It is currently a particularly exciting time for hydrogen peroxide (H2O2) redox research. On the one hand, the significance of oxidative stress by molecules such as H2O2 has become increasingly recognized to the point that it is discussed as a component of virtually every disease. On the other hand, H2O2’s role as a signaling molecule is now postulated to have an importance similar to other ubiquitous signaling molecules such as cAMP, nitric oxide and calcium. Understanding the regulation of cellular H2O2 and redox metabolism will aid in developing novel therapeutic tools. In support of the latter statement, it is the objective of this brief review to describe the role of this interesting molecule in nature and biology and to delineate recent scientific discoveries that moved H2O2 into the center of attention as cellular regulator for immune reactions.
H2O2 in Nature, Household and Medicine
Hydrogen peroxide (H2O2) is not very abundant in nature. It is formed mainly by the action of sunlight on water and is thus found in traces in rain and snow. H2O2 as a molecule is weakly reactive, but the single bond between the two oxygen atoms is easily broken, so that it readily fragments into a hydrogen and a hydroperoxyl radical or into two hydroxyl radicals (Fig. 1). Because these latter molecules are highly reactive (with hydroxyl radicals being the most reactive of all reactive oxygen species), H2O2 is used as a powerful oxidizing agent. As such, H2O2 has been a commercial product since the 1800’s. One of the earliest markets for the use of H2O2 was the bleaching of straw hats, important fashion items in Europe at that time. Today, over a billion pounds of H2O2 are produced annually in the United States (US Peroxide Inc.). H2O2 is used as a bleaching agent in paper and textile industries, for treatment of wastewater, and during metallurgical processes. In homes, H2O2 is used for cosmetic and medical purposes. It reacts with melanin in hair by destroying its double bonds and making it a colorless molecule. The result is blond hair, with keratin as the dominant hair color molecule. A 3% H2O2 solution in water is used as disinfectant for treatment of minor cuts and scratches because it inhibits and kills microorganisms.
Just as H2O2 has the ability to harm microorganisms, it also has the ability to kill our body’s cells. H2O2 per se causes only mild damage. The damaging power comes from the transition to highly reactive hydroxyl radicals (Fig. 1) that indiscriminately react with a wide variety of organic substrates causing peroxidation of lipids, cross-linking and inactivation of proteins, and mutations in DNA (Knievel, 2004). Hydroxyl radicals form when H2O2 is exposed to ultraviolet light or when it comes in contact with a range of transition metal ions, of which the most important is probably iron. It is widely accepted that H2O2 is a toxin in vivo at concentrations of about 50 µM and above. To counteract stress caused by H2O2, organisms have evolved a wide variety of defense mechanisms. For instance, the reactions leading to H2O2 are minimized by sequestering the metal ions that would otherwise act as catalysts into proteins. Ferritin, transferrin, hemosiderin and heme are examples of proteins that enclose iron and thus play a role in protecting the cell against oxidative damage (Nappi & Vass, 2000). Other tools in the combat against oxidative stress are enzymes that are employed to rapidly dismutate H2O2 to water. Superoxide dismutases (SOD), catalases, peroxidases (especially glutathione peroxidases), and thioredoxin-linked systems are examples of such enzymes (Droege, 2001, Arnér & Holmgren, 2000). Many other molecules serve as antioxidants including vitamins (e.g. vitamin E, vitamin C, provitamin A, also called beta-carotine), hormones (e.g. melatonin) and cofactors such as coenzyme Q (Brash & Havre, 2002; Reiter et al., 1995).
Reactive oxygen species (ROS) including H2O2 are produced in the body as a byproduct of oxidative metabolism (Fig. 2). Since the consumption of oxygen during cellular respiration is the fundamental pathway that sustains aerobic life, all higher life forms have to deal with the ROS that they produce in mitochondria during normal generation of energy and they usually do so effectively. The rates of ROS production and the rates of clearance are well balanced in a healthy body (Fig. 3). However, exogenous sources such as cigarette smoke or excessive caloric intake may significantly increase the endogenous oxidant load and shift the balance to an abnormally high level of ROS. Also, a characteristic of increasing age is a decline in the efficiency of metabolic processes such as cellular antioxidant mechanisms. Insufficient defense against oxidative stress is discussed as a major contributor to the problems encountered during aging (Beckman & Ames, 1998). It is interesting to note that in a recent study on centenarians none of the people who reached this advanced age were overweight and their diet was typically rich in antioxidant substances (Corliss & Lemonick, 2004). It is possible that their low and exemplary food intake contributed to a low rate of ROS production and a good redox homeostasis.
