Exercise results in many physiologic changes and altered gene expression and can induce both a limited and systemic inflammatory response in humans. While numerous organ and system responses (e.g., increased cardiac output, blood pressure changes) are induced, a myriad of cellular systems are activated to deal with the complex stresses of exercise. Among these cellular changes, heat shock proteins (HSP) are produced during exercise and may have important roles in modulating the effects of these heat and oxidative stresses to preserve cellular function. Heat shock gene expression, characterized by increased HSP in muscle and peripheral blood leukocytes, occurs following both eccentric exercise and exertion in the heat. This heat shock response may be adaptive against ongoing cellular stress and may also regulate important inflammatory mediators including nitric oxide (NO). NO production can have both protective and detrimental effects on the airways and circulation depending on concentrations, producing both bronchodilation and vascular leak.
In this review, we examine the role of HSP and NO production in exercise and in the T-helper 2 cell (TH2) environment of asthma, and we propose that an interrelated HSP and NO production in the lung during exercise plays a role in exercise-induced asthma.
HEAT SHOCK PROTEINS
The heat shock response is induced following alterations in various physical or chemical features of the environment in which there is a rapid increase in the intracellular concentrations of a set of evolutionarily conserved HSP. Elevated levels are observed following a variety of stresses to the host organism, including oxidative damage, thermal injury, viral infections, ischemia-reperfusion injury, and exercise (4,5,15). HSP function has been studied extensively and is reviewed in depth elsewhere. As a brief summary, in the absence of stress, HSP play a key role in maintaining cellular homeostasis, serving as chaperones during both protein assembly and membrane translocation. HSPs can stimulate an immune response characterized by increased T-helper 1 cell (TH1) and inflammatory cytokines and thus are typically associated with TH1-type disorders/responses. Asthma has been traditionally characterized as a TH2-type disease. Subjects with asthma, however, display increased levels of HSP when compared with those with no asthma; in particular, HSP70 is increased in the peripheral blood monocytes (24). This HSP70 overexpression in asthma results from a variety of complex interactions between environmental and genetic backgrounds but exists at baseline (1). Perhaps in a TH2 arena, HSP may also have an augmented role for propagating inflammation.
In response to stress, HSP genes are activated and induce HSP protein accumulation intracellularly, which in turn promotes tolerance in response to moderate physiologic stress and the cytotoxic effects of inflammatory cytokines or other heat stimulus, enhancing survival of the whole organism. HSP accumulation is important in cell survival under heat stress, but it may also be associated with physiologic tolerance and improved cell-to-cell function during hyperthermia. For instance, acute heat stress will protect rats from an endotoxin model of sepsis, improving survival and minimizing end-organ injury (22).
Our studies in in vitro epithelial monolayer systems reveal that heat conditioning sufficient to induce a heat shock response conferred tolerance to a subsequent, nonlethal heat stress as measured by an attenuation of the reduced epithelial resistance and a more rapid return of the epithelial barrier integrity. Further studies have demonstrated two potential HSP-related mechanisms: (i) HSP70 overexpression alone is sufficient to confer tolerance, and (ii) surprisingly, the activation of the heat shock transcription factor, HSF-1, directly induces the expression of the tight junction protein, occludin (3).
Thus, intracellular HSP as well as the activation of the HSP transcription machinery may help maintain epithelial barrier function and limit organ dysfunction under physiologic stresses in a variety of organisms and cell model systems. HSP induction has been demonstrated to protect human respiratory epithelium against NO-mediated cytotoxicity (27). The release or presentation of HSP70 extracellularly may lead to stimulation of inflammatory cytokine production however and thus would affect the host differently, possibly depending on the host milieu.
