Clinical Implications

A. Thermotolerance

Are the biochemical and functional characteristics of the heat shock response/ proteins as defined by cell culture experiments relevant to organs and tissues in the whole organism? Examination of tissues and organs subjected to various metabolic insults such as ischemia or fever revealed that the stress response does occur in different tissues/organs in vivo (reviewed in ref. 6). In addition, it has been observed that seizures and excitatory amino acids such as glutamate can induce a stress response in the brain (22,23). Increased synthesis of stress proteins was also observed in the rat heart subjected to hemodynamic overload (24). These findings that physiologically relevant insults can induce the stress response in vivo have led to studies examining whether the phenomenon of cellular thermotolerance is operative in the animal. Preliminary work from a number of laboratories appears promising in this regard. For example, rodents subjected to whole body hyperthermia (which was demonstrated to result in increased levels of the stress proteins in the heart) suffered less myocardial damage in response to a subsequent ischemia/reperfusion episode (25). Similar results were found in a rabbit model (26). In contrast, Yellon et al. did not observe such a protective effect, but their study used a longer ischemia/reperfusion insult (27). Taken together, these studies on the heart indicate that hyperthermic treatment can render the myocardium more resistant to ischemia/reperfusion-induced damage up to a certain point. Similarly, rat brains first rendered thermotolerant appeared less vulnerable to ischemia reperfusion-induced damage as well as to the deleterious effects of glutamate stimulation (23). In rodents first subjected to hyperthermia, the extent of retinal damage due to a subsequent intense light exposure was greatly diminished. Moreover, these investigators showed that maximal protection correlated with the overall levels of the highly stress-indue -ible hsp 72 in the retina (28). Last, in a rat model of the human adult respiratory distress syndrome, rats first made thermotolerant (via whole body hyperthermia) suffered no mortality as compared with 27% mortality observed for the nonheated control group (29).

Results such as these have stimulated efforts at possibly harnessing the protective effects of the thermotolerant phenotype in clinically relevant situations. One important issue is how to develop the thermotolerant phenotype in a timely and clinically relevant fashion. In vitro, the phenotype requires anywhere from 8 to 18 hr from the time of induction of the stress response to develop fully, which is likely the time period necessary to synthesize and accumulate maximal levels of the stress proteins. In the case of the heart, it is not possible to predict the occurrence of an acute myocardial infarction. Those individuals at high risk for an imminent infarction are typically already experiencing severe ischemia. The extent to which these ischemic episodes in humans induce a stress response remains an open question. Even if a pharmacological means of rapidly inducing the thermotolerant phenotype (on the order of minutes to perhaps a few hours) is developed, the applicability to acute clinical situations is unclear. Application of the stress response to clinical medicine is more promising in nonemergent situations such as scheduled surgery. For example, reconstructive surgeons have found that skin flap survival improves if the flap is first made thermotolerant (30). Other possible applications include making donor transplant organs thermotolerant and thereby possibly increasing the window of time that the organ can be transplanted and/or possibly even improving survivability once transplanted. The enthusiasm for exploiting the stress response in clinical medicine must be tempered, however, by the paucity of information regarding the price the cell pays for becoming thermotolerant. In particular, we still do not know all of the other cellular consequences associated with the acquisition of the thermotolerant phenotype.

B. Role of Stress Proteins in Cancer Therapy

The ability to modulate the stress response also has therapeutic implications as it relates to cancer. The increased expression of hsp 28 and hsp 72 has been associated with enhanced survival of tumor cells subjected to some cancer che-motherapeutic agents (reviewed in ref. 31). The increased expression of the multidrug resistance protein (MDR), whose corresponding gene contains an appropriate heat shock element, has been clearly shown to underlie the development of resistance to many cancer chemotherapeutic agents (reviewed in ref. 32). Thus, finding a way by which to downregulate or even prevent the expression of stress proteins in malignant cells may enhance the efficacy of many chemotherapeutic agents. Such an advance might allow the use of lower doses of chemotherapy, and thus perhaps minimize the toxic side effects of these agents on normal cells. In preliminary experiments, a flavonoid compound, querecetin, reported to be an inhibitor of protein kinases, was shown specifically to inhibit the expression of the stress proteins, although the mechanism by which such inhibition is manifested is not known (33).

