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Keywords:

  • cytokine secretion;
  • drug hypersensitivity;
  • sulfanilamide;
  • T cells;
  • β-lactam

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Background:  The most prevalent drug hypersensitivity reactions are T-cell mediated. The only established in vitro test for detecting T-cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug-sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well-documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T-cell sensitization to drugs.

Methods:  Peripheral blood mononuclear cell of 10 patients, five allergic to β-lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17-plex bead-based immunoassay kits.

Results:  Among the 17 cytokines/chemokines analysed, interleukin-2 (IL-2), IL-5, IL-13 and interferon-γ (IFN-γ) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide- and β-lactam-reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients.

Conclusion:  The measurement of IL-2, IL-5, IL-13 or IFN-γ or a combination thereof might be a useful in vitro tool for detection of T-cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.

Abbreviations
AMP

ampicillin

AMX

amoxicillin

DTH

delayed-type drug hypersensitivity reaction

IFN

interferon

IL

interleukin

LTT

lymphocyte transformation test

MPE

maculopapular exanthem

PBMC

peripheral blood mononuclear cells

PBS

phosphate-buffered saline

SMX

sulfamethoxazole

SPD

sulfapyridine

TT

tetanus toxoid

Adverse drug-induced reactions are one of the major causes of iatrogenic disease in public health (1). About 15–20% of adverse drug reactions are caused by immunological mechanisms, and can be mediated by IgE, occasionally IgG and T cells (2). The clinical picture of T-cell mediated, delayed-type drug hypersensitivity reactions (DTH) is very heterogeneous and different T-cell subsets are involved (3).

The diagnosis of T-cell mediated drug hypersensitivity reactions is a major challenge in daily clinical practice as patch-, prick- and intracutaneous tests often do not yield positive results even in patients with a clear history of DTH (4, 5). Challenge tests, which are considered to be the gold standard, are frequently not tolerated by patients, bear the risk of severe reactions and do not differentiate between allergic and nonallergic reactions (6).

The aim of drug hypersensitivity diagnosis is to confirm that the symptoms were caused by a drug hypersensitivity reaction, and to identify the culprit drug. Our group and others have previously shown that in vitro T-cell proliferation and activation tests can be applied to the diagnosis of drug hypersensitivities (7, 8). Furthermore, studies by other groups have shown that measurements of cytokine secretion, namely of interleukin-5 (IL-5) and interferon-γ (IFN-γ), are useful for drug hypersensitivity diagnosis (9–15).

Nevertheless, a systematic comparison of different cytokines produced in vitro after drug stimulation, which would identify the most suitable cytokine or combination of cytokines for diagnosis of drug hypersensitivity, has not yet been reported. In this study, we screened the supernatants of in vitro drug-stimulated peripheral blood mononuclear cell (PBMC) of 10 drug-allergic patients for 17 cytokines and chemokines, representing a broad spectrum of T-cell- and monocyte-derived cytokines/chemokines. Although DTHs are T-cell mediated, we could not exclude a T-cell-mediated activation or a direct activation of monocytes from allergic patients by drugs. The goal of our study was to determine (i) such of those cytokines, which are secreted predominantly upon exposure to the relevant drug in vitro; (ii) the shortest stimulation time of PBMC with drugs resulting in detectable concentrations of cytokines in supernatants; (iii) whether stimulation by cross-reactive drugs leads to an identical cytokine pattern; (iv) whether β-lactams and sulfanilamides induce different cytokine patterns and (v) whether there are characteristic cytokine secretion patterns for different forms of DTH. Our data show that IL-5, IFN-γ, IL-13 and IL-2 are specifically induced in response to sulfamethoxazole (SMX), sulfapyridine (SPD), ampicillin (AMP) or amoxicillin (AMX) stimulation in PBMC from β-lactams- or sulfanilamide-allergic patients.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Donor characteristics

A summary of the patients’ clinical characteristics is given in Table 1. The patients were analysed in clinical remission and were healthy at the time of analysis. Skin tests, lymphocyte transformation tests (LTT) and CD69 measurements were performed as described (7, 8, 16). Five healthy individuals without a history of adverse drug reactions but responsive to tetanus toxoid (TT) served as controls. All patients gave informed consent, and the study was approved by the local ethical committee.