HEAT SHOCK RESPONSE TO EXERCISE
Exercise in humans induces several physiologic changes ranging from cellular and tissue to whole organ system alterations. Physical exercise activates the stress response and induces the synthesis of several HSP in both cardiac and skeletal muscle and its release in the hepatosplanchnic viscera (4). Mechanisms underlying this increased synthesis may include elevated body temperature, as well as muscle damage and membrane stability changes that occur with exercise. Temperature increase, oxidative stress, and inflammatory responses after endurance exercise stimulate the synthesis of HSP in peripheral blood leukocytes as well as in pulmonary macrophages (6). For example, acute exercise increases HSP72 levels in the peripheral circulation, suggesting that these proteins may indeed have a systemic role during stress. The increase in serum HSP72 preceded any increase in HSP72 gene or protein expression in contracting muscle, suggesting that HSP72 was released from other tissues or organs (25). Regular endurance training influences HSP expression as demonstrated by Fehrenbach et al. (9), who compared the expression of a variety of HSP in the cytoplasm and on the surface of leukocytes in trained athletes before and after a half marathon to levels in untrained persons at rest. After the race, a significantly greater percentage of leukocytes in the athletes expressed cytoplasmic HSP27, HSP60, and HSP70, whereas heat shock cognate protein 70 (HSC70) and HSP90 remained unchanged. Strenuous exercise increased HSP expression in the blood immediately after the run, suggesting a protective role of HSP in leukocytes of athletes to maintain function after heavy exhaustive exercise. Interestingly, the trained athletes at rest demonstrated a down-regulation of HSP-positive cells when compared with the control population, which may reflect adaptation mechanisms to regular endurance training (6).
It is intriguing to speculate that HSP in the extracellular environment might actually participate in triggering the inflammatory response observed during exercise (21). The potential role of HSP in both generating tolerance to the stresses of exercise and in driving inflammation through the "leak" of HSP into the extracellular milieu leads to a number of important questions. What degree of exercise confers the most protection to the host? Does an overproduction of HSP due to profound muscle trauma induce a danger signal that is detrimental to the individual by greatly enhancing the proinflammatory cascade? Or, perhaps an exaggerated immune response to exercise may be detrimental to the host as might be postulated in the pathophysiology of asthma where there is an imbalance of TH1 and TH2 responses. Previously, we have demonstrated that monocytic cell culture models when cultured with TH2 cytokines produce increased amounts of cluster of differentiation 23 (CD23), the low-affinity immunoglobulin (Ig) E receptor, and that when stimulated with anti-IgE produced more inflammatory cytokines. Further stimulation with HSP70 in this microenvironment surprisingly significantly enhanced this receptor percentage on the cells and may be important in propagating the inflammation noted in asthma. HSP70 stimulation alone did not up-regulate CD23, suggesting that something specific about the TH2 environment as seen in asthma is important to see this response. The increased amount of HSP70 in asthma may be due to tumor necrosis factor α and NO providing a continued stimulus for HSP production (9). Although this is one hypothesis, the recent evidence that HSF-1 regulates non-HSP genes offers another mechanism for stress response-associated changes to affect dynamic processes.
NO, a major signaling molecule, has been extensively reviewed relating to its production and physiologic effects as well as its role in lung disease (2,7,8,23). In brief, NO is generated from arginine by the action of one of three NO synthases (NOS). In the vasculature, NO reacts with iron in the active site of the enzyme guanylyl cyclase, stimulating the production of the intracellular mediator cyclic guanosine monophosphate, and thus enhances the release of neurotransmitters resulting in smooth muscle relaxation and vasodilation causing hypotension as noted in shock states with elevated NO levels. Once released into the extracellular milieu, NO reacts with oxygen and water to form both nitrates and nitrites. NO toxicity is linked to its ability to combine with superoxide anions (O2−) to form peroxynitrite (ONOO−), an oxidizing free radical that causes DNA fragmentation, lipid oxidation, and airway epithelial injury. As previously reviewed, NO is produced by numerous cells including monocytes/macrophages, epithelial cells, and endothelial cells via three isoforms of NOS, and each specific response is related to the tissue, cell, and concentration of NO produced. In general, the constitutive form of NOS protects airways from excessive bronchoconstriction, whereas inducible NOS (iNOS) has a modulatory role in inflammatory disorders of the airways such as asthma and promotes airway edema, obstruction, and vascular leak.