In addition to the unwanted production of H2O2 in the body, ROS are also intentionally produced in the body and used for synthesis and detoxification processes as well as for immune defense (Fig. 3). H2O2 is produced in the thyroid gland as a substrate for thyroperoxidase, which catalyzes the attachment of iodine to thryoglobulin, an important protein for the synthesis of thyroid hormone. H2O2 is generated in peroxisomes to aid in the degradation of fatty acids and other molecules, and H2O2 is used for detoxification reactions involving the liver cytochrome P-450 system. Again, an excessive amount of these natural cellular processes due to disease or inappropriate lifestyle results in oxidative stress. Some cells, such as phagocytic leukocytes, have evolved the use of H2O2 as a bactericidal defense chemical, a phenomenon known as oxidative burst. In these inflammatory cells, NADPH oxidase associated with the plasma membrane reduces molecular oxygen to generate superoxide. Superoxide is spontaneously or enzymatically converted to H2O2 which can then freely pass through the membrane. While these oxidants are important in protecting us from infection, they can cause oxidative damage during chronic inflammatory activity. Inflammatory processes often overshoot in their reaction leading to excessive production of ROS such as H2O2, destruction of healthy body tissue, and development of autodestructive disease. The relationship of oxidative stress and inflammation is undisputed. Mounting evidence points to chronic inflammation as not only being the problem of well recognized inflammatory diseases such as tuberculosis, rheumatoid arthritis or inflammatory bowel disease (Hensley et al., 2000), but also as a contributor to a growing number of mechanistically unconnected illnesses such as atherosclerosis, Alzheimer disease, and some cancers (Forrester, 2004).
The fact that inflammation is involved in one way or another in almost all known diseases has led to the hypothesis that “inflammation may be the root of all evil” (Kreeger, 2003). Statements such as these contribute enormously to the newly increased interest in anti-oxidative treatments. Concurrently, during the last 10 years free radical research has been elevated to a mainstream research topic with amazing new discoveries as outlined in more detail below. The use of antioxidants in therapy has a long history. It is almost a central medical dogma that dietary or pharmacological practices that strengthen the body’s mechanisms to fight oxidative stress improve health. H2O2 treatment has been used by practicing physicians for more than a century and is still today offered in many treatments. However, H2O2 therapy has never become a widely accepted therapy in mainstream medicine and it is currently available as alternative therapy. Administered directly (orally or intravenously) or generated indirectly (e.g. by UV irradiation of blood), H2O2 is claimed to strengthen the body’s immune defense and thus to act effectively against a long list of ailments including some types of cancer, cardiovascular diseases, fractures, and depression. A major problem of antioxidant therapies is the lack of specificity. Extensive radical scavenging, or delivery of oxygen boosts via the blood, perturbs the body’s natural redox balance, and unwanted, often unpredictable, side effects are common. Additionally, the existing technology to monitor the redox state of patients during treatment and healing is poorly suited for routine clinical applications. Yet, the potential of redox therapies to become an important part of conventional medicine is recognized by many (Hensley et al., 2000; Mantovani et al., 2002).
H2O2 as Signaling Molecule
It should be evident by now that H2O2 is a member of the ROS family that plays an important role in nature and medicine. While the evidence supporting this statement so far has focused on the toxicological role of H2O2, it is time to introduce the second role of this amazing molecule, a developing image of ROS as important signaling molecules. Regulation of vascular tone, sensing of oxygen tension, and enhancement of membrane receptor signal transduction are only a few examples of non-detrimental processes that involve ROS (Droege, 2001; Lander, 1997). Most of the regulatory effects are not directly mediated by the most reactive members of ROS, such as O2.-, and .OH, but rather by their derivatives such as H2O2. In fact, H2O2 seems to be the most important ROS member that acts as a signaling molecule (Fig. 3). It has already been postulated that H2O2 is as important for cell functioning as other ubiquitous signaling molecules such as cAMP, nitric oxide and calcium. True or not, it is now certain that just as ROS and H2O2 can be considered “death molecules”, they also can be called “molecules of life” (Droege, 2001; Rhee et al., 2003). Organisms made a virtue of necessity by using the potentially dangerous ROS molecules to their advantage. The numerous, powerful enzymatic systems that are used by cells to prevent accumulation of toxic H2O2 levels also have a role in homeostatically maintaining H2O2 at levels where it serves as cellular regulator.
