In vitro analysis of metabolic predisposition to drug hypersensitivity reactions

Idiosyncratic hypersensitivity reactions may account for up to 25% of all adverse drug reactions, and pose a constant problem to physicians because of their unpredictable nature, potentially fatal outcome and resemblance to other disease processes. Current understanding of how drug allergy arises is based largely on the hapten hypothesis: since most drugs are not chemically reactive per se, they must be activated metabolically to reactive species which may become immunogenic through interactions with cellular macromolecules. The role of drug metabolism is thus pivotal to the hapten hypothesis both in activation of the parent compound and detoxification of the reactive species. Although conjugation reactions may occasionally produce potential immunogens (for example, the generation of acylglucuronides from non-steroidal anti-inflammatory drugs such as diclofenac), bioactivation is catalysed most frequently by cytochrome P450 (P450) enzymes. The multifactorial nature of hypersensitivity reactions, particularly the role of often unidentified, reactive drug metabolites in antigen generation, has hampered the routine diagnosis of these disorders by classical immunological methods designed to detect circulating antibodies or sensitized T cells. Similarly, species differences in drug metabolism and immune system regulation have largely precluded the establishment of appropriate animal models with which to examine the immunopathological mechanisms of these toxicities. However, the combined use of in vitro toxicity assays incorporating human tissues and in vivo phenotyping (or, ultimately, in vitro genotyping) methods for drug detoxification pathways may provide the metabolic basis for hypersensitivity reactions to several drugs. This brief review highlights recent efforts to unravel the bases for hypersensitivity reactions to these therapeutic agents (which include anticonvulsants and sulphonamides) using drug metabolism and Immunochemical approaches. In particular, examples are provided which illustrate breakthroughs in the identification of the chemical nature of the reactive metabolites which become bound to cellular macromolecules, the enzyme systems responsible for their generation and (possibly) detoxification, and the target proteins implicated in the subsequent immune response.     

Immune pathomechanism and classification of drug hypersensitivity

Drug hypersensitivity reactions (DHR) are based on distinct mechanisms and are clinically heterogeneous. Taking into account that also off‐target activities of drugs may lead to stimulations of immune or inflammatory cells, three forms of DHR were discriminated: the allergic‐immune mechanism relies on the covalent binding of drugs/chemicals to proteins, which thereby form new antigens, to which a humoural and/or cellular immune response can develop. In IgE‐mediated drug allergies, a possible tolerance mechanism to the drug during sensitization and the need of a covalent hapten‐carrier link for initiation, but not for elicitation of IgE‐mediated reactions is discussed. The p‐i (“pharmacological interaction with immune receptor”) concept represents an off‐target activity of drugs with immune receptors (HLA or TCR), which can result in unorthodox, alloimmune‐like stimulations of T cells. Some of these p‐i stimulations occur only in carriers of certain HLA alleles and can result in clinically severe reactions. The third form of DHR (“pseudo‐allergy”) is represented by drug interactions with receptors or enzymes of inflammatory cells, which may lead to their direct activation or enhanced levels of inflammatory products. Specific IgE or T cells are not involved. This classification is based on the action of drugs and is clinically useful, as it can explain differences in sensitizations, unusual clinical symptoms, dependence on drug concentrations, predictability and immunological and pharmacological cross‐reactivities in DHR.     

Attempts to modulate the immune response to a hapten-carrier complex with various hapten-containing compounds.

The immune response to a hapten-carrier conjugate appears to be a complex phenomenon where reactions of the T-cell population are not restricted to the carrier and where the reactions of the B-cell population are not limited to the hapten determinant of the antigen molecule. To get a better understanding of the different cell interactions during the immune response to a hapten-carrier complex, the effects of immunogenic or tolerogenic injections of various hapten-containing compounds on the responses induced by immunization with the same hapten coupled to protein carriers were studied. The results indicate that T cells involved in delayed hypersensitivity and T cells involved in contact dermatitis could belong to distinct subclasses and confirm that hapten and carrier moieties of the antigen molecule could compete, probably at the macrophage level, for both delayed hypersensitivity to the carrier and antibody synthesis to the hapten.     

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T‐cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p‐i concept appears to be more relevant than presentation of hapten‐modified peptides. In some HLA‐associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA‐B*15:02, certain T‐cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the ‘missing links’ in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T‐cell response by interacting specifically with HLA‐B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA‐B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.     