C. Neuroendocrine Mechanisms as Stress Protein Modulators

The recent evidence that the expression of the stress proteins also may be regulated, at least in part, by neuroendocrine mechanisms represents an exciting new development. Activation of the hypothalamic-pituitary axis was shown to induce specific expression of the most highly stress induced protein, hsp 72, in the rat adrenal cortex. Hypophysectomy ablated the response, and the addition of adrenocorticotropic hormone restored specific expression in the hypophysecto-mized rats. In contrast, the sympathetic nervous system appeared to be impor tant in the regulation of both hsp 72 and hsp 27 expression in the rat aorta. Adrenergic antagonists were found to block such expression, whereas adrenergic agonists induced their expression (34-36). Last, a dopamine agonist induced expression of hsp 72 in both the adrenal cortex and aorta of the rat (37). Clearly, these findings suggest that adrenergic and dopaminergic agents, as well as drugs that affect the hypothalamic-pituitary axis, have potential as modulators of the heat shock response in humans.

D. Role of Stress Proteins in Immunity

Investigation of possible clinical implications of the stress response/proteins gained added impetus as evidence began to accumulate that one or more of the stress proteins play a role in various aspects of the immune system (reviewed in ref. 6 and 38). First, genes encoding two members of the hsp 70 family were found to reside within the major histocompatibility complex (MHC). Moreover, computer modeling of the carboxy-terminal domain of hsp 70 family members, thought to be involved in the binding of both small peptides as well as unfolded polypeptides, revealed a possible binding motif very similar to the peptide binding cleft of the MHC class I proteins. Second, a peptide binding protein, termed PBP 74, was identified and shown to be related to the other members of the hsp 70 family. PBP 74 has been proposed be involved in peptide loading of MHC class II molecules. Third, deoxyspergulain, an immunosuppressant agent whose mechanism of action is unknown but appears to be distinct from that of both cyclosporin A and FK506 was found to specifically bind to hsp 73 (39). As mentioned earlier, FK506 appears to bind to hsp 56 (a protein with rotamase activity) and together the complex appears to have immunosuppressive activity (8). Finally, two of the 11 self-peptides isolated from purified class I HLA-B27 were shown to be peptides derived from hsp 90. Although these types of observations clearly are intriguing, they remain primarily phenomenological in nature, and they therefore will require further study to ascertain their biological relevance.

More compelling and scientifically developed is the observation that stress proteins from a variety of pathogens act as immunodominant antigens in animals (reviewed in refs. 40 and 41). For many years, it had been known that bacteria produced an approximately 60-kDa genus-specific protein; an antibody raised against this protein from one species tended to recognize the protein in all other species of the genus but not in any species from another genus. This protein was shown to be GroEL, the bacterial homologue of hsp 60, and it is a major target of the mammalian humoral response to bacterial infections. Interestingly, in many parasitic infections, it is the parasitic form of hsp 70, and in some cases hsp 90, which represents a major target for the humoral arm of the immune response. Recent evidence indicates that at least some parasitic and bacterial stress proteins can also induce a relatively strong T-cell response. Moreover, roughly 10-20% of y/8 T cells, a poorly understood population of T cells, have been shown to be specific for stress proteins of various pathogens (42). A particularly attractive hypothesis is that this class of T cells, which by lining the airway, gut, and epidermal epithelium, is well positioned to provide an early line of immune defense at major body-environment interfaces where pathogen entry into the host is likely to occur.

Does the immune response to bacterial and parasitic stress proteins protect the host from infection? A number of studies suggest that the answer is yes. Protection against chlamydial diseases was associated with an immune response to chlamydial DnaK, the bacterial homologue of hsp 70 (43). In a guinea pig model of legionnaire's disease, immunization with GroEL purified from Legionella pneumophila was effective in preventing disease (44). Immunization of mice with an 80-kDa protein from Histoplasma capsulatum, a protein related to hsp 70, resulted in improved resistance to infection (45). In a monkey model of malaria, immunization with Plasmodium falciparum hsp 70 prevented infection with blood stages of P. falciparum (46). In addition, infected hepatocytes appeared to express a cell surface epitope from this same hsp 70. An anti-hsp 70 antibody recognizing this epitope appeared to be effective in an antibody-depen-dent cytoloysis of the infected hepatocyte (47). Thus, these examples indicate that the immune response to stress proteins from various infectious pathogens is associated with protection for the host.

