The “Danger Hypothesis” and Innate Immune System - ACS Publications

MAY 1999 VOLUME 12, NUMBER 5 © Copyright 1999 by the American Chemical Society

Perspective New Concepts in Immunology Relevant to Idiosyncratic Drug Reactions: The “Danger Hypothesis” and Innate Immune System Jack P. Uetrecht* Faculties of Pharmacy and Medicine, University of Toronto, Toronto, Ontario, Canada M5S 2S2 Received November 16, 1998

Introduction Idiosyncratic drug reactions, sometimes referred to as hypersensitivity reactions or type B reactions, are adverse drug reactions that do not occur in most patients at any dose and do not involve known pharmacologic properties of the drug. Although they are often referred to as rare, with a typical incidence of from 1/100 to 1/100000, because of the total number of drugs involved and the number of patients treated, such reactions are actually common. Their unpredictable and serious nature makes them a significant clinical problem, and they also significantly hamper drug development. If we are ever to effectively deal with these adverse reactions, it is imperative that we come to better understanding of their underlying mechanism. The most prevalent hypothesis for the mechanism of idiosyncratic drug reactions is the hapten hypothesis (1-3). This hypothesis proposes that drugs, or more commonly reactive metabolites of drugs, act as haptens and irreversibly bind to proteins or other macromolecules. These altered proteins are “perceived” as foreign and induce an immune response. In most individuals, this immune response is asymptomatic, but in a few cases, it leads to pathology. There is a large amount of circumstantial evidence that supports the hypothesis that reactive metabolites are involved in idiosyncratic drug reactions (2, 4-11). For * To whom correspondence should be addressed. Telephone: (416) 917-8939. E-mail: [email protected].

example, halothane, which is associated with a relatively high incidence of serious idiosyncratic liver toxicity, is extensively metabolized to the reactive trifluoroacetyl chloride, and patients with halothane-induced hepatotoxicity have antibodies against trifluoroacetylated protein (12, 13). When the structure is modified to isoflurane or desflurane, which are metabolized to essentially the same reactive metabolite but to a lesser degree, the risk of liver toxicity is markedly reduced (14). Furthermore, the site of reactive metabolite formation usually correlates with the site of toxicity. For example, the oxidation of a C-H bond, as found in halothane, is essentially limited to cytochrome P450 with the result that most halothane oxidation occurs in the liver and the major toxicity is limited to the liver. Oxidation of vesnarinone to a reactive iminium ion occurs in neutrophils but not in the liver, and the dominant toxicity is agranulocytosis (15). Clozapine is oxidized to a reactive metabolite in both neutrophils and the liver, and clozapine is associated with both agranulocytosis and liver toxicity (16). These examples are illustrated in Figure 1. Many other such examples could be given. Idiosyncratic reactions involving the skin, which is a common site of such reactions, pose a conceptual problem because the concentrations of the enzymes most commonly involved in the formation of reactive metabolites are low, although they are not completely absent (17, 18). Furthermore, other cells that enter the skin can metabolize drugs (19). Another possible explanation is that,

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Figure 1. Illustration of the correlation between the amount and location of metabolism and the degree and type of idiosyncratic reaction.

although most reactive metabolites have a short biological half-life, some, such as acyl glucuronides, are reactive but freely circulate and may be responsible for idiosyncratic reactions in the skin (20). Others, such as the iminoquinone formed from carbamazepine, readily redox cycle and could undergo several such cycles before finally binding in the skin (21). Although the reactive metabolites of many drugs associated with a high incidence of idiosyncratic reactions have not been identified, many of the pathways can be postulated, and with LC/MS and the other sensitive analytical methods that are now available, it appears that most drugs form reactive metabolites to some degree. There seems to be a crude correlation between the amount of reactive metabolite formed and the risk that a drug will be associated with a high incidence of idiosyncratic drug reactions as illustrated by the comparison of halothane and isoflurane; however, this is difficult to prove because it is difficult to quantify the amount of reactive metabolite that is formed. The risk of an idiosyncratic drug reaction is often said to be independent of dose. Such reactions may appear to be independent of dose because most patients do not have an idiosyncratic reaction at any dose and the usual dose range is usually quite narrow; however, even with these confounding factors, a relationship between the dose and the incidence of an idiosyncratic reaction is often observed (22, 23). Obviously, if the total dose of a drug is low, the total amount of reactive metabolite that can be formed is limited, and drugs given at a daily dose of 10 mg or less are rarely if ever associated with a high incidence of idiosyncratic drug reactions. An alternative hypothesis to the reactive metabolite hypothesis has been proposed by Pichler in which a drug