Immune mechanism of drug hypersensitivity

Drug hypersensitivity reactions can lead to a great variety of different diseases. The main cause is a specific interaction of antibodies or T cells with a drug. In addition to the hapten concept, some drugs can bind directly to T-cell receptors and stimulate them. Based on recent investigation on different exanthemas, an extended classification of the Gell and Coombs type IV reaction is proposed.     

Long-lasting reactivity and high frequency of drug-specific T cells after severe systemic drug hypersensitivity reactions

BACKGROUND: Drug-reactive T cells are involved in most drug-induced hypersensitivity reactions. The frequency of such cells in peripheral blood of patients with drug allergy after remission is unclear. OBJECTIVE: We determined the frequency of drug-reactive T cells in the peripheral blood of patients 4 months to 12 years after severe delayed-type drug hypersensitivity reactions, and whether the frequency of these cell differs from the frequency of tetanus toxoid-reactive T cells. METHODS: We analyzed 5 patients with delayed-type drug hypersensitivity reactions, applying 2 methods: quantification of cytokine-secreting T cells by enzyme-linked immunospot (ELISpot), and fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity distribution analysis of drug-reactive T cells. RESULTS: Frequencies found were between 0.02% and 0.4% of CD4(+) T cells reacting to the respective drugs measured by CFSE analysis, and between 0.01% and 0.08% of T cells as determined by ELISpot. Reactivity was seen neither to drugs to which the patients were not sensitized nor in healthy individuals after stimulation with any of the drugs used. CONCLUSION: About 1:250 to 1:10,000 of T cells of patients with drug allergy are reactive to the relevant drugs. This frequency of drug-reactive T cells is higher than the frequency of T cells able to recognize recall antigens like tetanus toxoid in the same subjects. A substantial frequency could be observed as long as 12 years later in 1 patient even after strict drug avoidance. Patients with severe delayed drug hypersensitivity reactions are therefore potentially prone to react again to the incriminated drug even years after strict drug avoidance.     

Delayed drug hypersensitivity reactions – new concepts

Immune reactions to small molecular compounds such as drugs can cause a variety of diseases mainly involving skin, but also liver, kidney, lungs and other organs. In addition to the well-known immediate, IgE-mediated reactions to drugs, many drug-induced hypersensitivity reactions appear delayed. Recent data have shown that in these delayed reactions drug-specific CD4(+) and CD8(+) T cells recognize drugs through their T cell receptors (TCR) in an MHC-dependent way. Immunohistochemical and functional studies of drug-reactive T cells in patients with distinct forms of exanthems revealed that distinct T cell functions lead to different clinical phenotypes. Taken together, these data allow delayed hypersensitivity reactions (type IV) to be further subclassified into T cell reactions, which by releasing certain cytokines and chemokines preferentially activate and recruit monocytes (type IVa), eosinophils (type IVb), or neutrophils (type IVd). Moreover, cytotoxic functions by either CD4(+) or CD8(+) T cells (type IVc) seem to participate in all type IV reactions. Drugs are not only immunogenic because of their chemical reactivity, but also because they may bind in a labile way to available TCRs and possibly MHC-molecules. This seems to be sufficient to stimulate certain, probably preactivated T cells. The drug seems to bind first to the fitting TCR, which already exerts some activation. For full activation, an additional interaction of the TCR with the MHC molecules is needed. The drug binding to the receptor structures is reminiscent of a pharmacological interaction between a drug and its (immune) receptor and was thus termed the p-i concept. In some patients with drug hypersensitivity, such a response occurs within hours even upon the first exposure to the drug. The T cell reaction to the drug might thus not be due to a classical, primary response, but is due to peptide-specific T cells which happen to be stimulated by a drug. This new concept has major implications for understanding clinical and immunological features of drug hypersensitivity and a model to explain the frequent skin symptoms in drug hypersensitivity is proposed.     

Carrier and hapten functions in immune deviation. I. Contrary effects of hapten and carrier on cellular and helper functions of T cells.

Carrier and hapten functions have been studied in the immune deviation phenomenon. Delayed hypersensitivity to the carrier and anaphylaxis and Arthus hypersensitivities to the hapten and to the carrier were studied in guinea-pigs injected intravenously with large doses of carrier, homologous and heterologous hapten-carrier conjugates and subsequently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. Pretreatment with DNP-BSA or with HGG were found to modify, in opposite directions, the hypersensitivity reactions induced by DNP-HGG in adjuvant. It is suggested that the hapten and carrier moieties of the antigen molecule might have antagonistic effects on the T cells responsible for cellular immunity as well as on T cells involved in helper functions for B cells.     