NO Response in Exercise
NO production is increased during exercise primarily due to shear stress on vessel walls, and it effects vascular flow redistribution that occurs with exercise by causing vasodilation in the heart and skeletal muscles while preventing excessive vasoconstriction in the kidney and splanchnic circulations (8,11,12).
Green et al. (7) outlined the effect that exercise training has on endothelium-derived NO (7). NO is responsible for the decreased systemic vascular resistance and hypotension noted in extreme exercise. As the blood volume through the lungs increases with increased cardiac output, NO concentration increases. Laughlin et al. (17) have shown that exercise training induces endothelial NOS (eNOS) content along the coronary tree in humans; that is, interval sprint exercise training enhances endothelial function and increases eNOS content in some muscular arteries. This may be related to the shear stress or possibly due to the increased temperature associated with exercise. In any event, NO concentrations increase with exercise in a variety of species and cause the effects of hypotension, blood flow redistribution, and epithelial injury.
Hyperthermia can also enhance NO formation. The heat exchange in the airways also induces NO from epithelial cells, which in turn can cause airway injury.
Elevated levels of NO cause a variety of detrimental effects in both the cardiovascular and pulmonary systems, such as decreasing blood pressure and interfering with protein synthesis. Adaptation to heat can prevent NO overproduction, thus limiting the detrimental effects that could theoretically be induced with exercise; one such mechanism is via activation of HSP70 synthesis (20).
HSP and NO Interactions
HSP and NO seem to interact in a complex coupling fashion, reflecting the interaction between an important regulatory molecule and the body's innate defense mechanism. The balance between the production of each component defines the tissue or cellular response and can be different in tissue or vascular beds, depending on the species. For example, in cardiac H9c2 cells, heat shock-induced HSP90 has been shown to form a complex with eNOS and activated it to increase NO production (12). This NO binds to the respiratory chain in the mitochondria to down-regulate oxygen consumption in the heat-shocked cells. HSP90 actually interacts with all forms of NOS to facilitate phosphorylation, increasing NO production. In humans, eNOS activity is modulated by HSP90 in aortic endothelial cells such that hyperglycemia in vivo and in vitro translocates HSP90 outside these cells, resulting in a decrease in NO production (18). Up-regulation of HSP70 can restrict the overproduction of NO by limiting the action of iNOS and thus limit injury of tissue in an ischemia-reperfusion model (15). Treatment with geldanamycin in a murine model increases HSP70i by activating HSF-1 and reduces iNOS by inhibiting Kruppel-like factor 6 (KLF6) transcription factor (15). However, under these conditions, HSP70 accumulation was only partially decreased. NO participates in HSP70 induction, and together, they are important in regulating the antihypotensive effects of heat adaptation (20). Pretreatment with geranylgeranylacetone (GGA) improved the survival rate in a rat sepsis model (22). Once such mechanism includes the priming effect of GGA by enhancing HSF-1 activation to then enhance HSP70 induction. Overexpression of HSP70 reduced the amount of NO and other inflammatory mediators produced in this murine model of sepsis. One proposed mechanism for HSP's anti-inflammatory effect in respiratory epithelium is related to the stabilization of IκB, thus inhibiting the nuclear factor κB pathway (NF-κB) (28). Furthermore, HSF-1 has been demonstrated to interact with NF-κB pathway in a protective heat shock response in a cadmium-induced lung injury model, suggesting that HSF-1 may control the cytoprotective effects of HSP (26).
Exercise-induced bronchoconstriction (EIB) is present in most subjects with asthma, and for some individuals, it may be the only manifestation of airways disease. Might there be an interaction between HSP and NO that plays a role in this aspect of the disease?