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can act as a superantigen by noncovalently interacting with a major histocompatibility complex (MHC)1 molecule, resulting in a more general immune response (24, 25). They were able to generate clones of T cells from patients with idiosyncratic drug reactions that respond by proliferation on exposure to the responsible drug in the absence of metabolism. The observation that such cells can be found in patients with a recent history of an idiosyncratic reaction does not prove that such a noncovalent interaction can initiate an idiosyncratic reaction, and once an immune reaction is initiated by a covalent adduct of the drug, some of the cells that proliferate would probably also recognize drug alone. At this point, there is enough circumstantial evidence for the involvement of reactive metabolites to suggest that they are responsible for most idiosyncratic drug reactions. However, if evidence can be obtained that such cells are actually involved in the initiation of idiosyncratic drug reactions, it would provide a second pathway by which drugs could induce an immune response. There are some difficulties with the second half of the hapten hypothesis, specifically, that the damage is mediated by a classical antibody or cytotoxic T cell response to the hapten. Although there are notable examples in which antibodies that bind to the reactive metabolite acting as a hapten are found in patients with an idiosyncratic reaction to a drug (4, 12, 26, 27), in most cases such antibodies are not found. Even when such antibodies are found, as in the case of halothane hepatitis, it is not clear that these antibodies are pathogenic, and such antibodies could simply be a marker for an immune response (28). Helper T cells should be involved in either an antibody response or a cytotoxic T cell response, and this is the basis for the lymphocyte transformation test (29). The lymphocyte transformation test consists of taking cells from patients with a recent history of an idiosyncratic drug reaction and incubating them with the drug that is responsible for the reaction along with a source of antigen presenting cells and radiolabeled thymidine. In principle, T cells that are specific for the drug should be stimulated to proliferate and take up the thymidine. Except for patients with a history of reactions to drugs such as penicillin that are chemically reactive, this is an unreliable test with a high incidence of both false negative and false positive results (30-33). There are at least two possible reasons why this test could be falsely negative. One is that what the T cell recognizes is the reactive metabolite bound to protein acting as a hapten, and unless metabolism occurs in the cell incubation, the reactive metabolite would not be formed (34, 35). This would also explain why the test works better with drugs, like penicillin, that covalently bind without being metabolized. However, in studies by Kalish, using the reactive metabolite instead of the parent drug did not substantially increase the number of positive tests (36). The other factor that could lead to a false negative test is that the number of T cells specific for the drug is small (on the order of 1/100000) and it is difficult to detect proliferation of these cells in the midst of the other T cells (36). This problem can be approached with limiting dilution techniques in which varying numbers of lymphocytes are added to the wells of a multiwell plate and 1Abbreviations: MHC, major histocompatibility complex; APC, antigen presenting cell; TCR, T cell receptor.