Why does the immune system appear preferentially to target the stress proteins of infectious pathogens? Several possible reasons that are not mutually exclusive have been suggested (38,40,41). The stress proteins represent a relatively abundant set of proteins within the invading pathogen, and, therefore, on a purely statistical basis, the immune system may be more likely to recognize these "foreign" proteins rather than a less abundant one. In this regard, it also has been reported that infection not only induces a stress response in the host but also in the invading pathogen (thereby increasing the levels of "pathogenic stress proteins") (48). Alternatively, faced with a vast number of potential pathogens, the immune system may have simplified the problem of detection by taking advantage of the fact that stress proteins are essential components of any organism (i.e., most of the genes encoding the stress proteins are essential for growth) and they exhibit a high degree of homology. For example, recognizing genus-specific GroEL may have enabled the immune system to strike a balance between the need for sensitivity (i.e., the ability quickly to recognize the presence of a pathogen) and the need for specificity (i.e., responding only to pathogenic bacteria as opposed to normal commensal flora such as exists within the intestines). Similarly, by preferentially recognizing parasitic hsp 70, the immune system can distinguish between a bacterial versus a fungal infection.

The association of a protective humoral response against pathogen stress proteins lies at the crux of an apparent conundrum. Stress proteins have not been characterized as being localized to the cell surface or as being secreted. How then does the humoral response confer protection? One mechanism could be the earlier described antibody-dependent cell cytolysis of hepatocytes infected with P. falciparum which present the pathogen's hps 70 on the surface of the infected cell. Yet, many bacterial and parasitic pathogens produce their deleterious effects without ever entering host cells. One intriguing study suggested that GroEL from iSalmonella typhimurium mediated binding of the bacterium to intestinal mucus (49). Although GroEL appeared to be secreted, it apparently was also present on the cell surface. Interestingly, antibodies against GroEL blocked Salmonella aggregation on the intestinal mucus. No biochemical evidence was presented demonstrating a direct interaction between GroEL and the previously identified 15-kDa glycoprotein component of intestinal mucus that mediated binding of S. typhimurium. Nevertheless, this report suggests that GroEL, by a change in its locale to the cell surface and/or its secretion, may act as a "virulence factor" in 5. typhimurium infection. Other pathogens may have evolved similar mechanisms of infection which may account for the observation that the humoral immune response to stress proteins has been associated with protection. Indeed, chlamydial DnaK has been observed on the surface of elementary bodies, although its function at this site is unclear (43). As mentioned earlier, antibodies against chlamydial DnaK were associated with protection from the disease.

What about cell-mediated immunity against pathogenic stress proteins? Most bacterial and parasitic agents appear to induce such an immune response, but in some cases, rather than protecting the host, the response tends to exacerbate or contribute to the disease. For example, T-cell response to chlamydial GroEL has been associated with infection of the female reproductive tract as well as the respiratory system (50). Similarly, T cells that recognize GroEL from Borrel-ia burgdorferi, the etiological agent of Lyme disease, line the synovium of joints affected by the arthritic component of the disease (51). These types of observations have raised the question of whether these T cells were part of a general inflammatory response in these illnesses, or instead directly caused damage, perhaps by cross reacting with self-stress proteins. If the latter were true, it would imply that the well-conserved stress proteins contribute in some way to autoimmune diseases.

The idea that stress proteins play some role in different autoimmune diseases remains highly controversial (52). There are a few animal studies whose results are suggestive but by no means support a direct role for stress proteins in autoimmune disease. For example, T-cell reactivity to mycobacterial GroEL appeared to be important in the development of disease in the nonobese diabetic

(NOD) mouse model of insulin-dependent diabetes (reviewed in ref. 53). More recent work, however, suggested that these GroEL-reactive T cells somehow modulated the immune response rather than directly causing diabetes in the NOD mouse (54,55). Those T cells that recognized mycobacterial GroEL in the adjuvant-induced arthritis rat model probably play a similar modulatory role (reviewed in ref. 53). With regard to human autoimmune diseases and stress proteins, other than juvenile chronic arthritis (JCA), most studies have demonstrated guilt only by association. To date, only the synovial T cells from patients with JCA have been shown to respond strongly to human hsp 60 (56). Otherwise, the literature is filled with studies that are mainly of a phenomenological nature; either they refute (several) the presence of an association between stress proteins and most human autoimmune diseases, or they confirm an association (many) but do not answer the fundamental question of cause and effect.