We are only beginning of understanding how cells discriminate between beneficial and harmful H2O2 effects. To determine H2O2’s cellular role as either toxin or cellular mediator, it is necessary to estimate its local concentration. H2O2 is electrically neutral and it is not restrained by membranes. Therefore the rate of diffusion and the half-life have to be taken into account when estimating its local concentration. According to Fick’s equation, the rate at which a substance diffuses within a cell is simply a function of the concentration gradient and depends on the molecular properties of solute and solvent. However, a recent publication on the atomic-level structure of a channel that controls the transport of ammonia and carbon dioxide in and out of red blood cells (Khademi et al., 2004) is expected to trigger a flood of new information on the regulated movement of gases and other freely membrane-diffusible substances. It is thus possible that the near future might bring new insight into facilitated or hindered transport of H2O2 as regulative tool of redox signaling. In contrast to other small messenger molecules such as calcium, H2O2in vivo is short lived, in the range of milliseconds. For comparison, the half life for O2.- is estimated to be a microsecond and that for .OH a nanosecond. Reports on the in vivo lifetime of H2O2 are sparse and inconsistent due to the methodological limitations for ROS measurements (Batandier et al., 2002) and the uncertainty of influencing parameters. For instance, the stability of H2O2 is influenced by the pH. While the blood pH in a healthy body is known to be homeostatically regulated within a narrow range, information on the local pH in inflammatory tissues is limited. Findings range from variations up to two pH units in the lumbar disks of patients with low back pain (Nachemson, 1969) to more subtle changes of 0.5 pH units in a dermal pouch with inflammation from a rat (Punnia-Moorthy, 1987). The stability of H2O2 further depends on the cell’s redox state. H2O2 is more stable in an oxidizing environment, such as the extracellular space, than in a reducing environment like the cell interior. Therefore, H2O2 has also been proposed to act as an inter-cellular messenger (Reth, 2002). Biological parameters add to the complexity of correctly estimating local H2O2 concentrations. The net H2O2 concentration depends on the site and source of H2O2 production, the numerous H2O2 activities within the cell, the spontaneous and enzymatic dismutation of H2O2, as well as the concentration of protective agents.
Clearly, understanding the role of H2O2 as either beneficial signaling molecule or damaging cell agent requires detailed insight into H2O2 production as well as H2O2 elimination (Fig. 3). Studies investigating these events recently revealed amazing new discoveries. For instance, a whole new family of enzymes, peroxiredoxins, has recently been described. In addition to the two conventional enzymes, catalase and glutathione peroxidase, peroxiredoxins remove intracellular H2O2 by reducing it to water (Seo et al., 2000; Wood et al., 2003). Peroxiredoxins are found in almost all organisms. All members of this ubiquitous group of enzymes have a cysteine residue in common at their N-terminal region. This catalytic N-terminal cysteine is oxidized by H2O2 to cysteine-.OH (sulfenic acid). Many members of the family, including four of the six mammalian enzymes, contain another cysteine in addition to the N-terminal one. The oxidized N-terminal cysteine of one molecule and the second cysteine of another peroxiredoxin molecule form a disulfide bond, which is then reduced by thioredoxin. This reaction is highly specific since other thiols such as glutaredoxin, glutathione, and glutathione reductase cannot serve as reducing equivalents. Recent work increasingly supports the hypothesis that peroxiredoxins are not only antioxidant proteins, but also involved in redox signaling. For instance, it was shown that peroxiredoxin I and II can be regulated through Cdc2 protein kinase-dependent phosphorylation, a posttranslational modification process that is likely to occur in mitosis (Chang et al., 2002).