Table 1.   Patients’ characteristics
Patient’sID Age (years)/sexCulprit drugDiseaseSkin testLTT*CD69†Time interval‡
  1. MPE, maculopapular exanthema; DRESS, drug reaction with eosinophilia and systemic symptoms; EEM, erythema exsudativum multiforme; SJS, Stevens–Johnson syndrome; n.d., not determined.

  2. *LTT: a stimulation index of more than 2 is interpreted as positive.

  3. †CD69 upregulation on T cells: a stimulation factor of more than 2 is interpreted as positive.

  4. ‡Time interval between acute allergy and testing.

3264/mAMXMPENegativePositivePositive19 months
24926/fAMXMPEPositivePositiven.d.2 months
3135/mAMXMPENegativePositivePositive8 months
24435/fAMXMPENegativePositiven.d.5 month
35841/fAMXEEMPositivePositivePositive6 month
1145/mSMXMPE/MalaisePositivePositivePositive12 years
3848/mSPDDRESSNegativePositivePositive13 months
24571/mSMXSJSNegativePositivePositive2 months
3264/mSMXErythrodermiaNegativePositivePositive19 months
29541/fSPDDRESSNegativePositivePositive6 month

Isolation of PBMC from blood samples

Peripheral blood mononuclear cells were isolated from heparinized peripheral blood as described previously (17). Cells were cultured in RPMI-1640 (GIBCO®; Invitrogen, Basel, Switzerland) supplemented with 10% heat-inactivated human AB serum (Swiss Red Cross, Bern, Switzerland), 2mM l-glutamine (Biochrom, Berlin, Germany) and 25 μg/ml transferrin (Biotest, Dreieich, Germany). For some experiments, CD3+ cells were depleted by using human CD3 selection kits (Easy Sep®; StemCell Technologies, Grenoble, France) according to the manufacturer’s protocol.

Drugs and antigens

Tetanus toxoid (Serum Statens Institutet, Copenhagen, DK) was used at a final concentration of 5 μg/ml. Appropriate drug concentrations were evaluated previously in toxicity tests as described (7). The following nontoxic drug concentrations were used: AMX – 200, 500 μg/ml; AMP– 200, 500 μg/ml; SMX – 100, 200 μg/ml and SPD – 100, 200 μg/ml (all from Sigma, Basel, Switzerland). Lipopolysaccharide (LPS)-free drugs were from sterile containers.

Stimulation of PBMC and CD3− cells by drug antigens

A quantity of 2 × 105 PBMC were incubated for the time period indicated (mostly 72 h) with or without the drug, or with TT as a positive control in 100 μl cell culture medium. Supernatants were stored at −80 °C. Lymphocyte transformation test was performed as described previously (18). 3H-thymidine incorporation was measured by a Trace 96 counter (Inotech, Wohlen, Switzerland).

Cytokine/chemokine measurements

Seventeen cytokines/chemokines, namely IL1-β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-γ, CCL4, CCL2 and TNF-α, were measured in the supernatants by using commercially available 17-plex bead-based immunoassay kits (Bio-Rad Laboratories, Reinach, Switzerland) according to the manufacturer’s protocol. The detection range was approximately from 1 to 32 000 pg/ml. Cytokine/chemokine concentrations are either shown as pg/ml or as a ‘-fold increase’. The ‘fold increase’ is calculated by dividing the cytokine/chemokine concentration of the drug- or TT-stimulated PBMC cultures by the cytokine/chemokine concentration of the unstimulated PBMC culture.

Determination of assay conditions

In order to determine the minimal time interval yielding a robust detection of cytokines, PBMC of patients 11, 244 and 38 were stimulated with the eliciting drugs SMX, AMX and SPD for 24, 48 and 72 h respectively, and the concentrations of the 17 cytokines/chemokines were measured. Figure 1 presents the time kinetics for the five most relevant cytokines, IL-13, IFN-γ, IL-5, IL-2 and TNF-α (patient 38 not shown). An incubation time of 72 h resulted in the clearest increase, and this time interval was consequently used for all experiments.

image

Figure 1.  Time kinetics of drug-specific cytokine secretion of PBMC from allergic patients. PBMC from SMX-allergic patient 11 (top panel), and AMX-allergic patient 244 (bottom panel) were stimulated with the culprit and cross-reactive drugs (100 μg/ml SMX and SPD, or 500 μg/ml AMX and AMP, respectively), and with culture medium (CM) as negative or 5 μg/ml tetanus toxoid (TT) as positive control, respectively. Cytokine secretion was determined after 24, 48 and 72 h of stimulation.

