cDNA microarray analysis reveals novel candidate genes expressed in human peripheral blood following exhaustive exercise.

It is generally accepted that exhausting endurance exercise exhibits strong effects on the immune system. Such effects have been attributed to changes in the cellular composition of peripheral blood as well as to changes in the expression of plausible candidate genes. The list of candidate genes is far from being complete, since this issue has not yet been investigated in a systematic way. In this study, we used a custom-made cDNA microarray focused on inflammation as a screening approach to study gene expression in eight one-half marathon runners before, immediately after, and 24 h after exercise. Significant differential gene expression was verified by quantitative real-time PCR. Linear regression analysis showed that microarray expression analysis of cell type-specific surface molecules reflects the observed individual cellular shifts in peripheral blood cells with high statistical significance. In line with the results of former studies, we observed an upregulation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2), L-selectin, and IL-1 receptor antagonist (IL-1ra) after exhaustive exercise. The main results of this study report, for the first time, the downregulation of CD81; the upregulation of thioredoxin, which may play an important part in anti-oxidative defense; and, surprisingly, the downregulation of the anti-carcinogenic gene glutathione-S-transferase-3 (GSTM3) in peripheral blood. The study shows cDNA microarray expression analysis as a reliable systematic instrument to complete the list of candidate genes that may play a role in exhaustive exercise-induced modulation of the immune response.

[1]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[2]  J. Davis,et al.  Effects of high- vs moderate-intensity exercise on natural killer cell activity. , 1993, Medicine and science in sports and exercise.

[3]  Y. Soini,et al.  Antioxidant defense mechanisms in human neutrophils. , 2002, Antioxidants & redox signaling.

[4]  M. Yáñez-Mó,et al.  Cutting Edge: Dynamic Redistribution of Tetraspanin CD81 at the Central Zone of the Immune Synapse in Both T Lymphocytes and APC1 , 2002, The Journal of Immunology.

[5]  M. Lishner,et al.  Expression of tetraspanins in peripheral blood leukocytes: a comparison between normal and infectious conditions. , 2004, Tissue antigens.

[6]  R. Pieters,et al.  Expression of GSTM isoforms in childhood acute lymphoblastoid leukaemia , 2003 .

[7]  B. Pedersen,et al.  Enhanced plasma IL-6 and IL-1ra responses to repeated vs. single bouts of prolonged cycling in elite athletes. , 2002, Journal of applied physiology.

[8]  A. Zeiher,et al.  Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin‐1 expression , 2004, FEBS letters.

[9]  J. Yodoi,et al.  Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. , 2002, Molecular immunology.

[10]  J. Quadrilatero,et al.  Treadmill exercise in mice increases intestinal lymphocyte loss via apoptosis. , 2003, Acta physiologica Scandinavica.

[11]  G. W. Hatfield,et al.  Effects of exercise on gene expression in human peripheral blood mononuclear cells. , 2004, Journal of applied physiology.

[12]  C. Wolf,et al.  Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. , 2002, The Biochemical journal.

[13]  A. Víkingsson,et al.  Enumeration of IFN-γ producing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN-γ , 1994 .

[14]  H. Northoff,et al.  The cytokine response to strenuous exercise. , 1994, International journal of sports medicine.

[15]  S. Holm.  A Simple Sequentially Rejective Multiple Test Procedure , 1979 .

[16]  P. Shek,et al.  Exercise-induced changes in the expression of surface adhesion molecules on circulating granulocytes and lymphocytes subpopulations , 2004, European Journal of Applied Physiology and Occupational Physiology.

[17]  A. Holmgren,et al.  The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. , 1994, The Journal of biological chemistry.

[18]  Mutsuo Yamada,et al.  Systemic inflammatory response to exhaustive exercise. Cytokine kinetics. , 2002, Exercise immunology review.

[19]  D. Nieman,et al.  Effects of long-endurance running on immune system parameters and lymphocyte function in experienced marathoners. , 1989, International journal of sports medicine.

[20]  W. Cleveland.  Robust Locally Weighted Regression and Smoothing Scatterplots , 1979 .

[21]  M. Oh,et al.  Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. , 2001, Nucleic acids research.

[22]  M. Gorospe,et al.  Identification of a target RNA motif for RNA-binding protein HuR. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Yodoi,et al.  Redox-active protein thioredoxin prevents proinflammatory cytokine- or bleomycin-induced lung injury. , 2003, American journal of respiratory and critical care medicine.

[24]  W. Guggino,et al.  Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF. , 2003, American journal of physiology. Cell physiology.

[25]  L Hoffman-Goetz,et al.  Exercise and the immune system: a model of the stress response? , 1994, Immunology today.

[26]  J. Jordan,et al.  Moderate exercise leads to decreased expression of β1 and β2 integrins on leucocytes , 1997, European Journal of Applied Physiology and Occupational Physiology.

[27]  H. Northoff,et al.  Transcriptional and translational regulation of heat shock proteins in leukocytes of endurance runners. , 2000, Journal of applied physiology.

[28]  M. Radman,et al.  Mismatch Binding Protein-Based Mutation Detection Systems , 1995 .

