There are currently three mechanisms by which drugs are thought activate T-cells through their TCR; the hapten hypothesis, the Pi mechanism and the altered peptide hypothesis. A hapten is defined as a low molecular weight compound capable of binding to protein to create an immunogenic molecule. An immunogen is any antigen that is capable of inducing a humoral or cell-mediated immune response rather than immunological tolerance. Hence, an antigen is any substance that may be specifically bound by components of the immune system although not every antigen causes an immune response; ones that do are defined as immunogenic.
1.11.1.Hapten hypothesis
The hapten hypothesis states that drugs or low molecular weight compounds under 1000Da in weight must form stable adducts with endogenous protein to be able to induce an immune response (Park et al. 1998). Haptenation of proteins can occur either directly or through metabolic activation. In direct haptenation, like with the classic sensitising agents dinitrochlorobenzene (DNCB) and dinitroflourobenzene (DNFB), the haptenic molecule binds directly to protein in a relatively non-specific manner to modify lysine and cysteine residues generating the immunogenic protein (Knight and Green 1979) (Fig 1.4. A). β-lactam antibiotics are also known to be directly protein-reactive and mass spectrometric techniques have been used in the case of piperacillin (Whitaker et al. 2011) and
38 flucloxacillin (Jenkins et al. 2009) to determine the exact amino acid residues that are modified by the drugs. However, most drugs only become sufficiently haptenic after bioactivation and this bioactivation is thought to occur after the chemical structure of the molecule has been altered through the action of enzymes such as cytochrome P450 enzymes, which are usually responsible for the detoxification of compounds. However, the oxidation of certain drugs can, ironically, cause them to become protein-reactive. P450 enzymes are found most abundantly in liver but can be also found in skin, gut and kidney. Importantly, immune cells express only low levels of P450 enzyme. However, other enzymes known to cause drugs to become protein-reactive (e.g. NADPH oxidase and myeloperoxidase) are found in phagocytes and other APCs (Sezer et al. 2001).
1.11.2.The Pi concept
The pharmacological interaction or “Pi concept” states that drugs do not need to be bound covalently to protein to become immunogenic and that some drugs can in fact bind directly to the MHC binding grooves to cause T-cell-mediated immune reactions. The Pi concept, as opposed to the hapten hypothesis, states that parent drug is able to “structurally” fit into both the T-cell receptor and the antigen presenting cells-MHC groove to stimulate the T- cells to react (Pichler et al. 2006) (Adam et al. 2011) (Fig 1: 4. B). This concept was devised through experimentation with drug-specific T-cell clones to drugs like sulfamethoxazole where the T-cells were found to react to drugs in an MHC-T-cell receptor dependent way. Furthermore, specific T-cell clones were capable of reacting to drug-treated fixed APCs (Schnyder et al. 1997) (Zanni et al. 1998).
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1.11.3.Altered-self peptide repertoire hypothesis
One drug, abacavir, has recently been shown to directly bind to MHC molecules through its association with HLA-B*57:01 allele where up to 50% of patients carrying the allele will develop hypersensitivity reactions whilst patients not carrying the allele are prescribed the drug with absolutely no immunological hypersensitivity reactions predicted (Mallal et al. 2008). This association lead to the screening of patients before prescription of the drug, leading to the first successful example of personalised medicine (Yun et al. 2012). This particular mechanism of the Pi-concept has come to be named the “altered-peptide concept” (Fig 1:4. C). The interaction of abacavir with endogenous HLA-B*57:01 shows the binding interaction changes the confirmation of the peptide binding cleft, altering the repertoire of peptides displayed on the cell surface (Illing et al. 2012) (Ostrov et al. 2012) (Norcross et al. 2012). These data imply that pre-existing peptide-specific T-cells are activated indirectly by abacavir through the display of cross-reacting “altered-self” peptides.
1.11.4.The danger hypothesis
The danger hypothesis simply states that the body does not discriminate between non-self and self but only strives to protect against danger (Matzinger 1994) (Anderson and Matzinger 2000). It is thought that the immune system requires three signals to react fully to an antigen (Curtsinger et al. 1999). Signal 1 involves presentation of an antigen via the MHC to the T-cell receptor and if no other signals are received, then this will result in tolerance to the particular antigen. Signal 2 involves the action of co-stimulatory interaction between APC and T-cells and the release of pro-inflammatory cytokines like IFNγ to enhance presentation. Signal 3 involves the response of specific TH1 or TH2 cells through polarising
40 hypersensitivity reactions, drugs and/or protein-reactive metabolites have the ability to be presented as part of Signal 1 via a possible drug protein interaction or MHC-T-cell receptor interaction and Signal 2 and 3 can be derived from either surgical trauma, drug derived stress, infection (Uetrecht J. P. 1999), or necrotic or apoptotic cell death. It has been shown that only necrotic cells can activate dendritic cells (Gallucci et al. 1999) but apoptotic cells have been shown to activate cytotoxic T-cells (Shi et al. 2000). It is well documented that patients with viral infections are more susceptible to adverse drug reactions (Sullivan and Shear 2001), however, with respect to danger signals in drug hypersensitivity reactions the specific nature and origin of the danger signals from drug and non-drug signalling is not well defined and more research is needed.
41 Fig 1.4. The hapten hypothesis, the “Pi-concept”, and the altered peptide concept. (A) Hapten hypothesis where the peptide embedded in the MHC I molecule, is covalently bound and altered by protein-reactive drug (e.g. flucloxacillin), creating an immunogenic antigen. (B) The “pi-concept” where the drug is capable of binding directly to the TCR during MHC presentation (e.g. SMX, carbamazepine). (C) Altered peptide concept where the drug is capable of non-covalent binding directly to particular HLA-B associated MHC I molecules, allowing for presentation of “altered-self” peptides to CD8+ T-cells and subsequent sensitisation (e.g. only abacavir to date). Adapted from Adam et al (2011).