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Data analysis

All experiments were performed twice. If the cytokine concentration was under/over the detectable range, the lowest/highest value of the standard curve was used for calculations. Statistical significance was determined by the two-sided Mann–Whitney test for unpaired data, and the Wilcoxon signed-rank test for paired data. A P-value < 0.05 was considered to be statistically significant. Cut-off values were calculated as mean +2 SD as reported elsewhere (19, 20).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Characterization of patients

Ten drug-allergic patients with well-documented and typical drug-related histories and symptoms, positive skin tests in three of 10, positive LTT in 10/10 and CD69 upregulation in eight of eight patients in response to either AMX, SMX or SPD were included in this study (Table 1). Four of five AMX-allergic patients had a maculopapular exanthem (MPE), and one an erythema exsudativum multiforme. Two of the sulfanilamide-allergic patients showed a drug rash with eosinophilia and systemic symptoms, one had a Stevens–Johnson syndrome, one an erythrodermia and one a severe MPE. Of note, patient 32 reacted with MPE to AMX and with erythrodermia to SMX. All five healthy donors had no history of drug allergy, had previously tolerated AMX and were negative in the LTT to all tested drugs.

Baseline cytokine secretion is comparable for patients in remission and healthy controls

All experiments were performed after 2 months–13 years of occurrence of the DTH. At the time of blood sample collection, all symptoms of DTH had subsided. Therefore, we expected the baseline cytokine/chemokine secretion in healthy controls and patients to be comparable. Cytokine release (pg/ml) from both patients’ and healthy donors’ PBMC incubated for 72 h without any drug was low for the cytokines (mostly <500 pg/ml), but high for the chemokines (mostly 500–20 000 pg/ml) (Table 2).

Table 2.   Baseline cytokine secretion of patients in remission and healthy controls in pg/ml
PatientT-cell-derived
 Monocyte/macrophage-derived
IL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL4†G-CSFCXCL8CCL2†IL-1βIL-12IL-7*
(A) SMX/SPD-allergic patients
 1140151710242551222451017840322
 3821901361698250511963622456184844931
 32312124103103415141081621244
 24531407135254872438711197711173551040926833
 295710428295354635831148221848108075176852
 Median319015104633505415364510104094933
PatientIL-5IL-13IFN-γIL-2IL-4IL-17IL-6†IL-10TNF-αGM-CSFCCL-4†G-CSFCXCL-8CCL2†IL-1βIL-12IL-7*
(B) AMX-allergic patients
 2441239471115203012326177852695346558814444
 3124487321634351140135534633857521
 32215144333194357167681431332
 3581646691621220353169424133071057633
 2492124369617168519924791190534646756233
 Median226974121634551440135534633856233
ControlsIL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL-4G-CSFCXCL-8CCL2IL-1βIL-12IL-7*
  1. SMX, sulfamethoxazole; SPD, sulfapyridine; AMX, amoxicillin.

  2. Data represent the mean of 2 independent experiments. Cytokine values in the two independent experiments were comparable.

  3. *Produced by stromal cells in many tissues.

  4. †Signficantly increased when compared with healthy controls. P < 0.05, Wilcoxon signed-rank test.

(C) Healthy controls
 380151542162433193616411610059166310811733
 6121879110121191153351271442733
 3252254816353106212051271114633
 32638501514140220629293962560933
 6031108122791331113539876811042033
 Median32871217913209353351271104933

In the group of SMX/SPD-allergic patients, only the baseline levels of the chemokines CCL2 and CCL4 were significantly elevated when compared with healthy controls (P < 0.05). The other 15 cytokines and chemokines were not elevated (Table 2A,C). Comparing the group of AMX-allergic patients to the healthy control group, the baseline levels of the chemokines CCL2 and CCL4 and additionally IL-6 were significantly increased (P < 0.05). The other 14 cytokines and chemokines were not elevated (P > 0.05) (Table 2B,C).

Interestingly, we could not observe a consistently higher baseline cytokine secretion in patients in remission for shorter periods (2–8 months) when compared with patients in remission for 1 year or longer.

