Cells respond to stressful stimuli (e.g., heat shock) by increasing HSP gene expression in proportion to the severity of the stress—a temperature threshold of 4°C-8°C above the normal growing temperature is required for induction of HSP expression (3). The cellular “sensor” for heat stress appears to be alterations of protein structure; heat stress leads to protein unfolding, and it is the intracellular accumulation of unfolded proteins, rather than temperature per se, that directly activates the heat shock response. Accordingly, the same response can be activated by any stimulus that generates intracellular accumulation of unfolded proteins (e.g., oxidant stress, ischemia, and heavy metals). Experimental support for this mechanism is shown by the fact that microinjection of denatured proteins into cells results in the upregulation of HSP expression.

A family of transcription factors, known as heat shock factors (HSFs), regulates HSP gene expression (3). Three HSFs are present in humans (HSF1, -2, and -4), of which HSF1 appears to be the most important stress-inducible HSF. The HSFs are characterized by a highly conserved amino-terminal, helix-turn-helix DNA binding domain, and a carboxy-terminal transactivation domain. HSF1 is present as a constitutively phosphorylated monomer during the resting, unstressed state. HSP70, a product of HSF1 activation, associates with HSF1 via protein-protein interaction and retains HSF1 in the cytoplasm, in an inactive, monomeric form. In response to heat shock, HSP70 disassociates from HSF1 and allows HSF1 trimerization, and these HSF1 trimers rapidly translocate into the nucleus. While trimerization is sufficient for DNA binding, transactivation requires inducible serine phosphorylation. HSF1 binds to heat shock elements (HSEs) within the promoter regions of HSP genes, defined by a tandem repeat of the pentamer nGAAn (“n” denoting a less conserved sequence) arranged in an alternating orientation either “head to head” (e.g., 5′-nGAAnnTTCn-3′) or “tail to tail” (e.g., 5′-nTTCnnGAAn-3′), resulting in the upregulation of HSP gene expression.

The most well-known biologic function of HSPs is their ability to serve as molecular chaperones (1,2). In this role, HSPs serve to fold, transport, and stabilize intracellular proteins. Since many forms of cellular injury lead to misfolding of intracellular proteins and to defects of intracellular protein processing and trafficking, the molecular chaperone properties of HSPs are thought to play a major role in the mechanisms by which the heat shock response confers protection in such diverse forms of cellular injury. Experimentally, the heat shock response can be induced either by thermal stress or through pharmacologic induction. In either case, it is well established that induction of the heat shock response confers protection in various animal models of critical illness, including septic shock, acute lung injury, oxidant stress, and ischemia-reperfusion injury (11,12). That HSP72 plays a major role in cytoprotection is evident by studies in which HSP72 is overexpressed genetically, and the experimental animals are afforded similar protection to that seen by induction of HSP72 through thermal stress or pharmacologic induction. For example, adenovirus-mediated transfer of the HSP72 gene to the lung epithelium confers protection in an animal model of acute lung injury (13).

Another mechanism by which the heat shock response confers cytoprotection is through its ability to modulate cellular proinflammatory responses. Numerous studies have demonstrated that induction of the heat shock response inhibits subsequent production of cytokines, chemokines, and nitric oxide (NO) when cells are exposed to proinflammatory stimuli. This has been demonstrated in both in vitro and in vivo experimental models. One major mechanism by which the heat shock response inhibits cellular proinflammatory responses is through inhibition of the nuclear factor-κB (NF-κB) signal transduction pathway (14). NF-κB is a transcription factor that regulates the expression of many genes involved in inflammation (see Chapter 19). Heat shock-mediated inhibition of the NF-κB pathway involves inhibition of IκB kinase and increased de novo expression of the endogenous NF-κB inhibitory protein, IκBα. These inhibitory effects of heat shock on cellular proinflammatory responses and the NF-κB pathway appear to be relatively specific, rather than a global downregulation of cellular function and gene expression. Furthermore, genome-level studies indicate that the mononuclear cell response to heat stress is highly divergent compared with the mononuclear cell response to endotoxin (15).

The visible transformation of a common bruise is an ancient colorimetric reaction that is dependent upon the enzyme HO. HO is the first and rate-limiting step in the degradation of heme (purple hue) to biliverdin (green hue), and finally to bilirubin (yellow hue) (Fig. 20.2). Three known isoforms of HO exist: HO-1, -2, and -3. In the context of cellular adaptation to stress, HO-1 appears to be the most relevant isoform. HO-1 is identical to HSP32 and is highly inducible by a variety of cellular stressors and stimuli, including heme, NO, cytokines, heavy metals, hyperoxia, hypoxia, endotoxin, heavy metals, and heat shock (16).

HO-1 activity is present in virtually all organs. The importance of HO-1 in human health and disease was recently demonstrated by the description of a 6-year-old boy with complete HO-1 deficiency (17). This patient’s clinical condition was characterized by severe growth retardation, hemolytic anemia, tissue iron deposits, widespread evidence of endothelial cell damage, and increased susceptibility to oxidant injury. These findings are remarkably similar to the phenotype commonly observed in HO-1 null mice.

