regulation and maintenance of the cellular redox status by intracellular redox-regulating molecules such as TRX become important to maintain tissue homeostasis also after intensive exercise. The TRX enzyme may represent an important anti-oxidative protection mechanism in peripheral blood cells after exercise stress. It is important to mention, however, that ROS generated through cellular metabolism do have positive functions as cellular second messengers through the regulation of numerous signal transduction pathways. The level of ROS needs to be carefully balanced to fulfill their positive tasks without producing undue damage. An upregulation of TRX has already been shown to enable the maintenance of such low and protective ROS levels (19).
Interestingly, another part of the oxidative stress response is GSTM3, which we demonstrate for the first time to be downregulated on gene expression level after endurance exercise. Glutathione transferases (GSTs) form a group of multigene isoenzymes involved in the cellular detoxification of both xenobiotic and endobiotic compounds. More than a dozen genes are known to encode GSTs. In this context, GSTs bind to or catalyze the inactivation of a variety of carcinogens (8). GST gene expression studies in humans have shown that polymorphisms in members of the GST gene family are associated with an increased risk of lung cancer (51). Levels of GST isoform expression have been shown to vary between different tissue and cell types. Within the peripheral blood, expression of GSTM3 mRNA has so far only been demonstrated in patients with acute lymphoblastic leukemia, where its expression was related to good prognosis (27). The role of GSTM3 in exercise immunology remains to be determined.
An additional gene that may also be viewed as part of the cellular protection mechanism is IL-1ra. The main assignment of IL-1ra is the regulation and the control of the effects caused by IL-1ß by a competitive inhibition of the receptor. An overbalance of IL-1ß at the receptor leads to inflammation and tissue destruction. This anti-inflammatory cytokine is known to be increased in serum by prolonged exercise (46, 52, 57). Upregulation of IL-1ra mRNA in our study confirms that IL-1ra as opposed to IL-6 is generated in peripheral blood cells. Changes of IL-1ra mRNA in PBMC after exercise have already been shown (10).
The upregulation of IL-1ra may play an important role for the balance between the proinflammatory and the anti-inflammatory cytokines in the immune system.
In conclusion, we have investigated gene expression in peripheral blood of eight marathon runners using a cDNA microarray focused on inflammatory markers. A number of genes regulated in response to exercise have been identified. We presently have no information about corresponding protein changes. Some of these genes may have been altered as a consequence of cellular shifts. Furthermore, three genes have been found of particular interest that have not yet been described in relation to exercise. These are downregulated CD81, which may attenuate inflammatory reaction; upregulated TRX, which may play an important part in anti-oxidative stress reaction; and downregulated GSTM3, another gene involved in balancing the redox system.
It is beyond the scope of this study to carry out additional investigations on up- or downstream genes. Moreover, correlations of gene transcript abundance with corresponding protein levels will have to be done in the future.
Altogether, the reliability of our microarray results is confirmed by the good reproducibility in real-time PCR measurements. The nice reflection of cellular shifts in some of the transcript changes and the confirmation of several previously known effects strongly support the notion that such microarrays may serve as a suitable screening tool for exercise monitoring. Microarray analysis of separated cell subsets, excluding cell shift-associated changes, should be performed in the future.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Zieker, Dept. of Transfusions Medicine, Univ. of Tuebingen, Otfried-Mueller-Str. 4/1, D-72076 Tuebingen, Germany (e-mail : dzieker{at}web.de)
* D. Zieker, E. Fehrenbach, and J. Dietzsch contributed equally to this study.
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REFERENCES |
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