Drugs can induce a marginal increase in cytokine secretion by PBMC of healthy individuals

To determine the ‘unspecific’ background cytokine secretion of PBMC in response to the drugs, we measured the cytokine release from PBMC of healthy donors after a 72-h stimulation with all relevant drugs. We observed that the unspecific secretion of cytokines/chemokines of healthy donors was dependent on the drug antigen used for stimulation (Table 3). In the group of sulfanilamides, SMX generally did not induce any cytokine/chemokine secretion in healthy controls (Table 3A). In contrast, SPD increased the median secretion of IFN-γ, IL-2, IL-13, IL-17, CCL4, IL-10, TNF-α, G-CSF and IL-1β by two- to 13-fold (Table 3B). However, statistical analysis revealed that only IL-1β secretion was significantly increased after stimulation by SPD when compared with nonstimulated PBMC cultures of healthy individuals (P < 0.05).

Table 3.   Healthy donors, increase in cytokine secretion after PBMC stimulation with AMX, AMP, SMX and SPD (fold increase)
AntigenDonorIL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL4G-CSFCXCL8CCL2IL-1βIL-12IL-7
(A)
 SMX38010101111213311211
 SMX6112111111112111111
 SMX32510111161111111111
 SMX32611211121111112111
 SMX6011101111011111111
 Median 11111111111111111
AntigenDonorIL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL4G-CSFCXCL8CCL2IL-1β*IL-12IL-7
(B)
 SPD380171212135131311611
 SPD61184222122122313112121
 SPD32511111110111111111
 SPD326136141036731023121421
 SPD6013331121111111811
 Median 132212135131311811
AntigenDonorIL-5IL-13IFN-γIL-2IL-4IL-17IL-6*IL-10TNF-αGM-CSFCCL4G-CSFCXCL8CCL2IL-1βIL-12IL-7
(C)
 AMX3801021113412111625911
 AMX611210311501212122111
 AMX32510111010111111111
 AMX32611221161011123111
 AMX6012321152112111311
 Median 12221151111122311
AntigenDonorIL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL4G-CSFCXCL8CCL2IL-1βIL-12IL-7
  1. PBMC, peripheral blood mononuclear cell; AMX, amoxicillin; AMP, ampicillin; SMX, sulfamethoxazole; SPD, sulfapyridine.

  2. Fold increase in cytokine/chemokine secretion (concentration of stimulated/concentration of nonstimulated) from healthy donors after stimulation by (A) 100 μg/ml SMX, (B) 100 μg/ml SPD, (C) 500 μg/ml AMX and (D) 500 μg/ml AMP. Data represent the mean of 2 independent experiments. Cytokine values in the two independent experiments were comparable.

  3. *Significantly increased when compared with unstimulated control. P < 0.05, Wilcoxon signed rank test.

(D)
 AMP38010011111111111111
 AMP6111114119121131911411
 AMP32510111121112111111
 AMP32611212231211213311
 AMP6012201111111111111
 Median 11211121111111111

Similarly to sulfanilamides, we observed differences in cytokine secretion in response to the β-lactams AMX and AMP from PBMC of healthy donors. Amoxicillin induced a median increase in secretion of IL-1β, CXCL8, CCL2, IL-2, IL-13, IFN-γ and IL-6 by two- to fivefold, but only the increase in IL-6 secretion was statistically significant (P < 0.05) (Table 3C). On the other hand, PBMC from healthy donors reacted to AMP stimulation with increased median secretion of IFN-γ and IL-6 (Table 3D). However, statistical analysis showed that the increase of these cytokines was not significant.

In summary, the tested drugs generally either did not or only weakly increased the cytokine/chemokine secretion in healthy donors, with the exception of IL-6 and IL-1β, which were statistically increased after stimulation by AMX and SPD respectively.

Cytokine secretion pattern of PBMC from drug-allergic patients in response to sulfanilamides and penicillins

We measured the cytokine/chemokine release from PBMC of drug-allergic donors after a 72-h stimulation with the culprit or cross-reactive drug. Fold increase was again calculated as cytokine/chemokine concentration of stimulated PBMC divided by cytokine/chemokine concentration of nonstimulated PBMC (Table 4).

