The oxidant molecules produced by the immune system to kill invading organisms may activate at least two important families of proteins that are sensitive to changes in cellular redox state. The families are nuclear transcription factor kappa B (NFkB) and activator protein 1 (API). These transcription factors act as "control switches" for biological processes, not all of which, as illustrated earlier, are of advantage to the individual. NFkB is present in the cytosol in an inactive form, by virtue of being bound to IkB. Phosphorylation and dissociation of IkB renders the remaining NFkB dimer active. Activation of NFkB can be brought about by a wide-range of stimuli including pro-inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and viruses and their related products, and UV and ionizing radiations. The dissociated IkB is degraded and the active NFkB is translocated to the nucleus where it binds to response elements in the enhancer and promoter regions of genes. A similar translocation of API, a transcription factor composed of the proto-oncogenes c-fos and c-jun, from cytosol to nucleus, also occurs in the presence of oxidant stress. Binding of the transcription factors is implicated in activation of a wide-range of genes associated with inflammation and the immune response, including those encoding cytokines, cytokine receptors, cell adhesion molecules, acute phase proteins, and growth factors (35) (Fig. 5.4).
Unfortunately, NFkB also activates transcription of the genes of some viruses, such as HIV. This sequence of events, in the case of HIV, accounts for the ability of minor infections to speed the progression of individuals who are infected with HIV towards AIDS because, if anti-oxidant defenses are poor, each encounter with general infections results in cytokine and oxidant production, NFkB activation, and an increase in viral replication. It is thus unfortunate that reduced cellular concentrations of GSH are a common feature of asymptomatic HIV infection (15).
Inflammatory stimuli LPS, oxidants,stress
Oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. This situation may also lead to upregulation of NFkB activity, as the transcription factor has been shown to be activated in endothelial cells cultured with linoleic acid, the main dietary n-6 poly-unsaturated fatty acid, an effect inhibited by vitamin E and NAC (36). The interaction between oxidant stress and an impaired ability to synthesize glutathione that results in enhanced inflammation is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize GSH (37). In this study, an inverse relationship between glutathione concentration and the ability of monocytes to produce IL-1, IL-8, and TNF-a was observed. Furthermore, treatment of the patients with the GSH prodrug, oxothiazalidine-4-carboxylate (procysteine), increased monocyte GSH content and reduced IL-1, IL-8, and TNF-a production (Fig. 5.5). Thus, anti-oxidants might act, to prevent NFkB activation, by quenching oxidants. However, NFkB and API may not respond to changes in cell redox state in the same way. When rats were subjected to depletion of effective tissue GSH pools by administration of diethyl maleate, there was a significant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (38). An increase in inflammatory stress would be expected in this study. In an in vitro study using HeLa cells and cells from human embryonic kidney, both TNF and hydrogen peroxide resulted in activation of NFkB and API (39). Addition of the anti-oxidant sorbitol
to the medium suppressed NFkB activation (as expected) but (unexpectedly) activated AP1. Thus, the anti-oxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g., NFkB) and cellular proliferation (e.g., AP1). Evidence for this biphasic effect was seen when glutathione was incubated with immune cells from young adults (40). A rise in cellular glutathione content was accompanied by an increase in IL-2 production, and lymphocyte proliferation, and a decrease in production of the inflammatory mediators, PGE2 and LTB4. Modification of the glutathione content of liver, lung, spleen, and thymus in young rats, by feeding diets containing a range of casein (a protein with a low sulfur amino acid content) concentrations, changed immune cell numbers in lung (41). It was found that in unstressed animals, the number of lung neutrophils decreased as dietary protein intake and tissue glutathione content fell. However, in animals, given an inflammatory challenge (endotoxin) liver and lung GSH concentrations increased directly in relation to dietary protein intake. Lung neutrophils, however, became related inversely with tissue glutathione content. Addition of methionine to the protein deficient diets normalized tissue glutathione content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet of adequate protein content (Fig. 5.6).
Thus, it can be hypothesized that anti-oxidants exert an immuno-enhancing effect by activating transcription factors that are strongly associated with cell proliferation (e.g., AP1) and an anti-inflammatory effect by preventing activation of NFkB by oxidants produced during the inflammatory response.
The molecular mechanisms that underlie the increased inflammatory and oxidant stress that accompanies aging has been examined in aged mice. The role of changes in PPAR-a activity in the process has been examined by Poynter and Daynes (42). Their findings suggest a role for PPAR-a in the maintenance of redox balance during the aging process.
GSH (umol/g lung tissue)
Figure 5.6 The relationship between lung neutrophil and glutathione content in animals fed diets with sulfur amino acid contents ranging from 2.2 to 6.5 g/kg and then given either a control saline or lipopolysaccharide injection intraperitoneally. Note: Neutrophils were counted in total lung sections and are expressed as the total number x 100 observed per subdivision of the graticule field.
In aged mice, the administration of agents capable of activating the alpha isoform of the PPAR-a receptor was able to restore the cellular redox balance. Evidence for this effect came from the observation that tissue lipid peroxidation was decreased, NF-kB activity decreased, and spontaneous inflammatory cytokine production was reduced. Aged animals bearing a null mutation in PPAR-a failed to elicit these changes following treatment with PPAR-a activators, but remained responsive to vitamin-E supplementation, thereby highlighting independent effects of anti-oxidants and PPAR-a on inflammation. Aged mice were also found to express reduced transcript levels of PPAR-a and the peroxisome-associated genes, acyl-CoA oxidase and catalase. Supplementation of aged mice with PPAR-a activators or with vitamin E caused elevations in these transcripts to levels seen in young animals. A number of natural endogenous molecules have been found that are capable of activating PPARs. For example, 15-deoxy-D12,14 prostaglandin J2 represents a natural PPAR-y ligand (43,44). Many specific fatty acid species and their derivatives, especially polyunsaturated fatty acids (45-48), the leukotriene B4 (49), and the eicosanoid 8(S)-hydroxyeicosatetraenoic acid (50), have been shown to be ligands for PPAR-a. Activation of PPARs has been demonstrated to antagonize signaling through an array of important pathways, including STATs, AP-1, and NF-kB (45,51-55). Poynter and Daynes (42) demonstrated that NF-kB is present in an active state in the macrophages and lymphocytes that reside in the spleens of aged mice (56). This active NF-kB correlated with the expression of the
NF-KB-regulated genes IL-6, IL-12, macrophage migration inhibitory factor, cyclooxygenase-2, and TNF (56). The administration of specific PPAR-« activators, or vitamin E, to aged rodents effectively reduced the elevated levels of active NF-kB, re-established control over pro-inflammatory cytokine production, and reduced lipid peroxide levels in various tissues (56).
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