Perspective

incubated with the drug responsible for the reaction (37). The cells are cultured for a longer period of time so that cells that are not stimulated by the drug die. From the number of wells that are positive, i.e., in which colonies form, and the number of cells that were originally placed in these wells, the incidence of the cells in the blood that are specific for the drug can be calculated. Even with this refinement, the lymphocyte transformation test is often negative; however, there have not been many studies published in which the use of reactive metabolite and limiting dilution techniques have been combined (36). It has been suggested that adding a prostaglandin synthase inhibitor increases the sensitivity of the test, but again even with the addition of such an inhibitor, almost half of the tests were still negative (38). There is another characteristic of some idiosyncratic drug reactions that strongly suggests that these reactions are not mediated by classical antibody- or cell-mediated immune reactions. Specifically, we have been puzzled by the fact that if a patient with a recent history of clozapine-induced agranulocytosis is rechallenged with clozapine, it usually takes just as long (about 6 weeks) for the onset of agranulocytosis as it did during the initial exposure (39, 40). This is very difficult to reconcile with an amnestic response of the immune system. Alternatively, clozapine-induced agranulocytosis could be due to direct cytotoxicity. However, the characteristics of the reaction are very difficult to explain on the basis of a direct cytotoxic reaction. Specifically, the reaction is idiosyncratic in humans; it has not been successfully reproduced in animals, and there is usually a delay of more than 1 month between starting the drug and the onset of agranulocytosis. It might be possible to explain the delay in onset on a slow buildup of a toxic agent or the slow depletion of some critical factor. However, if such a mechanism were responsible for the delay in onset, it should take a long time for the bone marrow to recover when the drug is stopped, but in general, it recovers very rapidly. Specifically, the average time it takes for the neutrophil count to return to normal in the circulation is 1 week (41), and therefore, the marrow must recover almost immediately because it takes about 1 week for neutrophil precursors in the bone marrow to mature and reach the circulation. There also appears to be a delay in the onset of agranulocytosis on reexposure to phenothiazines, and this has been used as evidence that it is a toxic reaction (42); however, these reactions are idiosyncratic, and their other characteristics do not really fit a simple cytotoxic mechanism. Many other idiosyncratic drug reactions may be associated with a delay on reexposure, but this is not well documented because, in most cases, patients are not rechallenged with a drug believed to be responsible for an idiosyncratic drug reaction. The major characteristic of idiosyncratic drug reactions that make them especially difficult to deal with is simply their idiosyncratic nature. It has been impossible to predict which patients will have an idiosyncratic reaction to a specific drug. Although the formation of a reactive metabolite appears to be necessary for the reaction, as illustrated by halothane, it also appears that most patients and animals form the relevant reactive metabolite without a clinically significant adverse reaction (43). Furthermore, although it is probably a risk factor in some patients, attempts to find differences in the formation or detoxication of a specific metabolite that would predict who will have an idiosyncratic reaction to a drug have

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not yet been successful (44-46). In the few cases where covalent binding can be detected in humans, it also occurs in patients who do not have an idiosyncratic reaction to the drug (47). It has also been postulated that since the reactions are likely immune-mediated, certain MHC genotypes should be linked to an increased risk of a specific idiosyncratic drug reaction. Here again, although weak associations with specific MHC genotypes have been detected, most such studies have been disappointing (48-50). Thus, we remain unable to predict individual susceptibility to idiosyncratic drug reactions, and a better understanding of the mechanism of these reactions will be required to make progress in this area. If, as is suggested by their characteristics, most idiosyncratic drug reactions are immune-mediated, it is likely that a better understanding of the mechanism will depend on advances in our understanding of immune-mediated reactions. The purpose of this perspective is to highlight relatively new concepts from immunology that have implications for the mechanisms of these difficult adverse reactions.

Danger Hypothesis For a long time, immunologists presumed that a major function of the immune system was to differentiate “self” from “nonself” and to respond to self with tolerance and to mount a response against nonself. In some cases, the immune system seemed to do it incorrectly and mounted a response against self, resulting in an autoimmune reaction. More recently, Matzinger (51) argued that it is difficult and inefficient to try to differentiate self from nonself and proposed an alternative hypothesis in which the immune system responded with tolerance to most antigens, and what triggers an immune response is presentation of an antigen in the context of a “danger signal” rather than the foreignness of the antigen. The exact nature and range of stimuli that can act as the danger signal remain to be determined, but certainly, cell damage must be a major stimulus for the production of the danger signal. In the more traditional view, tolerance of self is achieved, in part, by deletion of T cells that recognize self-antigen present in the thymus during maturation (52, 53). Another aspect of tolerance in the more traditional view is that lymphocytes require a second signal as well as signal 1, and without signal 2, the system becomes tolerant to the antigen. Signal 1 is the “recognition” by T cells of antigen. This recognition involves interaction between processed antigen imbedded in the MHC on antigen-presenting cells (APCs) and the T cell receptor (TCR) on T cells (54). The nature of signal 2 is not completely defined, but a principal component consists of the binding of CD28 on T cells to B7 (there are actually two B7 molecules, B7-1 and B7-2) on APCs (55). Other signaling molecules and soluble cytokines also play a role in mediating signal 2 (56). However, the requirement for signal 2 does not help to explain how tolerance to self antigens is achieved because self-antigens are presented by APCs in the same manner as foreign antigens. This view does, however, require that the APC be activated to produce an immune response to an antigen because activation of APC leads to the release of cytokines and the expression of B7 on the surface of the APC. This concept is similar to an essential aspect of the Danger Hypothesis.