Table 4.   Allergic patients increase in cytokine secretion after PBMC stimulation with culprit and cross-reactive drug, and TT (fold increase)
 PatientIL-5IL-13IFN-γIL-2IL-4IL-17IL-6IL-10TNF-αGM-CSFCCL4G-CSFCXCL8CCL2IL-1βIL-12IL-7
  1. SMX, sulfamethoxazole; SPD, sulfapyridine; TT, tetanus toxoid; AMX, amoxicillin; AMP, ampicillin.

  2. Fold increase in cytokine/chemokine secretion (concentration of stimulated/concentration of nonstimulated) after stimulation by culprit drug (cu), cross-reactive drug (cr) and positive control TT (pos.) in (A) sulfanilamide-allergic and (B) amoxicillin-allergic patients. Drug concentrations are 100 μg/ml SMX and SPD, 500 μg/ml AMX and AMP. Data represent the mean of two independent experiments. Cytokine values in the two independent experiments were comparable.

(A) Sulfanilamide-allergic patients
cu
 SMX112364937081956719922713261011931
 SPD3814265580171116731211311
 SMX32123131111111111111
 SMX2455112404751153218111111
 SPD2954231336916895121915611321
 Median 4424019517531278611311
cr
 SPD112306821224141174414252117911
 SMX3811630711111312311111
 SPD3211171111111111111
 SPD24554281111112011111
 SMX29500535482416122911511
 Median 14581111312311111
pos.
 TT1116011591961594129041331915727119011
 TT38112127497771132437140111731
 TT32277631181312125211
 TT2458992115417962373215386352399114411
 TT295221611922123211111
 Median 21212763478432197140111711
(B) Amoxicillin-allergic patients
cu
 AMX24412155019313471141411411
 AMX3175484653131133414111311
 AMX3213131151911121111
 AMX358398313366259801515171301
 AMX249154245181192977131312111811
 Median 1248245313434141411411
cr
 AMP24415113142124213311211
 AMP31521221018721121912111111
 AMP32n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
 AMP3581563301259424381301612161311
 AMP24957231215321211211
 Median 861771022251113311211
pos.
 TT24421301031737338119511311
 TT3110054813646122261171111511
 TT32113261191211124111
 TT3583719710981166561390612213172401
 TT2491237510602531612822016711185511451
 Median 214810325373226119121311

In the group of sulfanilamides, the culprit drug strongly induced IL-13, IFN-γ and IL-2 with a median fold increase of 40–195 (Table 4A). A weak increase (median increase three- to 12-fold) was observed for IL-5, IL-1β, IL-10, IL-17, IL-6, TNF-α, GM-CSF, CCL4 and G-CSF (Table 4A). The cross-reactive drugs of the sulfanilamide group induced a weaker cytokine/chemokine secretion. Only IL-13, IFN-γ, IL-2, TNF-α and G-CSF were increased (median three- to eightfold). The median secretion of these cytokines in drug-allergic patients was higher when compared with cytokines secreted by PBMC from nonallergic patients, however (Table 3).

Upon in vitro stimulation with the culprit AMX, a strong secretion of IL-5, IL-13, IFN-γ, TNF-α and G-CSF (median increase 12- to 48-fold), and a weaker secretion of IL-2, IL-4, IL-6, IL-10, CCL4 and IL-1β (median increase three- to fivefold) was induced (Table 4B). Similar to AMX, AMP induced a strong secretion of IL-5 and IL-13 (median increase 17- to 86-fold) and a weak median increase (five- to 11-fold) of IL-2, IL-10, CCL4, G-CSF, IFN-γ and TNF-α (Table 4B).

Peripheral blood mononuclear cells stimulated with TT revealed a cytokine/chemokine secretion pattern similar to the drugs. Predominantly secreted cytokines were IFN-γ, IL-2, IL-13, IL-17, TNF-α and CCL4 for both patient groups (median increase seven- to 140-fold). Further secreted were G-CSF, GM-CSF, IL-4, IL-5, IL-6, IL-10 and IL-1β (Table 4).

In summary, IL-2, IL-5, IFN-γ, IL-13 and TNF-α, induced by both culprit and cross-reactive drugs, were increased most consistently among the 17 cytokines/chemokines in patients, when compared with healthy controls.