In vitro studies involving gene transfection or gene transfer approaches have provided clear evidence that HO-1 confers cytoprotection (16,18). For example, overexpression of HO-1 conferred protection against oxygen toxicity, hemoglobin toxicity, tumor necrosis factor (TNF)-α-mediated apoptosis, and Pseudomonas-mediated cellular injury and apoptosis. Experiments in animal models, involving either pharmacologic induction of HO-1 or genetic overexpression of HO-1, confirm that HO-1 confers cytoprotection in vivo. Induction of HO-1, by intravenous hemoglobin administration, protected rats against the lethal effects of endotoxemia. Lung epithelial overexpression of HO-1, via an adenovirus vector, conferred protection in rats exposed to hyperoxia; cardiac-specific overexpression conferred protection in a murine model of ischemia. There is also interest in HO-1-mediated cytoprotection in the field of transplant biology. In a cardiac xenograft transplantation model, increased expression of HO-1 improved graft survival.

The by-products of HO enzymatic activity include carbon monoxide (CO), bilirubin, and ferritin (Fig. 20.2), and each of these by-products has been postulated to play a role in cytoprotection. Ferritin is known to protect against oxidant stress, and bilirubin can function as a potent antioxidant. The most recent work in the field implicates CO-related cell signaling as the key component of HO-1-mediated cytoprotection (19,20,21). For example, HO-1-derived CO appears to play an important role in the host defense to severe infection in a murine model of polymicrobial sepsis (22). CO shares a variety of properties with NO, including neurotransmission, regulation of vascular tone, and activation of soluble guanylate cyclase. The reported biologic effects of CO include potent anti-inflammatory effects (via the mitogen-activated protein kinase [MAPK] pathway), anti-apoptotic effects, and antioxidant effects. In vivo administration of low concentrations of inhaled CO protected rats against hyperoxia-mediated acute lung injury, and administration of exogenous CO to cardiac tissue protected the tissue against ischemia-reperfusion injury following transplantation. Several experimental studies have confirmed these initial results, demonstrating that CO inhalation or pharmacologic administration of CO-releasing drugs is cytoprotective in several different animal models of sepsis and acute lung injury (23,24). These studies are particularly intriguing because the amount of CO administered in these experiments is within the range administered to patients undergoing lung diffusion scans. Unfortunately, a recent study in nonhuman primates showed that the doses of inhalational CO that produced anti-inflammatory effects also resulted in relatively high and potentially toxic levels of carboxyhemoglobin (CO-Hb) levels (>30%) (25). However, the anti-inflammatory effects of CO were recently examined in a randomized, doubleblind, placebo-controlled, two-way crossover trial in which healthy volunteers were injected with a 2 ng/kg dose of lipopolysaccharide (LPS). Inhalation of 500 ppm CO versus air had no effect on the inflammatory cytokine production, though no adverse side effects were observed (CO-Hb levels increased to as high as 7%) (26). Conversely, inhalation of CO by patients with stable Chronic Obstructive Pulmonary Disease (COPD) at doses of 100-125 ppm for 2 hours/day for 4 days was effective in improving lung inflammation and function (27). Clearly, further studies are required. In addition, as yet there have been no clinical trials of CO-releasing agents in humans.

HSPs have been classically regarded as exclusively intracellular proteins. Studies over the last decade, however, clearly illustrate that HSPs can also exist in the extracellular compartment. For example, adult patients suffering from major trauma have increased serum levels of HSP72 (4). Critically ill children with septic shock also have increased serum levels of HSP72, and the absolute levels are much higher than that reported for critically ill adult patients following multisystem trauma (5). The latter may be reflective of an increased capacity of children to express HSPs compared with adults, consistent with the experimental literature previously mentioned.

Whether increased levels of extracellular HSPs represent active release/secretion of HSPs or a nonspecific release of HSPs from dying cells remains to be determined. Current evidence indicates that both processes are operative (28). Regardless of how HSPs enter the extracellular compartment, emerging evidence indicates that extracellular HSPs are biologically active. For example, HSP72 has been demonstrated to activate proinflammatory and antibacterial responses in macrophages (28,29,30,31,32). In this context, extracellular HSPs are said to serve as “danger signals” for the innate immune system.

The biologic role, if any, of extracellular HSPs in critical illness remains to be defined and is currently an active area of investigation. It is possible that extracellular HSPs are simply an epiphenomenon of illness severity reflecting induction of the heat shock response in critically ill patients. The experimental data mentioned earlier, however, indicate that extracellular HSPs are capable of modulating the innate immune system, and this biologic effect is of obvious potential significance to the critically ill patient. The difficulty of elucidating this significance is highlighted by the observations that increased extracellular HSPs correlate with improved survival in adult patients with trauma (4), but correlate with illness severity and mortality in children with septic shock (5). In addition, a recently developed multi-biomarker-based risk model to predict outcome in children with septic shock includes HSP72 as one of the major decision rules, with increased HSP72 serum concentrations being an indicator of increased mortality risk (33). Finally, there are now multiple studies in critically ill patients with diverse conditions that have shown a significant correlation between extracellular HSP72 levels and increased morbidity and mortality (34,35,36,37,38

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