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A major aspect of the Danger Model proposed by Matzinger is the laws of lymphotics (51). The first law states that a lymphocyte will die if it receives signal 1 without signal 2, and the second law states that a lymphocyte will accept signal 2 only from APCs. She goes on to postulate that thymocytes are unable to receive signal 2 from any source, and so if they encounter antigen they would be deleted, which is similar to the more traditional view. Additional laws are as follows. (1) Inexperienced or “virgin” T cells can only receive signal 2 from “professional” APCs (i.e., dendritic cells), while experienced T cells (those that have responded at least once to antigen) can also receive signal 2 from other APCs such as B cells or macrophages. (2) Resting B cells only accept signal 2 from experienced or effector, i.e., activated, T cells. (3) Effector B or T cells respond to signal 1 without requiring signal 2. (4) The effector cells then either revert to a resting state or die after a relatively short period of time so that the response does not get out of hand. The central determinant of an immune response in this model is the activation of APC, and Matzinger proposes that APCs are activated in the presence of “bad death” (i.e., necrosis) or cell stress (57). She proposes that the default response of the immune system is tolerance, and it is “danger” rather than nonself that leads to an immune response. In this model, apoptosis is not bad death and would lead to tolerance. Although the model presented by Matzinger is slightly more complex than this, it is likely that the truth will be found to be even more complex than presented by the Danger Model. However, the Danger Model may be very useful for understanding many aspects of idiosyncratic drug reactions as described below. There are many examples in which specific situations that could mediate a danger signal are associated with an increase in the risk of an idiosyncratic drug reaction. For example, it has been known for a long time that when ampicillin is given to patients with mononucleosis, there is a marked increase in the risk of an idiosyncratic reaction that approaches 100% (58). More recently, it has been observed that patients who are HIV positive have a much higher risk of idiosyncratic reactions to sulfonamides and other drugs (59, 60). Even an influenza vaccination appears to increase the risk of idiosyncratic drug reactions as revealed by studies of vesnarinoneinduced agranulocytosis (15). Open-heart surgery, which clearly should produce a danger signal, appears to increase the risk of procainamide-induced agranulocytosis by a factor of 10 (61, 62). Many other such factors that represent a danger signal probably remain to be discovered. Especially relevant to adverse drug reactions, since reactive metabolites are necessary for the formation of haptens, these reactive metabolites could also cause sufficient cell stress or necrosis to result in a danger signal without causing serious direct toxicity. It is also very important to point out that although surgery appeared to increase the incidence of procainamide-induced agranulocytosis by a factor of 10, 95% of the patients still did not develop agranulocytosis. This suggests that there is a mechanism or mechanisms by which the immune system can downregulate potentially harmful immune responses before they do too much damage. Such mechanisms are probably under genetic control. As proposed by the Danger Hypothesis, the usual response to a hapten is tolerance. Many drugs, e.g.,

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acetaminophen, form reactive metabolites but at usual doses are not associated with a significant incidence of idiosyncratic drug reactions. It also appears that starting with a low dose of a drug and then later increasing to a therapeutic dose increases tolerance (63). The lower dose may lead to tolerance because the resultant decrease in the amount of reactive metabolite formed may result in apoptosis rather than in necrosis or simply less cell stress, and therefore, there is no danger signal. Another observation that can be explained by the Danger Hypothesis is the time course of drug-induced autoimmune reactions. Drugs, such as R-methyldopa, cause an autoimmune hemolytic anemia (64, 65), and other drugs, such as procainamide, cause a lupus-like syndrome that is associated with a broader range of autoantibodies (66). As implied by the term autoantibody, these antibodies bind to self-antigens in the absence of drug. Therefore, even if the administration of the drug is stopped, the antigenic stimulus remains, and one might expect the reaction to continue. In fact, although antibodies may be detected for some time, as soon as the drug is discontinued the clinical syndrome usually is resolved quite rapidly (67). This can be explained on the basis of the Danger Hypothesis where the drug, presumably due to a reactive metabolite, is responsible for the danger signal. According to the laws of lymphotics, effector cells either revert to a resting state or die after a relatively short period of time; therefore, in the absence of a danger signal, the model predicts rapid resolution. Although the immune response may kill cells, cell death mediated by the immune system is usually apoptotic, and this should not contribute to a danger signal.