Hapten and carrier specificities of cellular and humoral responses to highly substituted dinitrophenyl-human gamma-globulins in guinea-pigs.

Delayed hypersensitivity (DH) and antibody reactions to the carrier and to the hapten have been studied in guinea-pigs immunized with different doses of highly substituted dinitrophenyl-human gamma-globulins (DNP56-HGG) in Freund's complete adjuvant (FCA). The results confirm that DH reactions are specific for the carrier while antibody-mediated reactions are specific for the hapten in the early stages of the immune response. Later in the response, however, DH reactions to the hapten as well as a transient humoral reaction to the carrier could be observed. T cells specific for the hapten and B cells specific for the carrier are therefore triggered after a single infection of a highly substituted hapten-carrier conjugate in FCA. Their regulatory functions in the immune response to hapten-carrier conjugates are discussed.     

Responses of the immune system to injury

Three categories of immunotoxic effects are identified: direct immunotoxicity, hypersensitivity, and autoimmunity. Direct immunotoxicity consists of immunosuppression and immunostimulation. Total abrogation of the immune response (immunosuppression) results in more frequent, severe, and often atypical and relapsing infections and lymphomas. Immunostimulation is associated with febrile reactions, the induction/facilitation of autoimmune diseases and allergic reactions to unrelated allergens, and impaired hepatic drug biotransformation. Hypersensitivity is manifested by a variety of symptoms involving either antigen-specific or non-antigen-specific humoral and cellular adverse responses. Autoimmune reactions are divided into organ-specific and systemic reactions. Because of the involvement of many redundant mechanisms, it is difficult to predict responses of the immune system to a given immunotoxic injury. In laboratory animals, histologic but also functional changes are necessary to show evidence of and to predict such adverse responses.     

Stimulation and inhibition of macrophages prior to immunization with a hapten-carrier conjugate.

Delayed hypersensitivity reactions and antibody-mediated reactions to the carrier and to the hapten have been studied in animals treated with BCG or/and trypan blue before immunization with a hapten-carrier complex. The results show that it is possible to induce, with BCG, at the beginning of the response, an antigenic competition between hapten and carrier moieties of the antigen molecule. Macrophages which modulate the response to the hapten-carrier complex by acting both on T cells involved in cellular immunity and those involved in antibody synthesis, appear to be the target of antigenic competition.     

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine σ-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.     

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions

Delayed-type, T cell–mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.     

Pathogenesis of drug allergy – current concepts and recent insights

Drug hypersensitivity reactions (DHRs) may be caused by immunologic and non‐immunologic mechanisms. According to the World Allergy Organization, drug allergy (DA) encompasses the subgroup of immunologic DHRs which are mediated either by specific antibodies or specific T lymphocytes. Due to the immunologic memory, DA reactions bear an increased risk for dramatically enhanced reactions on re‐exposure. Some current concepts of DA were described decades ago. Drug allergies to soluble macromolecular protein drugs such as biopharmaceuticals are predominantly T cell‐dependent drug‐specific antibody responses leading to IgE‐or IgG‐mediated allergy. However, most drugs are too small to be directly recognized by specific B and T cells. Immune reactions to low‐molecular drugs have been explained by the hapten model: a hapten drug can bind covalently to soluble autologous proteins (e.g. serum albumin). Resulting compounds may then be recognized by matching B cell receptors (BCRs) and induce a specific T cell‐dependent IgE‐or IgG‐antibody production. Drug haptens may bind to extra‐ or intracellular proteins, which are processed and presented by various professional antigen‐presenting cells (APCs). Depending on the APC, they may induce not only specific antibody production, but also non‐immediate T cell‐mediated DA. More recently, a supplementary effector mechanism for non‐immediate DA to low‐molecular drugs has been described, namely the pharmacological interaction of native low‐molecular drugs with immune receptors (p‐i‐concept). Low‐molecular drugs may directly and reversibly attach to immune receptors. These non‐covalent interactions may modify the affinity between autologous major histocompatibility complex (MHC), presented peptides and specifically primed T cell receptors (TCRs) and thereby stimulate T cells. A special type of p‐i‐reaction has been recently described between the antiviral drug abacavir and the F pocket of HLA‐B*57:01. This interaction causes an alteration of the MHC‐presented self‐peptide repertoire and may consecutively lead to a kind of auto‐reactivity. Such types of reactions can explain the strong MHC‐HLA associations which have been found for some T cell‐mediated DHRs.     

