A positive lymphocyte transformation test to β-lactams (β-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to β-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-γ with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-γ but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-γ, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-γ. These data suggest that cytokines produced by Th2 cells play an important role in all β-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable.

β-lactams (β-L)3 are the antibiotics that most frequently induce adverse drug reactions (ADR) related to specific immunological mechanisms (1, 2). ADR to β-L are clin- ically and pathogenically heterogeneous, and they have been classified as immediate, accelerated, and delayed, depending on the latency between drug administration and appearance of symptoms (within 1 h, between 1 and 72 h, or after 72 h, respectively) (3, 4). So far, most studies have dealt with immediate reactions to β-L, which are considered the most serious for the patient. The model of penicillin allergy has greatly contributed to the understanding of mechanisms responsible for immediate reactions in which drug-specific IgE Abs play an essential role (1, 2, 5, 6, 7, 8, 9). The mechanisms responsible for nonimmediate reactions are less known, inasmuch as different immunological effector processes may be involved.

There is in vitro evidence that T cells participate in any type of ADR to β-L, since PBMC isolated from subjects suffering from hypersensitivity reactions can be specifically stimulated in culture with the appropriate drug or its metabolites (10). More recently, the generation of drug-specific T cell lines and T cell clones has provided a useful tool to better investigate the characteristics of T cells, including their specificity, as well as the molecular mechanisms involved in drug recognition by the TCR. Thus, it has been demonstrated that recognition of these nonpeptide Ags is MHC restricted but does not necessarily require prior uptake and processing of haptenated proteins by APC (11, 12, 13). However, conflicting results have been reported with regard to the pattern of cytokines produced by β-L-specific T cells during the effector response. Penicillin G (pen G)-specific T cell clones have been found to produce mainly high amounts of IFN-γ, and rather variable levels of IL-4 and IL-5, thus showing a prevalent Th1-like pattern (14, 15). In a different study, T cell clones generated from pen-induced bullous exanthems exhibited a CD8+ Th1-like phenotype, whereas T cell lines derived from peripheral blood of patients with pen-induced urticarial exanthems were predominantly CD4+ with a Th2-like pattern of cytokine production, thus suggesting heterogeneity of effector responses according to the type of clinical manifestation (10).

In the present study, we first examined the lymphocyte proliferative response to β-L in a group of subjects, who had suffered from ADR, by using a classic lymphocyte transformation test (LTT). Then, we assessed the cytokine profile of short-term T cell lines specific for pen G, amoxicillin (amox), ampicillin (amp), streptokinase (SK), and Dermatophagoides pteronyssinus group 1 (Der p 1), generated from PBMC of these subjects and nonatopic or atopic donors with negative history of ADR to β-L. Only a proportion of subjects with history of ADR to β-L showed positive LTT to one or more of these drugs, and this was not apparently related to either the type and latency of clinical manifestation or the presence in the serum of β-L-specific IgE Abs. As expected, SK-specific T cells from all groups of subjects showed intracellular expression of IFN-γ with poor or no IL-4, while Der p 1-specific T cells exhibited prevalent IFN-γ expression in nonatopic and remarkable IL-4 expression in atopic donors. On the contrary, the majority of cells from all pen G-specific short-term T cell lines and T cell clones generated from patients with ADR to β-L showed high production of IL-4, IL-5, and IL-13, but poor or no expression of IFN-γ, thus exhibiting a cytokine profile even more Th2-polarized than Der p 1-specific T cell lines derived from atopic donors. These data suggest that immune responses to β-L are generally characterized by the activation of Th2 cells irrespective of the type of ADR induced by these drugs.

Purified pen G, amp, amox, PMA, ionomycin, brefeldin A, and saponin were obtained from Sigma (St. Louis, MO). PHA and BSA were purchased from Life Technologies (Grand Island, NY). Recombinant IL-2 was a kind gift of Eurocetus (Milano, Italy). Affinity-purified Der p 1 allergen was kindly provided by Lofarma Allergeni (Milan, Italy). SK was purchased from Behring (Behringwerke, Marburg, Germany). The anti-CD3 (UCHT1) mAb was purchased from PharMingen (San Diego, CA). PE-conjugated anti-IL-4 (3010.211, IgG1), FITC-conjugated anti-IFN-γ (25723.11, IgG2b), peridinin chlorophyll protein-conjugated anti-CD8 (SK1, IgG1), and allophycocyanin-conjugated anti-CD3 (SK7, IgG1) mAbs were purchased from Becton Dickinson (San Jose, CA). The PE-conjugated anti-IL-5 (TRFK5, IgG1) and anti-IL-13 (Jes10–5A2, IgG1) were purchased from PharMingen. Fluorochrome-conjugated IgG1 and IgG2b isotype control mAbs were purchased from Southern Biotechnology Associates (Birmingham, AL).

