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Medicine & Science in Sports & Exercise:
BASIC SCIENCES: Original Investigations

HSP expression in human leukocytes is modulated by endurance exercise

FEHRENBACH, ELVIRA; PASSEK, FRANK; NIESS, ANDREAS MICHAEL; POHLA, HEIKE; WEINSTOCK, CHRISTOF; DICKHUTH, HANS-HERRMANN; NORTHOFF, HINNAK

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Abstract

FEHRENBACH, E., F. PASSEK, A. M. NIESS, H. POHLA, C. WEINSTOCK, H.-H. DICKHUTH, and H. NORTHOFF. HSP expression in human leukocytes is modulated by endurance exercise. Med. Sci. Sports Exerc., Vol. 32, No. 3, pp. 592–600, 2000.

Purpose: Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression.

Methods: The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control.

Results: After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01).

Conclusions: Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

Cells from virtually all organisms respond to a variety of environmental stress factors by the rapid transcription and subsequent translation of a unique, highly conserved set of polypeptides termed heat shock or stress proteins (HSP). Mammalian cells are known to synthesize HSP in vitro after brief exposures to temperatures of 3–5°C above normal (heat shock (HS)) (23). Earlier studies demonstrated that, after recovery from mild temperature increases, thermotolerance was conferred to the cells, which allowed survival during subsequent exposures to otherwise lethal temperatures and suggested a protective function for HSP (23). The mechanism of thermotolerance is still not completely understood.

HSP expression is also altered during glucose depletion and oxidative stress (27). Cells that are starved for glucose overproduce a set of proteins called glucose-regulated proteins (GRP) (23). Similarly, cells exposed to low levels of oxidative stress such as H2O2 exhibit protection against subsequent exposure to higher, normally lethal, oxidative stress levels (27). This induction of oxidative stress resistance is thought to be linked to the overexpression of genes that encode the oxidative stress proteins (OSP) (27). The functions of HSP, GRP, and OSP are incompletely understood, but evidence suggests that many stress proteins are enzymes that either provide immediate stress protection or conduct cellular repair processes (19). Furthermore, they seem to be responsible for stress tolerance after repeated stress situations (23,27).

The metabolic changes caused by exercise are similar to those known to induce stress protein synthesis (9,16). Physical exercise can elevate core temperature to 44°C and muscle temperatures up to 45°C (23). Exercise also causes oxidative stress via an increased generation of reactive oxy- gen intermediates (ROI) (5). Activation of blood neutrophils described after exercise and an increase of lipid peroxidation products suggest that oxidative stress plays a role in exercise-induced changes in the blood compartment (5,26). Furthermore, sustained physical activity results in the progressive depletion of glucose and glycogen stores, a phenomenon that is highly correlated with fatigue. Heavy exercise also induces an inflammatory reaction that includes leukocytosis and increases in host defense mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNFα) (30). Given all these factors, it seems to us that exercise is an excellent tool for studying the effects of stress on gene expression and the physiological significance of stress proteins.

Recently, there has been increasing interest in a family of 70-kDa heat shock proteins (HSP70) that appear to play a role in protein translocation and assembly processes. In mammalian cells, there are three different types of HSP70-like proteins: stress-inducible HSP70, constitutively expressed cognate HSC70, and glucose regulated proteins (GRP) (19). Although structurally related, members of this gene family appear to be functionally distinct. HSC are constitutively expressed in several cell types of different organisms. The main feature of these genes is that, unlike HSP genes, their transcription is at most only moderately responsive to heat. HSC have been shown to provide essential functions needed for normal growth, and recently they have been assigned a role in translocation of proteins across membranes (13,31).

Of particular interest is that the highly stress-inducible HSP70 may be required for the transport of nuclear-encoded polypeptides that are destined for processing and assembly in mitochondria (6). In muscle, one of the major effects of regular exercise training or conditioning is an increased mitochondrial biogenesis in which the mitochondrial content can actually be doubled (4). Because precursor polypeptides from the nucleus are required for the assembly of most complete mitochondrial proteins, HSP70 could provide a vital link in the mechanism of exercise-induced mitochondrial biogenesis (23). This raises the question of whether inducible HSP70 in leukocytes is also influenced by exercise.

The regulation of expression and phosphorylation of HSP27 by the inflammatory cytokines TNFα and IL-1 (11) may be meaningful for the stress response to intensive exercise.

Mammalian HSP60 apparently functions to facilitate proper oligomeric assembly of proteins within the matrix of mitochondria (31), and HSP60 is associated with inflammation and autoimmunity (8). T cells that are reactive with HSP60, possibly triggered by high local concentration of HSP, could be involved in the initiation or perpetuation of the inflammatory reaction. An inflammatory reaction is also induced by the physiological stimulus of extensive exercise (30).

HSP90 is a very abundant protein in all cells that are grown under normal conditions, and its synthesis increases three- to fivefold after heat shock. In cells deprived of glucose or oxygen, or treated with agents that perturb calcium homeostasis, synthesis of HSP90 declines concomitantly with an increased synthesis of GRP and HSP70 (31).

Only a few studies have examined mammalian stress proteins in vivo, and the methods used primarily involved artificially raising the temperature of animal or in vitro culture models (12,16,20,21). A more natural stress that has not been adequately investigated as a possible inducer of HSP synthesis in human blood is strenuous physical exercise. Given the many similarities between the conditions observed with strenuous exercise and the factors that may cause HSP synthesis in vitro, it was of great interest to determine whether HSP synthesis is induced in immune cells of athletes undergoing strenuous endurance exercise in vivo.

The present study was designed to investigate for the first time the differential expression of HSP27, HSP60, HSP70, HSC70, and HSP90 in circulating human leukocyte subpopulations (lymphocytes, monocytes, and granulocytes) of half-marathon runners before and at different time points after the competition. Furthermore, HSP expression in the blood of trained athletes at rest compared with that of nontrained individuals was analyzed to study the influence of regular training on HSP expression in immunocompetent cells.

©2000The American College of Sports Medicine

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