1Department of Molecular Biology, University of Salzburg, 2Department of Neurology, Paracelsus Medical University, 3Department of Dermatology, Paracelsus Medical University, 4Bühlmann Laboratories, 5Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg
Steiner, M., Harrer, A., Lang, R., Schneider, M., Ferreira, F., Hawranek, T., et al. Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity. J. Vis. Exp. (55), e3263, doi:10.3791/3263 (2011).
Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain.
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3.
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.
1. Preparation of drug conjugates
2. Determine coupling rate of drug conjugates (Figure 2)
3. BAT using drug conjugates
4. Representative Results:
This experiment evaluates BAT as a beneficial tool to detect IgE-dependent drug hypersensitivity. Protein conjugates of NSAIDs are used for activation of basophils. By HPSEC with RI and UV detection aggregation state, coupling degree, and effective yield of the conjugates are determined. As Figure 4 shows, 43.5% of the DF conjugates remained monomeric with a coupling degree of 5.0 DF/HSA. For the aggregates a coupling degree of 9.5 was determined. The propyphenazone conjugate was shown to consist of 68.5% monomers with a coupling degree of 21.2. The coupling degree of the aggregates was 27.9. In total, the determined coupling degree of DF conjugates was 7.6 DF and that of PP conjugates was 23.2.
BAT performed with conjugates under optimized conditions (concentration of conjugates, stimulation time) allowed investigation of IgE-dependent reactions in NSAID hypersensitivity. As shown in Figure 5, only the PP conjugate was able to trigger basophil activation visualized by an upregulation in CD63 surface expression: 20.6% of basophils were activated characterized by a log-shift in fluorescence intensity. The MFI increased by a factor of 4.9 from 386 (negative control) to 1893. In contrast, using the DF conjugate only 3.1% of the basophils were activated which was below the 5% cut-off. Also the MFI increased only by a factor 1.1 (limit 2.0) from 197 (negative control) to 210. Assay validity is given by (i) <5% basophil activation in the negative control, (ii) a log-shift in fluorescence intensity of a basophil subpopulation, and (iii) >50% activated basophils (positive control).
Figure 1. Coupling of DF and PP derivative to HSA. After dissolving the drugs, water, MES buffer, and EDC (coupling reagent) are added. The samples are vortexed for 1 minute before HSA is pipetted to the drug solutions. Within 2 hours shaking at room temperature the drugs covalently bind to HSA. Monomers as well as aggregates may result.
Figure 2. Calculation of coupling degrees. First, the constants kRI and kUV are determined. Therefore, HSA standard with known concentration is injected into the HPSEC system. (dn/dc) and (dA/dc) of HSA are given values of 0.185 and 0.518, respectively 5. The peak areas are used for calculating the constants. Second, for each drug three standards are injected to determine drug- and concentration-specific RI and UV areas. Since the kRI and kUV constants are known from the steps above, the drug-specific (dn/dc) and (dA/dc) derivatives can be calculated. Third, drug-HSA conjugates are injected into HPSEC. The resulting peak areas are used to calculate the concentrations of drug and HSA per peak by solving the two equations with two unknown variables.
Figure 3. Basophil gating. Cells predicted as lymphocytes are gated side scatter versus forward scatter (SSC-FSC plot). The basophils are identified as CCR3hi SSClo from the gated lymphocytes (SSC-CCR3-PE plot). Basophils are analyzed for activation (CD63hi) in the CD63-FITC-CCR3-PE plot. The negative control is used to set the cut-off of maximum 2.5% basophils remaining CD63hi.
Figure 4. Determined coupling degrees of drug conjugates. RI and UV signals show two peaks representing aggregates (eluting at low retention volume) and monomers (eluting at high retention volume) of drug-HSA conjugates. Percentages of monomers and aggregates (determined from RI signals) and their coupling degrees are depicted (n=1).
Figure 5. Basophil activation test. Results of drug conjugate-activated samples, negative controls, and positive controls are shown in CD63-FITC-CCR3-PE plots (n=1).