NO plays a key role in airway bronchial tone and vascular physiology and may indeed play a role in exercise-induced airway and vascular changes. NO is released from the airway epithelium during the regulation of respiratory heat exchange and can be measured noninvasively in the exhaled breath. An elevation in the fraction of exhaled NO (FeNO) is demonstrated in exhaled breath of a patient with asthma compared with the subject with no asthma, as more NO is present at baseline either from chronic airway inflammation or structural changes. An increase in FeNO levels of subjects with asthma may be an early and more reliable predictor of asthma exacerbation and thus increasing airway inflammation (10). Both subacute inflammation and remodeling that are present in asthma influence NO in the lung; airway wall remodeling is associated with increased levels of exhaled NO independent of airway inflammation (19). Persistent elevation in FeNO in a small subset of subjects with asthma after treatment with corticosteroids for an exacerbation may be attributed to underlying airway remodeling rather than solely continued acute inflammation (14). Suffice it to say that subjects with asthma have higher NO levels in the lung than their counterpart with no asthma, possibly because of the immune response at baseline, and this may have an effect when under stress or during exercise.
A differential pattern of FeNO is observed in subjects with asthma and those with no asthma after exercise (16). During hyperventilation as seen in exercise, airway temperature falls so that bronchial blood flow increases and NO production is augmented (16). This may be a response of an increase in eNOS or iNOS after airway irritation and cytokine production. Excessive NO production may be associated with EIB and can contribute to the prolonged airway obstruction observed in subjects with asthma as well as the increased airway vascular permeability, which correlates with the severity of EIB in subjects with asthma (13). NO plays an intimate role in the development of airway obstruction in thermal challenge; subjects with asthma display higher FeNO during hyperpnea than subjects with no asthma, and this continued as they developed airflow limitation (16). In further evaluating the role of NO in EIB, De Gouw et al. (2) pretreated the patients with asthma with an inhaled inhibitor of NO, N-monomethyl-L-arginine, and noted that EIB was slightly attenuated but not significantly compared with NO substrate administration. This suggests that other factors may be important in regulating NO in EIB.
Perhaps, the TH2 cytokine milieu observed in the subject with asthma alters the set point for interactions between HSP and NO in the lung. Does this environment in exercise-induced bronchospasm confer more susceptibility to effects of NO, or is it that more NO is produced at baseline? Although expressed at higher degrees than subjects without asthma, a balance between the defense mechanisms afforded by HSP production and the regulatory molecule NO in asthma exists to prevent the worsening effects of their overproduction. The lung of the exercising person with asthma must find this fine balance to protect the host from continual airway insult. Exercise may induce other system changes in addition to HSP production to complete the inflammatory immune response.
The interaction of HSP and NOS may be a novel mechanism for continued protection from organ dysfunction under extreme stress. As a clinician who sees a variety of trained college athletes with EIB, and with our previous work evaluating inciting factors for airway inflammation in asthma, it was interesting to note that HSP may have a role in this disease state. I propose a potential model for the interaction between HSP and NO in this setting as outlined in the Figure. As most subjects with asthma experience EIB, and this condition has higher levels of HSP and NO production, perhaps the normal-regulation aspects are not present and amounts of NO produced exceed HSP protective role to cause the symptoms. The NO produced may be altered because of the TH2 cytokine predominance as seen in asthma. The mechanism for this effect is not defined, but the recent data demonstrating HSF-1's direct transcriptional activation of genes as diverse as occludin 1 and the antiapoptotic protein BAG3 suggest a number of interesting avenues for further investigation. Cold exposures also induce HSP, and this effect might be important in exercise-induced asthma if breathing in relatively cold air during exercise incites bronchospasm. A slow warm-up period for the EIB subjects with asthma will decrease the severity of their reaction and thus may allow them to build up tolerance to stress possibly by enhancing HSP production or HSF-1 generation.
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