H2O2 is well suited to act as cellular messenger since it does not randomly react with all molecules, as most other ROS do, but instead primarily targets cysteine residues. It oxidizes the -SH group of cysteine to –.OH which is then reduced by cellular reducing agents such as glutathione and thioredoxin. However, these reactions are only possible when the cysteine is deproteinated (-S-), which is not often the case at physiological pH. Only a positively charged amino acid in the vicinity of the cysteine can keep it in an oxidizable form, which means that only selected proteins are targets for H2O2 (Reth, 2002). The H2O2-dependent modifications of the target proteins can cause their activation or inactivation. For instance, H2O2 downregulates transcription factors such as p53, Jun, and Fos, but leads to activation of NF-kappa B and c-Jun N-terminal kinase (JNK) pathways (Chen et al., 2001; Livolsi et al., 2001; Reth, 2002). An important group of molecules that are downregulated by H2O2 is the protein tyrosine phosphatase (PTP) group, evolutionarily conserved molecules that play a central role for transmitting signals from cell surface receptors to the nucleus. It was shown that stimulation of cells from a human epidermal cell line by endothelial growth factor (EGF) caused H2O2 oxidation of PTP-1B in a reversible way, with maximal activation 10 minutes after receptor stimulation (Lee et al., 1998). The phosphorylation state, and thus activation state, of any cellular protein is the net effect of tyrosine kinase activity, which phosphorylates the protein, and corresponding PTP activity, which reverses the reaction. Thus, activation of tyrosine kinase by EGF binding to the EGF receptor, and inhibition of PTP activity by H2O2 can lead to similar effects, promotion of growth factor signaling. While PTPs are traditionally viewed as terminators of signaling processes, they could also be seen as active initiators of cellular signaling. If PTP activity is inhibited, the basal level of tyrosine kinase activity will initiate signal pathways in the absence of receptor stimulation. Elegant experiments provided additional in vivo evidence of the reversible regulation of other growth factor receptors such as PDGF and insulin by H2O2 (Li & Dixon, 2000; Meng et al., 2002). It seems accepted that PTP oxidation by H2O2 is an important physiological signaling event. However, many studies are still necessary to unfold the network of countless cellular events that are potentially regulated by H2O2 to predict the long-term consequences for particular cells. Dependent on the presence and interaction of the redox sensitive molecules in the cell, and dependent on the activation state of the cell, H2O2 signaling can lead to cell activation or cell inhibition. For instance, at sites of inflammation H2O2 appears to have the potential to augment or inhibit T cell signal pathways outside of normal receptor control as presented in more detail below.
New discoveries on the elimination of H2O2 and the proteins that are targeted by H2O2 are accompanied by new findings for the cellular production of H2O2. NADPH oxidase in phagocytes (Phox) is a well-characterized enzyme that participates in the killing of microorganisms through the generation of toxic oxygen radicals. The enzyme is rapidly activated under inflammatory conditions and produces superoxide and H2O2. Its activation involves the assembly of several soluble regulatory components with the membrane-associated enzyme, called gp91phox. In many non-phagocytotic cells, including epithelial, endothelial, and smooth muscle cells, new proteins that are homologous to gp91-phox have been described. They seem to play the role that NADPH oxidase plays in phagocytotic cells, i.e. intentional production of ROS (Hoidal, 2001; Lambeth, 2004). The NOX family is involved in regulation of cell growth and proliferation (NOX 1, NOX 5), participates in host defense (NOX 1) and mediates oxygen sensing (NOX 3/4). A second family of non-phagocytic NADPH oxidase similar enzymes has a dual redox function and is thus called DUOX enzymes. These enzymes contain not only an ROS-generating domain, but additionally an N-terminal region with similarities to known peroxidases including myeloperoxidase, thyroid peroxidases, and others. This means that DUOX enzymes can immediately process the H2O2 that is produced at the gp91phox homology domain to oxidize other substrates. It was shown that DUOX 1 and 2 play a role in thyroid hormone synthesis.