BAT is a well-established although not yet routinely used method for diagnosis of IgE-mediated allergic disease 6,7. For drug hypersensitivity, however, its applicability is compromised as low molecular weight compounds are not able to crosslink IgE receptors, a prerequisite for basophil activation 8. Therefore, drugs under investigation need to be covalently coupled to suitable carrier proteins (e.g. HSA). Importantly, the coupling degree (i.e. number of drug molecules per carrier protein) needs to be controlled to guarantee immunological activity (i.e. IgE receptor cross-linking) of conjugates. Theoretically, two haptens per carrier molecule should be sufficient for IgE receptor cross-linking and as few as five DF molecules per HSA have been shown to trigger mediator release in a cell-based assay4. Both conjugates, PP-HSA and DF-HSA, have been determined to display a sufficiently high coupling degree (HPSEC) and to be immunologically active making them suitable reagents for use in BAT. Another issue may be that drug metabolites may play a role, as a metabolite instead of the parent drug may cause the hypersensitivity reaction. In case of DF, this possibility has been evaluated in detail previously using five major Phase I metabolites and one linkage variant4. Important factors for mastering the described technique include quality of blood sample (<12 hours since blood collection, number of detected basophils >500) and optimization of stimulation conditions (time, dose). In addition, so-called non-responders who do not even react with the positive control (antibody directed against the IgE receptor) have to be identified. Therefore, validation criteria have to include an anti-FcεRI antibody as positive control. An important advantage of using drug conjugates is the fact that they appear non-toxic in contrast to pure drugs, as shown for DF-HSA conjugate by co-stimulation with anti-FcεRI antibody in BAT4. Pure DF, in contrast, displays cytotoxic effects at a concentration of 1.25 mg/ml causing problems with the interpretability of BAT9. Generally, testing for potential cytotoxicity is strongly recommended for every newly produced drug conjugate.
Here, we have shown the potential of PP-HSA conjugate used in BAT to reveal IgE-mediated hypersensitivity. In contrast, DF hypersensitivity is not associated with IgE demonstrated by the lack of basophil activation in response to DF-HSA conjugate. Importantly, in case of uncertainty concerning an IgE-mediated mechanism, conjugates have to be evaluated for immunological activity by in vitro cell-based assay systems as has been shown for DF4.
Using the described setup of conjugating low molecular weight drugs covalently to suitable protein carrier molecules a number of hypersensitivity reactions against drugs including antibiotics, other NSAIDs, radiocontrast media, muscle relaxants, anesthetics, etc. may be investigated for an involvement of an IgE-mediated mechanism. Hence, BAT may serve as an addition to the existing methods for diagnosis (skin test, oral provocation testing).
No conflicts of interest declared.
This work was funded by the Austrian Science Fund (FWF) grant P18820-B13.
The study was approved (PLUS_Ethik_090514) by the Ethics Committee for Experiments Involving Humans and/or Animals at the University of Salzburg complying with the Helsinki Declaraction as revised in 1983 and all patients participating gave their written informed consent.
|7.8 x 300 mm TSK Gel -G2000SWXL column||Tosoh Corp.||08540||-|
|BD FACSCanto II||BD Biosciences||338962||-|
|Bench top centrifuge||Sigma-Aldrich||-||-|
|Diclofenac sodium salt||Sigma-Aldrich||D6899||-|
|Flow2 CAST Kit||Bühlmann Laboratories||FK-CCR||Effective June 14, 2011 Flow2 CAST kit is now Flow CAST|
|HP1100 HPLC system||Hewlett-Packard||-||-|
|Human Serum Albumin||Sigma-Aldrich||A9511||-|
|N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)||Sigma-Aldrich||E6383||-|
|Propyphenazone derivative||University of Salzburg||-||-|
|Sodium chloride (NaCl)||Sigma-Aldrich||S1679||-|
|Sodium hydrogen carbonate||Sigma-Aldrich||90421C||-|
|Triple detector array||Viscotek||TDA302||-|
|Personal BioVortex V-1 plus||Peqlab||90-V-1||-|
|Water bath 1012||GFL||1012||-|