Statistical analysis showed that the release of IL-5, IFN-γ, IL-13, IL-2, but not of TNF-α, induced by the culprit drug was significantly higher in SMX/SPD-allergic patients when compared with drug-stimulated healthy donors. In contrast, upon stimulation with the cross-reactive drug, none of the five cytokines except IL-2 as mentioned above was secreted in statistically significant higher amounts when compared with controls (Fig. 2, top panel). In AMX-allergic patients, the culprit (AMX) drug induced significantly higher amounts of IL-5, IFN-γ, IL-13, IL-2 and TNF-α when compared with nonallergic individuals, whereas the cross-reactive drug (AMP) did not induce a statistically significant increase in TNF-α (Fig. 2, bottom panel). With a cut-off value (calculated as mean + 2 SD of healthy donors) of 10 for IFN-γ and IL-13, two for IL-5, five for IL-2, six of 10 patients were positive for IL-5, eight of 10 for IFN-γ, IL-13 and IL-2.

image

Figure 2.  Drug-induced secretion of IFN-γ, IL-2, IL-13, IL-5 and TNF-α from drug-allergic patients and healthy controls. Top panels show cytokine secretion in response to 100 μg/ml SMX and SPD, and bottom panels to 500 μg/ml AMX and AMP. PBMC of patients and healthy donors were stimulated either with culprit drug (cu), cross-reactive drug (cr), culture medium (cm) or tetanus toxoid (not shown). Results are shown as fold increase of negative control. Open and filled symbols represent two independent experiments, respectively. *P < 0.05; **P < 0.01; ***P < 0.001.

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Cytokine/chemokine secretion is T-cell dependent

Delayed-type drug hypersensitivity reaction have been shown to be T-cell mediated (3). To demonstrate that the secretion of IL-5, IFN-γ, IL-13 and IL-2 was T-cell dependent, we depleted PBMC of SMX-allergic patient 11 and AMX-allergic patient 244 (Fig. S1A,B) from CD3+ cells. When CD3− cells were stimulated in parallel to PBMC by the culprit drugs, we observed that the secretion of IL-5, IFN-γ, IL-13 and IL-2 was absent in CD3− cell cultures. Further, we could show that the occasional secretion of monocyte-derived CCL-4 and TNF-α in patient 11 was abolished if CD3+ cells were depleted from PBMC (Fig. S1C).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

In the present study, we show a drug-specific increase in secretion of IL-2, IL-13, IFN-γ and IL-5 in PBMC cultures of 10 drug-allergic patients. Cells were stimulated for 72 h as this was the minimal time interval still resulting in robust secretion of IL-2, IL-13, IL-5 and IFN-γ, all of which were significantly increased in response to both culprit sulfanilamides and β-lactams. TNF-α secretion reached statistical significance only in response to culprit β-lactams. Cross-reactive drugs-induced secretion patterns that were comparable to the culprit drug, but quantitatively diminished.

Assaying cytokine secretion patterns has previously been postulated as a promising in vitro read-out system of drug hypersensitivity (21), and several studies have suggested to measure IL-5 or IFN-γ in the supernatant of drug-stimulated PBMC (10, 14, 22). The reported patterns of cytokine secretion were proposed to be more sensitive than proliferation assays and to be better correlated with the pathogenetic events. However, a systematic comparison of different cytokines produced in vitro after drug stimulation to identify the most suitable cytokine/chemokine for diagnosis of drug hypersensitivity, has not yet been reported. We addressed this question by screening the release of 17 cytokines/chemokines from PBMC of either drug-allergic patients in remission or healthy controls after stimulation with the relevant drugs. The baseline cytokine/chemokine secretion of patients in remission and of healthy controls was comparable.

To determine the most suitable cytokines/chemokines for diagnosing drug sensitization, it is important to determine the unspecific background cytokine secretion induced by the respective drugs. We observed either absence of or only weak drug-induced secretion of cytokines/chemokines in nonallergic individuals. However, not all drugs had the same stimulatory effect: while SMX- and AMP-induced cytokine/chemokine secretion did not induce any significant unspecific cytokine secretion, AMX induced a statistically significant increase of IL-6 in nonallergic individuals. Sulfapyridine-induced cytokine/chemokine secretion in healthy donors more frequently than any of the other drugs, even though only IL-1β was significantly increased. Overall, we could not observe any difference in induction of cytokine secretion by sulfanilamides when compared with penicillins. Rather, differences in cytokine secretion induced by any one drug seemed to be dependent on each of the healthy individuals. Of note, no cellular proliferation was observed in LTT with cells from any of the healthy donors. The relevance and potential role of this stimulatory effect on healthy donors’ PBMC, notably observed at nontoxic drug concentrations, remain unclear at present. However, this drug-dependent cytokine secretion in healthy individuals needs to be considered in the interpretation of cytokine release from drug-allergic patients when applied as a potential diagnostic tool.