H2O2 Regulation of T Cells
It is of particular interest that one incorporates the new knowledge of H2O2 as signaling molecule into the big picture of inflammation. While the normal process of inflammation allows organisms to survive threats due to injury and infection, uncontrolled, pathological inflammation may lead to debilitating and life threatening diseases such as rheumatoid arthritis, heart disease, and cancer. Consequently, therapies for these diseases often involve the down-regulation of inflammation. T cells are central regulators of inflammatory cascades, and induction of apoptosis in overactive T cells is a popular therapeutic strategy to limit inflammation. The most promising approaches for novel inflammatory treatments are biologic therapies in which the cellular production of natural substances is therapeutically controlled to direct the cells down a preferred metabolic pathway. Since this could perhaps be accomplished by regulating H2O2 metabolism in T cells, there is much interest in understanding the regulation of redox signaling in T cells. T cells are exposed to ROS at sites of inflammation where activated macrophages and neutrophils produce large amounts of superoxide and its derivatives via the phagocytic isoform of NADPH oxidase (Fig. 4). It is estimated that during this oxidative burst the local H2O2 concentration may reach 10-100 µM in the vicinity of T cells (Nathan & Root, 1977). A large body of evidence, accumulated since the late seventies, indicates that T cells are able to sense these redox changes and to respond by activation of certain T signal cascades and transcription factors (Droege, 2001). Most of these results were considered as the response of T cells to oxidative stress. With the recent change of perspective on the role of ROS towards active second messenger molecules, it is now the time to revisit old evidence and to gather new evidence to improve the understanding of the role of H2O2 in T cells.
It has been known for some time that H2O2 at low concentrations has T cell mitogenic effects and promotes IL-2 production (Roth & Droege, 1987; Hehner et al., 2000). T cell responses in general are highly regulated by tyrosine phosphorylation. It is thus not astonishing that H2O2 affects T cell activity by balancing the activities of tyrosine kinases and phosphatases. For instance, H2O2 results in the activation of the SRC-family protein tyrosine kinase p56(lck) and of the SYK-family protein tyrosine kinase ZAP-70, similar to the signaling events that take place after stimulation of the T cell receptor (Livolsi et al., 2001). The activation of T cell receptor signal pathways (Fig. 4) seems to occur via inhibition of PTPs and to result in activation of all three members of the MAPK family: ERK, p38, and JNK (Lee & Esselman. 2002). It is proposed that the oxidative activation of T cells not only occurs during pathological conditions of chronic oxidative stress, but also regularly as a very early response of the immune system to an invading pathogen (Hehner et al., 2000). It may be that T cells sense small quantities of ROS long before inflammatory stages are reached. Changes in the redox environment could explain how T cells are informed early in the development of inflammation that they soon might or might not be needed. They respond by activating signaling processes that can result in promotion of their proliferation or death, dependent on the progression of the approaching immunological challenge and their best course of action. One problem for treatment of inflammatory diseases is the lack of knowledge of when and why a nonspecific inflammatory response becomes a more polarized, specific inflammatory response. Therapeutic regulation of early inflammatory stages would be helpful in avoiding the transition to chronic debilitating inflammation. Thus, monitoring of redox related T cell events could become a diagnostic tool for early detection of inflammation at a time before damage is done.
Initially, the response of T cells to H2O2 was believed to be solely a response to its inflammatory environment, where activated macrophages and neutrophils increase the local H2O2 concentration. More recently, there is evidence that T cells themselves produce H2O2 upon stimulation of their antigen receptor [Devadas et al., 2002; Williams & Kwon, 2004]. Interestingly, it is proposed that not only H2O2 is produced, but also superoxide anion (O2.-). Both then act as second messenger, each activating different T cell receptor signaling pathways. In these studies using the transformed Jurkat T cell line, O2.- activated an apoptotic signaling pathway, while H2O2 activated a proliferative pathway. The T cell receptor dependent H2O2 production by Jurkat cells was reproduced in activated proliferating human T blast cells (Kwon et al., 2003). However, in the non-transformed human cells the production of H2O2 resulted in cellular inhibition, specifically an inhibition of the duration of ERK activation. The discrepancies between Jurkat cells and cycling human T cells cannot yet be explained. They might be a sign of the complexity of redox signaling processes for immune regulation. One explanation is that a negative feedback loop exists dampening the initial activation of ERK. It is also possible that H2O2 that is produced at different times after receptor activation targets different ERK pathways and leads to different endpoints. Furthermore, different cells and different cell states vary in their activity of co-stimulatory receptor-initiated pathways such as CD4 or CD28 pathways, which by themselves are sensitive to redox changes. It is thus likely that the overall response of cells to changes in the cells’ redox status will very much depend on the metabolic cell state in which the changes occurred.