Innate Immune System For several decades, immunologists have concentrated on the adaptive immune system in which antigen is processed and presented in the context of MHC-II to helper T cells by APCs (54). These cells, in turn, stimulate B cells to differentiate and proliferate into antibodyproducing plasma cells and/or stimulate cytotoxic T cells that recognize the same antigen, although in the form of peptides derived from the antigen presented in the context of MHC-I instead of MHC-II. With the aid of gene rearrangements, this system is able to respond to an almost infinite number of antigens in a very specific manner. Almost forgotten during these studies was the innate immune system. The innate immune system was viewed as a primitive system present in invertebrates but less important in mammals, which also possess an adaptive immune system. However, invertebrates, which do not possess an adaptive immune system, deal quite effectively with pathogens. Furthermore, there has been a recent realization that the adaptive immune system does not operate in isolation and the innate immune system is probably also very important in mammals (68). The innate immune system can only respond to stimuli that have been encoded in an organism’s DNA because the gene rearrangements that make an adaptive response possible do not occur in the cells of the innate system (69). The cells most important for an innate response are granulocytes, macrophages, NK cells, and γδ T cells (70). The types of constant structures that stimulate an innate response are molecules, such as lipopolysaccharides, that are present in the cell wall of many bacteria, viral DNA, which is hypomethylated, and several nonpeptidic phos-

Perspective

phoantigens that are derived from various infectious agents (69, 70). However, the full range of structures to which the innate immune system can respond has yet to be determined, and it is an active field of research at the present time. The innate system also differs from the adaptive system in that antigen can be recognized by γδ T cells in a non-MHC-dependent manner (71). However, it appears that cell-surface receptors for MHC-I molecules do play an important role in the control of the innate response (71). Another factor that appears to stimulate an innate response is cell stress (72). This would seem to be an important feature in invertebrates for the detection of cells infected by viruses or other damaged cells that could be harmful because the innate system is not able to respond to new and varied antigenic structures. Although the innate immune system can deal with pathogens and damaged cells directly, in mammals, another important function of the innate system may be to determine whether the adaptive immune system will respond to a stimulus with tolerance or an active immune response (69, 70). Thus, it may be the innate immune system that delivers the danger signal described in the previous section. Adjuvants often contain agents such as lipopolysaccharide that stimulate the innate immune system. Janeway referred to adjuvants as the immunologist’s “dirty little secret”, because without them pure antigens usually produce little or no response (73). The innate system has several mechanisms by which it can stimulate an adaptive response. An innate response can lead to the secretion of cytokines by macrophages and NK cells, the attachment of complement to antigen, and the expression of B7 on APCs (72, 73). However, there is no need to postulate that the innate immune system is required to provide the danger signal, and APCs may be able to detect danger without the involvement of the innate immune system. Furthermore, Matzinger believes that the danger signal originated even before the innate immune system (57). When the immune system is understood in detail, it is likely that the innate and adaptive immune systems will be found to be closely interdependent, and the distinction between them may begin to blur. It seems likely that the innate immune system could be involved in the mechanism of some idiosyncratic drug reactions, yet this does not appear to have been considered previously. The characteristics of the innate immune system could easily explain the perplexing time course of clozapine-induced agranulocytosis on reexposure, i.e., the lack of a rapid amnestic response, because there are no memory cells in the innate immune system (73). Since stressed cells seem to be able to stimulate the innate immune system, it is obvious how a drug that produces reactive metabolites could lead to an innate immune response. There are several other examples of idiosyncratic reactions that have characteristics that suggest mediation by the innate immune system. For example, the aminopenicillins, ampicillin and amoxicillin, cause a rash in children that is often described as a “toxic” rash (74). However, these rashes are idiosyncratic, and there is usually a delay between starting the drug and the onset of the rash which makes it unlikely that they are true toxic reactions. These rashes are often seen in patients who have a viral infection; they often do not recur if the patient takes the drug again, and skin tests are usually negative (75). In some cases, these may be