Nonatopic donors were healthy volunteers with negative family and personal history for atopy and negative skin prick tests for a panel of common environmental allergens, including Der p. Atopic donors were identified on the basis of clinical symptoms (rhinitis and/or bronchial asthma), positive skin prick tests, and presence of serum IgE Abs specific for Der p alone or Der p plus other allergens. Twenty-nine subjects with ADR to β-L, 7 atopic patients showing immediate-type hypersensitivity to Der p, and 17 nonatopic donors, all matched for sex and age, were used throughout the study. All of them were informed about the purpose of the study and gave their written consent. Sixteen subjects with ADR to β-L showed mild skin-localized reactions (rash, urticaria, angioedema), whereas 13 showed extensive severe reactions (anaphylactic shock, laryngeal and/or pharyngeal edema, bronchial asthma attack, or Stevens-Johnson syndrome). Twelve of them had immediate reaction (within 30 min after drug administration) and the other 17 had late reactions (more than 1 h after drug administration). Two subjects with ADR to β-L also showed a history of atopy with positive skin prick tests and presence of serum IgE to Der p, whereas the other 27 had no history of atopy, had negative skin prick tests to a panel of common environmental allergens, and exhibited an absence of serum IgE Abs specific for inhaled or food allergens. At the time of sampling, 13 of 29 subjects with ADR to β-L exhibited serum IgE Abs specific for pen G, amp, or amox, alone or in combination.

IgE Abs specific for β-L (penicilloyl G, penicilloyl V, ampicilloyl, amoxicilloyl), inhaled or food allergens were measured in the serum by commercial kits (CAP System; Pharmacia, Uppsala, Sweden).

PBMC from subjects with ADR and nonatopic donors were isolated by Ficoll-Hypaque density gradient (Pharmacia), and 2 × 105 cells were cultured in triplicate for 6 days in antibiotic-free RPMI 1640 medium (Seromed, Berlin, Germany), supplemented with 2 × 10−5 M 2-ME, 2 mM l-glutamine, 1% nonessential amino acids, 1% sodium pyruvate (complete medium; all from Life Technologies), and 5% autologous serum in 96 round-bottom microwell plates (Nunclon; Nunc, Roskilde, Denmark) without or with different concentrations of β-L (2.5, 0.5, 0.1, 0.02, and 0.004 mg/ml). Sixteen hours before harvesting, 0.5 μCi [3H]TdR (Amersham, U.K.) was added to each well, and radionuclide uptake was measured by scintillation counting (Tomtec; Wallac, Turku, Finland). The mitogenic index (MI) was calculated as the ratio between the mean values of cpm obtained in stimulated cultures and those obtained in the presence of medium alone. LTT was considered as positive when the MI was ≥3 at least in two sequential doses of the drug.

Drug-specific short-term T cell lines and T cell clones were generated by a technique previously described in detail (16). Briefly, 106/ml PBMC obtained from subjects with ADR and healthy controls were stimulated for 6 days with pen G, amp, or amox (0.5 mg/ml) in complete medium, supplemented with 5% autologous serum. Subsequently, rIL-2 (10 U/ml) was added, and the cells were kept in culture for an additional 8 days. Thereafter, T cell blasts were washed and assessed for drug specificity. Parallel short-term T cell lines specific for Der p 1 or SK were generated from PBMC of patients with ADR, healthy controls, and atopic donors.

For the cloning procedure, T cell blasts from pen G- and SK-specific T cell lines from two drug-sensitive patients were seeded under limiting-dilution conditions (0.3 cell/well) in round-bottom microwell plates (Nunc), containing 105 irradiated (6000 rad) allogeneic PBMC as feeder cells, 1% PHA (v/v), and rIL-2 (20 U/ml), as reported (17). Growing microcultures were then expanded at weekly intervals with rIL-2 (20 U/ml) and 105 irradiated feeder cells.

The specificity of both short-term T cell lines and clones was assessed, as described (16). Briefly, 5 × 104 T cell blasts were incubated in triplicate for 48 h in 0.2 ml volume with 5 × 104 irradiated (6000 rad) autologous PBMC (as APC) in the presence of the appropriate drug (0.5 mg/ml) or Ag (Der p 1, 5 μg/ml; SK, 200 U/ml). After 16-h pulse with 0.5 μCi [3H]TdR (Amersham), cultures were harvested, and radionuclide uptake was measured by scintillation counting. T cell lines and clones were considered as specific when MI was ≥3.