Discoveries that T cell receptors catalytically generate substantial amounts of H2O2 are seminal observations (Fig. 4). However, the way T cells produce H2O2 is still largely unknown. There are distinct differences in the expression of the proteins that are involved in cellular redox regulation between phagocytic leukocytes and T lymphocytes (Reth, 2002). For instance, it was believed that T cells do not express the necessary components of the phagocyte-type NADPH oxidase, however, Jackson et al. (2004) recently showed the activation of such an enzyme after T cell receptor stimulation and recruitment of apoptotic signals such as Fas ligand and Fas. Kinetic studies indicate that in addition to this sustained H2O2 production, there seems to be a rapid transient H2O2 production, independent of Fas or NADPH oxidase. Since H2O2 production can be measured before O2.- is detected, and since O2.- generation can be measured even when H2O2 is dismutated by catalase or thioredoxin peroxidase, H2O2 does not seem to be derived from O2.- as is believed to be the case in most other known systems (Devadas et al., 2002). A potential source for the unique production of H2O2 is the T cell receptor itself. This idea comes from studies with isolated antibodies, which have the ability to catalyze a light-dependent reaction between molecular oxygen and water that leads to the production of H2O2 (Wentworth et al., 2000; 2001). These events occur in all antibodies, regardless of source or antigenic specificity, and are being investigated at the molecular level. The reaction is initiated by singlet oxygen that reacts with H2O to ultimately produce H2O2 via intermediates such as H2O3 and ozone (Wentworth et al., 2003). The catalytic site of this reaction is at the interface between the variable and constant Ig domains, where H2O2 molecules are trapped in a hydrophobic pocket (Zhu et al., 2004). When Wentworth and colleagues tested the ability of a wide variety of non-antibody proteins to catalyze H2O2 production, the only other protein that produced H2O2 at a physiologically significant rate was the purified αβ T cell receptor. Employing a real-time monitor for H2O2 that we developed and tested by reproducing the antibody catalyzed water oxidation (Sharma et al., 2003; Nindl et al., 2004), we accumulated evidence that transformed Jurkat T cells and human T cell blasts indeed produce H2O2 under metabolically favorable conditions in a membrane-associated, light dependent event (unpublished data).
Conclusions
Hydrogen peroxide is a molecule that is well known in industry and in the household. In the body, H2O2 plays a dual role, as both a dangerous toxin and as a valuable signaling molecule. This is a fundamental change in perspective, since for many years H2O2 was primarily recognized as a toxin. It is produced in the body as a byproduct of oxidative metabolism, and most organisms have evolved powerful mechanisms to eliminate it. During inflammation, H2O2 is produced by white blood cells as a first line of defense against microorganisms that have invaded the body. Inflammatory processes often lead to excessive production of reactive oxygen species (including H2O2), destruction of healthy body tissue, and development of autodestructive disease. The significance of oxidative stress has become increasingly recognized to the point that it is discussed as a component of virtually every disease.
Besides being a toxin, H2O2 has another, opposing, role as an important and beneficial cellular signaling molecule. Evidence suggesting H2O2 as a cell signaling molecule comes from different cell types indicating that H2O2 may play a ubiquitous role as an intracellular and/or intercellular second messenger much like nitric oxide or calcium. ROS and H2O2 seem to be used by cells mainly as regulators of signal transduction by cell surface receptors. It is also possible that a physiologically significant role for H2O2 as cell regulator is restricted to certain cells, especially cells involved in inflammatory events.
T lymphocytes are especially interesting candidates for redox regulation. T cells are vital in regulating inflammatory responses and they are exposed to exogenous H2O2 produced by activated inflammatory phagocytic leukocytes. Studies show that H2O2 is able to promote either proliferation or death of inflammatory T cells, depending upon the circumstances. It seems that multiple steps in various T cell signal pathways are redox sensitive, so that the overall outcome of oxidative stimulation for the cells would be a weighted balance of the individual signaling activities. There is also substantial evidence that T cells can produce H2O2 in response to various activating stimuli. One source of H2O2 production is the phagocyte-type NADPH oxidase, but other sources such as the T cell receptor itself are likely. An improved understanding of the actions of H2O2 in T cells will have enormous therapeutic implications since down-regulation of inflammatory T cells is the treatment of choice for many inflammatory diseases.
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