Regulatory effects of osteoprotegerin on cellular and humoral immune responses

The interaction between receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK has been reported to regulate immunity in addition to bone metabolism. The aim of this study was to determine if osteoprotegerin (OPG), an inhibitor of the RANKL-RANK interaction and possibly a new drug against osteoporosis, would adversely affect immunity. OPG was used to treat mice developing different models of cellular and humoral immune responses and also in vitro in T and B cell assays. In mice, OPG does not affect cell-mediated reactions such as contact hypersensitivity to the hapten oxazolone and liver damage, granuloma formation, and infectious load induced by mycobacterial infection. However, OPG increases humoral reactions such as the production of IgM, IgG, and IgE against the T cell dependent antigen keyhole limpet hemocyanin and the production of IgM against the T cell independent antigen Pneumovax. In vitro, OPG modestly co-stimulates T cells but does not affect the proliferation of B cells. OPG has modest immunoregulatory effects that seem to be confined to the humoral response to specific antigens.     

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

Background:  T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable.
Methods:  Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry.
Results:  All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5–3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines.
Conclusions:  CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.     

Drug-Specific Th2 Cells and IgE Antibodies in a Patient with Anaphylaxis to Rituximab

Rituximab (RTX) is currently used in the treatment of lymphoproliferative diseases and of several rheumatologic disorders and is a frequent cause of acute infusion reactions, usually classified as cytokine release syndrome (CRS). Some infusion reactions to RTX raise concern for immediate type I hypersensitivity, even if to date RTX-specific IgE antibodies have not been reported. To improve knowledge of the mechanisms of reactions to RTX, we investigated humoral and cellular immune responses to this drug in a patient suffering from rheumatoid arthritis who displayed two immediate infusion-related reactions. RTX-exposed tolerant patients and healthy untreated subjects were used as controls. Non-isotype-specific and IgE anti-RTX antibodies were positive in the serum samples collected from the reactive patient but not in those from the control groups. Only the reactive patient also displayed skin testing positivity with RTX. More importantly, RTX-stimulated peripheral blood mononuclear cells from the reactive patient, but not from the controls, displayed a dose-dependent proliferative response associated with a Th2 cytokine production profile. Our results show the presence of RTX-specific Th2-type cells and IgE antibodies, thus suggesting that type I hypersensitivity may be an additional mechanism to CRS in the development of RTX reactions.     

T lymphocyte-mediated immune responses to chemical haptens and metal ions: implications for allergic and autoimmune disease

Chemical haptens and metal ions interact with proteins and thereby become recognizable by T and B lymphocytes. They induce the production of proinflammatory cytokines and chemokines by various cell types due to triggering of innate immune responses. This is an important prerequisite for the activation of the adaptive immune system and the development of diseases like allergic contact dermatitis and adverse drug and autoimmune reactions. Our increasing knowledge about the molecular basis of hapten and metal ion recognition by T cells and about the pathomechanisms of contact hypersensitivity and chemical-induced autoimmune reactions allows concomitant progress in the development of modern strategies for immunotherapy and will hopefully enable more specific intervention in hapten- and metal ion-induced human diseases in the future.     

Detection of human IgE to sulfamethoxazole by skin testing with sulfamethoxazoyl-poly-L-tyrosine

Adverse reactions to sulfamethoxazole (SMX) occur in 4% to 6% of normal individuals. Many of these reactions resemble immunopathologic reactions, but skin test or in vitro evidence of a role for IgE is limited. Earlier RAST studies in our laboratory provided evidence that the N4-SMX hapten was a major determinant in immediate hypersensitivity reactions to SMX. We tested the hypothesis that IgE to this hapten is present on the mast cells of patients who have experienced immediate hypersensitivity reactions temporally related to exposure to SMX. A multivalent skin test reagent, SMX168-poly-L-tyrosine, and a univalent hapten, SMX-tyrosine, were synthesized. Forty-four patients with histories of allergic reactions to SMX and six subjects who had been exposed to the drug, but who had not reacted, were skin tested. Twenty-seven percent of the history-positive patients were skin test positive. None of the control individuals was positive. The immunologic responses to SMX in three patients who had experienced allergic reactions during SMX/trimethoprim therapy were analyzed in serial skin test and RAST assessments. One to three years after the clinical reactions, IgE to SMX could be demonstrated by skin testing in all three patients with a SMX-poly-L-tyrosine skin test reagent. Skin test reactions were inhibited by the monovalent reagent, SMX-tyrosine, in a dose-dependent manner. SMX-specific IgE antibodies could also be detected by RAST in serum obtained within days of the reactions from two of the three individuals.(ABSTRACT TRUNCATED AT 250 WORDS)