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delayed-type hypersensitivity reactions (75), but in our experience, even in adults, the skin tests are usually negative and the reaction does not usually occur on reexposure. Such characteristics may be explained by an innate immune response. The adverse reactions of HIV positive patients to sulfamethoxazole and other drugs also may be mediated by the innate immune system because they often do not recur on reexposure and occur in the context of a danger signal (76). However, as mentioned above, the distinction between an innate reaction and an adaptive response may blur because a reaction mediated by the innate system may stimulate an adaptive response. The adaptive response may contribute to pathogenesis or may simply be an epiphenomenon. A model of idiosyncratic drug reactions that incorporates the Danger Hypothesis and the innate immune system is shown in Figure 2. In this figure, the drug is denoted by the letter R and the electrophilic metabolite that binds to protein is denoted by R+. If only a small amount of reactive metabolite is formed, a significant response is unlikely. If more reactive metabolite is formed, when the cell undergoes apoptosis, either because of senescence or because the reactive metabolite led to apoptosis, the cell will undergo phagocytosis. The haptenized proteins will be processed and presented as haptenized peptides to T cells in the absence of signal 2 as shown in the left arm of the figure. (The processed peptides bound to MHC-II are shown in Figure 2 as a shorter structure than the original haptenized protein.) Presentation in the absence of signal 2 is likely to lead to immune tolerance to the drug, or more accurately, tolerance to the reactive metabolite of the drug acting as a hapten. If the reactive metabolite is more cytotoxic, either because of the amount formed or because of the nature of the reactive metabolite, it may lead to cell stress or necrosis. This would lead to a danger signal and upregulation of B7 on the APC as illustrated in the central arm of Figure 2. Although in the Matzinger Danger Model apoptosis leads to tolerance while necrosis induces an immune response, there is evidence that antigen from apoptotic cells can induce an immune response (77). There could also be some environmental agent, such as an infection, that acts as a danger signal and upregulates B7. Alternatively, cells of the innate immune system could detect cell stress or some other danger signal, and they could produce cytokines or other factors that could upregulate B7 as illustrated in the right arm of Figure 2. Alternatively, and not shown in Figure 2, the innate system-derived factors might provide signal 2 by directly stimulating the helper T cells. In Figure 2, it is implied that the immune response, either antibody-mediated or cell-mediated, would be against the haptenized peptide; however, it is common to find antibodies against native proteins that have not been modified by reactive metabolite (78-80). One mechanism by which this could happen is one in which haptenization can lead to a change in the processing of proteins and the presentation of peptides not usually formed by the protein. When peptides from self-proteins are presented to the immune system that have not been presented before, or have been presented in much smaller quantities that would not have induced tolerance, these peptides are called cryptic peptides (81, 82). In the Matzinger model, the concept of self-proteins is not used,

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Figure 2. Illustration of the proposed mechanisms of idiosyncratic drug reactions that involve the danger hypothesis and the innate immune system. See the text for details.

but the important concept is that these cryptic peptides would not have previously induced tolerance and could, in the presence of a danger signal, induce an immune response. This may lead to an immune response directed against the native protein. It is also possible that a hapten-modified protein resembles an infectious agent, leading to a break in the tolerance to the protein. This is called molecular mimicry (46, 83, 84). By either mechanism, true autoimmune responses could be initiated. Finally, the danger signal may stimulate cells of the innate immune system, and these cells might directly mediate an idiosyncratic reaction. Exactly how this could occur is unknown, but a simple hypothesis is that macrophages, NK cells, and/or γδ T cells might detect a danger signal on cells that have been modified by reactive metabolite. These cells would become the target cells,

either because they formed a large amount of reactive metabolite or because they were more sensitive to the toxic effects of the reactive metabolite. The danger signal detected by cells of the innate immune system would not have to be the same as that detected by APCs in the adaptive immune system. The cells of the innate immune system could directly induce apoptosis in the affected target cells. Although the involvement of the innate immune system in idiosyncratic reactions has not been demonstrated, there is evidence that the innate immune system may be involved in other types of xenobiotic toxicity. Specifically, agents such as acetaminophen and carbon tetrachloride cause liver toxicity that is not idiosyncratic. Although there is a large amount of evidence to support the involvement of reactive metabolites in such reactions, despite extensive investigation, the