The discovery that T cells reactive to self stress proteins are present normally in otherwise healthy individual led to the proposal that these T cells may recognize other cells undergoing a stress response (e.g., due to some type of an infection or transformation) and thereby help to eliminate these cells (38). Whether a particular stress event, be it infection, transformation, or some other type of metabolic insult, results in the processing and presentation of peptides derived from self stress proteins to the immune system remains an extremely interesting but unanswered question. Studies with established cell lines have demonstrated that the intracellular locale of many stress proteins changes in response to stress, but localization to the cell surface, either as an intact protein or as a peptide associated with the histocompatibility complex, has not been clearly demonstrated. However, those observations mentioned earlier in which 2 of 11 self-peptides present within the MHC class I molecule were derived from hsp 90 implies that cells may normally present self peptides derived from stress proteins on a routine basis. That different forms of stress in vivo may lead to an increase and/or alteration in the presentation of self stress peptides, thereby providing for some type of activating signal to the immune system is an interesting question which deserves additional study.

Further fueling the idea that stress proteins are important in immune cell recognition are studies reporting that both grp 94 and hsp 73 behaved as tumor-rejection antigens. Both of these stress proteins, when purified from a particular tumor and subsequently used as an immunogen, conferred protection to challenge from that same tumor but not to an antigenically distinct tumor. Grp 94 and hsp 73 from normal cells provided no such tumor immunity, and sequence analysis of the purified stress proteins isolated from the tumors revealed no differences when compared with the proteins isolated from nontumorigenic cells. Subsequent work, however, reported that both hsp 73 and grp 94 bound to a heterogeneous population of peptides (57,58). Presumably, the associated peptides found with grp 94 and hsp 73 from the tumor cells were responsible for the successful tumor immunity, but the direct demonstration that the bound peptides, when used as the immunogen, could confer tumor resistance remains to be shown. Thus, these data suggest that stress proteins are involved in processing proteins for antigenic presentation rather than being immunogenic themselves.

E. Stress Proteins as Vaccines and Adjuvants

There is increasing excitement and enthusiasm for the use of mycobacterial and parasitic forms of the stress proteins as novel acellular vaccines and/or carrier-free adjuvants. For example, mice primed with BCG and then immunized with a hapten conjugated to tuberculin PPD produced long-lasting and high titers of anithapten antibodies without the use of adjuvants (59). The same effect was observed if the hapten was conjugated directly to mycobacterial GroEL or DnaK. Importantly, this effect occurred even in the animal showing high titers of antibody against the mycobacterial GroEL or DnaK stress proteins used as the adjuvant. In fact, high doses of GroEL were shown to be as effective a primer as BCG. However, the most provocative and exciting finding was that an effective T cell-mediated response could be induced by hapten conjugated to DnaK, without the need for either an adjuvant or for previous priming (60). Clearly, these results have broad implications for vaccine development against infectious diseases and tumors.

F. Role of Stress Proteins in Toxicology

Although too extensive in scope to adequately present here, we should mention briefly that changes in stress protein expression may prove useful as it relates to toxicology. In the hope of developing rapid assays as well as reducing the use of animals, toxicologists are exploring the use of changes in the expression of one or more of the stress proteins, in cells grown in vitro, as a sensitive and reliable indicator of the possible toxic effects of different compounds. As a further extension of this type of technology, investigators are developing transgenic stress reporter organisms. Using well-defined heat shock promoter elements to drive the expression of a reporter gene (luciferase, B-galactosidase, chloramphenicol acetyl transferase), the reporter organism carrying such a construct would be employed, for example, in the monitoring of environmental pollutants (61). Although such approaches are still at an exploratory stage, requiring substantial validation efforts, exploiting changes in the expression of the stress proteins as well as other gene products associated with cellular injury (metallothionines, cytochrome 450 system) may revolutionize the field of toxicology.

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