[29]  Keiichiro Suzuki,et al.  Induction of Thioredoxin Reductase Gene Expression by Peroxynitrite in Human Umbilical Vein Endothelial Cells , 2002, Biological chemistry.

[30]  H. Northoff,et al.  Expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes. , 1999, Free radical biology & medicine.

[31]  G. Freeman,et al.  Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses. , 2002, International immunology.

[32]  D. Nieman,et al.  The immune response to exercise. , 1994, Seminars in hematology.

[33]  J. Eberwine,et al.  Amplified RNA synthesized from limited quantities of heterogeneous cDNA. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[34]  H. Northoff,et al.  Effect of exhaustive exercise stress on the cytokine response. , 1997, Medicine and science in sports and exercise.

[35]  J. Zierath,et al.  Marathon running increases ERK1/2 and p38 MAP kinase signalling to downstream targets in human skeletal muscle , 2001, The Journal of physiology.

[36]  Chris E Cooper,et al.  Exercise-Induced Oxidative Stress , 2005, Sports medicine.

[37]  H. Northoff,et al.  Expression of the inducible nitric oxide synthase (iNOS) in human leukocytes: responses to running exercise. , 2000, Medicine and science in sports and exercise.

[38]  Yongchao Ge.  Resampling-based Multiple Testing for Microarray Data Analysis , 2003 .

[39]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[40]  Y. Benjamini,et al.  THE CONTROL OF THE FALSE DISCOVERY RATE IN MULTIPLE TESTING UNDER DEPENDENCY , 2001 .

[41]  Z. Šidák.  Rectangular Confidence Regions for the Means of Multivariate Normal Distributions , 1967 .

[42]  A. Steensberg,et al.  IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. , 2003, American journal of physiology. Endocrinology and metabolism.

[43]  Shunichi Kumagai,et al.  [Pathological roles of oxidative stress in autoimmune diseases]. , 2003, Rinsho byori. The Japanese journal of clinical pathology.

[44]  Daniel Bernstein,et al.  Exercise assessment of transgenic models of human cardiovascular disease. , 2003, Physiological genomics.

[45]  H. Northoff,et al.  Free radicals and oxidative stress in exercise--immunological aspects. , 1999, Exercise immunology review.

[46]  Peter Schjerling,et al.  Pro‐ and anti‐inflammatory cytokine balance in strenuous exercise in humans , 1999, The Journal of physiology.

[47]  E. Fehrenbach,et al.  Role of heat shock proteins in the exercise response. , 1999, Exercise immunology review.

[48]  K. Miyazawa,et al.  Regulation of Interleukin-1β-induced Interleukin-6 Gene Expression in Human Fibroblast-like Synoviocytes by p38 Mitogen-activated Protein Kinase* , 1998, The Journal of Biological Chemistry.

[49]  Terence P. Speed,et al.  Normalization for cDNA microarry data , 2001, SPIE BiOS.

[50]  Assessing the efficiency of dye-swap normalization to remove systematic bias from two-color microarray data , 2004 .

[51]  M. Colonna,et al.  2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism. , 2000, Human immunology.

[52]  B. Ames,et al.  Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants. , 2000, Journal of applied physiology.

[53]  Douglas M. Hawkins,et al.  A variance-stabilizing transformation for gene-expression microarray data , 2002, ISMB.

[54]  T. Lyberg,et al.  Long‐Distance Running Modulates the Expression of Leucocyte and Endothelial Adhesion Molecules , 2004, Scandinavian journal of immunology.

[55]  A. Lechtermann,et al.  Exercise-induced apoptosis of lymphocytes depends on training status. , 2004, Medicine and science in sports and exercise.

[56]  Terence P. Speed,et al.  A comparison of normalization methods for high density oligonucleotide array data based on variance and bias , 2003, Bioinform..

[57]  R. Polanowska-Grabowska,et al.  Heat-shock proteins and platelet function. , 2000, Platelets.

[58]  E. Reszka,et al.  Significance of genetic polymorphisms in glutathione S-transferase multigene family and lung cancer risk. , 2001, International journal of occupational medicine and environmental health.

[59]  L Hoffman-Goetz,et al.  Exercise and the immune system: regulation, integration, and adaptation. , 2000, Physiological reviews.

[60]  M. Djavaheri-Mergny,et al.  NF‐κB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFα‐treated Ewing sarcoma cells , 2004 .

[61]  Michael N Sawka,et al.  Exertional heat injury and gene expression changes: a DNA microarray analysis study. , 2004, Journal of applied physiology.

[62]  A. Holmgren,et al.  [21] Thioredoxin and thioredoxin reductase , 1995 .

[63]  A. Holmgren,et al.  Thioredoxin and thioredoxin reductase. , 1995, Methods in enzymology.

[64]  S. Dudoit,et al.  STATISTICAL METHODS FOR IDENTIFYING DIFFERENTIALLY EXPRESSED GENES IN REPLICATED cDNA MICROARRAY EXPERIMENTS , 2002 .

[65]  R. Pieters,et al.  Mu class glutathione S‐transferase mRNA isoform expression in acute lymphoblastic leukaemia , 2003, British journal of haematology.