Perspective

final pathway leading to cell death has not been elucidated. One observation in such reactions is that the toxicity is prevented if Kupffer cells, i.e., liver macrophages, are inhibited by agents such as gadolinium chloride (85, 86). Since macrophages are an important part of the innate immune system and there is no evidence of involvement of the adaptive immune system in such reactions, this may be an example of the involvement of the innate immune system in the mechanism of drug toxicity. However, these are not idiosyncratic reactions. It follows from this hypothesis that, in addition to acting as a hapten, a reactive metabolite may have to cause cell damage that leads to a danger signal if it is to induce an idiosyncratic drug reaction. This might explain why not all drugs that form reactive metabolites are associated with a high incidence of idiosyncratic drug reactions. Furthermore, if the reactive metabolite of a drug caused more toxicity to the cells of a specific individual, that individual may be at increased risk of having an idiosyncratic reaction to that drug. This would explain the observation that the cells from a patient who has had an idiosyncratic adverse reaction to a drug will often be more sensitive to the toxic effects of the reactive metabolite of that drug than cells from controls. This was noted by Spielberg some years ago with anticonvulsants (87), confirmed by other investigators (88), and also observed with sulfonamides (89) and also more recently by us with cells from patients with a history of clozapineinduced agranulocytosis (90). This was ascribed to a defect in the ability of the cells to detoxify the reactive metabolite; however, there is no evidence to support this hypothesis, and there are many other possible mechanisms that would cause these cells to be more sensitive to the toxicity of the reactive metabolite. It has long been a puzzle to us why an acute cytotoxic response of a patient’s cells would predict what appears to be a delayed immune-mediated reaction in these patients; however, it does fit the Danger Hypothesis. Unfortunately, although there are clear differences between the populations who have had an idiosyncratic reaction to these drugs and controls, there is overlap between the two populations and such a response is not a reliable test of who will have a reaction to a specific drug. However, the exaggerated response to a reactive metabolite appears to provide clues as to where to look for the basis for the increased susceptibility. First, the difference appears to be genetic because first-degree relatives also usually have an increased response to the same reactive metabolite (91). In general, the response is usually specific for a given drug so those patients whose cells are more susceptible to the reactive metabolite of phenytoin are not more sensitive to the reactive metabolite of sulfamethoxazole. However, for some unknown reason, there is a high degree of cross-reactivity between the anticonvulsants phenytoin, carbamazepine, and phenobarbital (92). It may require gene chip technology to determine what genes are important in the determination of the genetic component of an increased risk of an idiosyncratic reaction to a specific drug. It must also be repeated that even when a drug is given in the context of a clear danger signal, such as surgery or an HIV infection, although the incidence of idiosyncratic reactions increases, most patients do not have an idiosyncratic reaction to a drug given at that time. This may be due to mechanisms, probably under genetic control, that downregulate po-

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tentially harmful immune reactions. In addition to environmental factors, such as infections, that influence the risk of an idiosyncratic reaction, there is the effect of dose on tolerance. As mentioned above, a low dose of a drug appears to increase the probability of tolerance to a higher dose. Once tolerance is induced by any mechanism, it is likely to be more difficult to break tolerance if a patient is exposed to some environmental factor that could increase the risk of an idiosyncratic reaction. However, if exposure to the drug is not continuous, tolerance may be lost. Such a complex interplay of genetic and environmental factors may explain why it is so difficult to confidently predict who will have an idiosyncratic drug reaction to a specific drug and so difficult to reproduce idiosyncratic drug reactions in animals. If the concepts presented in this perspective are relevant to idiosyncratic drug reactions, they may help attempts to understand the underlying mechanisms and deal with these difficult adverse reactions.

Note Added in Proof Earlier in vitro studies suggested that there is very little oxidation of vesnarinone to a reactive metabolite in the liver; however, we have just found evidence in rats in vivo using an antibody against vesnarinone that there is significant covalent binding in the liver.

Acknowledgment. The research was supported by the Medical Research Council of Canada (Grants MT9336 and MT13478).

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