For IL-5 we could not observe a sensitivity as high as previously reported (12) as only six of 10 patients were clearly positive for IL-5 in our assay. Drug-specific secretion of IL-2, IL-13 and IFN-γ was observed in eight of 10 patients. Thus IL-2, IL-13 and IFN-γ seem to be more sensitive parameters for drug sensitization than IL-5. The specificity of these three cytokines seems lower, however, as a four- to 10-fold increase of IL-2, IL-13 and IFN-γ was observed in one to two healthy controls. Therefore, a combination of two cytokines, namely IL-5 as a very specific marker, and either IL-2, IL-13 or IFN-γ as more sensitive parameter(s) would presumably be sufficient to detect sensitizations to drugs. By depleting CD3+ cells, we could further show that the cytokines that characterize patients with DTH in vitro are T-cell dependent, and that this assay would therefore detect drug-reacting T cells.

All other cytokines/chemokines, were only occasionally increased after drug stimulation in allergic patients, and do not seem suitable for diagnostic purposes. It is noteworthy that monocyte-derived cytokines such as TNF-α, G-CSF, IL-1β, CCL-4 and IL-12 were occasionally increased in some patients and healthy donors. This secretion was not consistent over repeated experiments. However, we could show in patient 11 that CCL4 and TNF-α secretion were abolished if T cells were depleted. This suggests that the secretion of non-T-cell-derived cytokines is T-cell dependent and probably not a direct effect of the drug.

According to our data, the cross-reactive drugs cause a similar cytokine/chemokine pattern as the culprit drugs, even though quantitatively diminished especially after sulfanilamide stimulation. The very similar cytokine secretion pattern between AMX and AMP is most probably attributable to their chemical similarity, the difference being one aryl-hydroxy group in the side-chain only.

The in vitro drug-stimulated PBMC showed a Th0 pattern as reported previously (17, 23), which was similar to TT. The control antigen TT generally induced a stronger cytokine secretion than any drug and was similar to the culprit drug-induced pattern, even though it was varying between individual patients. It seems that the cytokine secretion depends more on an ‘inherent’ reactivity of in vitro stimulated PBMC than on the type of antigen or clinical phenotype (17). This implies that it is difficult to associate the in vitro secretion of a certain cytokine with a clinical picture.

In conclusion, the measurement of IL-5 as a very specific marker in combination with either IFN-γ, IL-13 or IL-2 as a more sensitive marker might be a useful tool for the in vitro detection of T-cell sensitization to drugs. For β-lactam sensitization, TNF-α might be suitable as well. The secretion of IL-5, IFN-γ, IL-13 and IL-2 seems to be independent of the type of drug and the phenotype of the drug reaction. A prospective study with a larger number of suspected drug-allergic patients and healthy individuals will be needed to provide an accurate threshold for positivity and quantitative data on the sensitivity and specificity of these tests for drug hypersensitivity diagnosis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

This work was supported by Swiss National Science Foundation grant no. 310000-113744/1 to W.J. Pichler, and no. 3100A0-116113/1 to B.O. Gerber, by the Kamillo Eisner Foundation, and an unrestricted grant by Pfizer.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Figure S1. Secretion of IFN-γ, IL-2, IL-13, IL-5, TNF-α and CCL-4 from drug-allergic patients is T-cell-dependent. (A) Patient 11, secretion of IL-5, IL-13, IFN-γ and IL-2 from PBMC or CD3-depleted PBMC (CD3−) stimulated by SMX (100 μg/ml), TT (5 μg/ml, positive control) or CM only (negative control). (B) Patient 244, secretion of IL-5, IL-13, IFN-γ and IL-2 from PBMC or CD3-depleted PBMC (CD3−) stimulated by AMX (500 μg/ml), TT (5 μg/ml, positive control) or CM only (negative control). (C) Patient 11, secretion of TNF-α and CCL4 from PBMC or CD3-depleted PBMC (CD3−) stimulated by SMX (100 μg/ml), TT (5 μg/ml, positive control) or CM only (negative control).

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