Intracytofluorometric analysis of IL-4, IL-5, IL-13, and IFN-γ synthesis at single cell level was performed as described (18). Briefly, T cell blasts were stimulated with PMA (10 ng/ml) plus ionomycin (1 μM) for 4 h, the last two of which in the presence of brefeldin A (5 μg/ml). After incubation, cells were washed twice with PBS (pH 7.2), fixed for 15 min with formaldehyde (2% in PBS (pH 7.2)), washed twice in PBS (pH 7.2), permeabilized with PBS (pH 7.2) containing 0.5% BSA and 0.5% saponin, then incubated with the appropriate mAbs. Cells were analyzed on a FACScalibur cytofluorometer using CellQuest software (Becton Dickinson). The area of positivity was determined using an isotype-matched mAb. In all cytofluorometric analyses, a total of 104 events gated as CD3+ cells for each sample was acquired.

The ability of drug- or Ag-specific T cell clones to produce cytokines was evaluated after stimulation of 106/ml viable T cell blasts for 36 h with PMA (20 ng/ml) plus anti-CD3 mAb (50 ng/ml). For the quantitation of IL-4 and IFN-γ, a house-made capture ELISA was performed, as described (16). Cytokine levels 5 SD over the mean levels of control supernatants (irradiated feeder cells alone) were regarded as positive.

For statistical analysis t and χ2 tests were used.

The results of the in vitro LTT to pen G, amox, and amp in the three groups of subjects studied are summarized in Table I. Twelve of 29 subjects with ADR to β-L (41%) showed positive LTT to at least one of the three drugs tested. LTT was positive in 6 of 16 (37%) subjects with mild reactions, and in 6 of 13 (46%) subjects with extensive reactions. Six of 12 subjects with immediate reaction (50%), and 6 of 17 subjects with late reaction (35%) to β-L also showed positive LTT. Cross-reactivity in the LTT to at least two of the three β-L was observed in 9 of 12 subjects (data not shown). By contrast, none of the 17 control subjects tested under the same experimental conditions showed positive LTT to any of the three drugs (Table I).

Table I.

LTT in subjects with ADR to β-L

DiagnosisType of ReactionaNo. of CasesNo. of Cases with Positive LTT (%)b
ADR  29 12 (41%) 
 Mild 16 6 (37%) 
 Extensive 13 6 (46%) 
 Immediate 12 6 (50%) 
 Late 17 6 (35%) 
Healthy controls  17 0 (—) 
DiagnosisType of ReactionaNo. of CasesNo. of Cases with Positive LTT (%)b
ADR  29 12 (41%) 
 Mild 16 6 (37%) 
 Extensive 13 6 (46%) 
 Immediate 12 6 (50%) 
 Late 17 6 (35%) 
Healthy controls  17 0 (—) 
a

Subjects with ADR to β-L were subdivided into two groups depending on the severity of symptoms or the latency from drug administration. Mild reactions included limited skin lesions showing different morphological pattern (rash/urticaria/angioedema). Extensive reactions included anaphylactic shock, laryngeal or pharyngeal edema, asthma, or Stevens-Johnson syndrome. Immediate ADR were defined as those occurring within 30 min and late ADR as those occurring at least 1 h after drug administration.

b

PBMC from each subject were stimulated for 6 days with pen G, amp, or amox. LTT was considered as positive when mitogenic index was ≥3 at least with two sequential drug doses, as reported in Materials and Methods.

The relationship between LTT response and the presence of β-L-specific serum IgE Abs was also investigated. No correlation between the positivity of LTT and the presence of β-L-specific serum IgE Abs was found (Table II). It is of note that 10 of 12 subjects showing positive LTT to β-L had no history of atopy and negative skin tests to, and no detectable levels of serum IgE Abs specific for, a panel of common environmental inhalant and food allergens. Only two subjects showed positive skin tests to, and detectable levels of serum IgE Abs specific for, Der p.

Table II.

Lack of correlation between positivity of LTT to β-L and presence of β-L-specific serum IgE Abs in subjects with ADR

Subjects with ADR Showing:No. of CasesNo. of Cases with Positive LTT (%)a
β-L-specific IgEb 13 6 (46%) 
No β-L-specific IgEb 16 6 (37%) 
Subjects with ADR Showing:No. of CasesNo. of Cases with Positive LTT (%)a
β-L-specific IgEb 13 6 (46%) 
No β-L-specific IgEb 16 6 (37%) 
a

LTT was considered as positive according to the criteria reported in Table I.

b

β-L (penicilloyl G, penicilloyl V, ampicilloyl, amoxicilloyl)-specific IgE Abs were detected in the sera by CAP System.

To evaluate the cytokine profile of β-l-specific T cells, short-term T cell lines were generated from PBMC of 29 patients with ADR and 17 control subjects by using pen G, Der p 1, or SK as stimulants. In the same cases, amox and amp were also used. T cell lines specific for Der p 1 and SK were generated under the same experimental conditions in seven atopic Der p-sensitive donors showing no history of ADR. After 14 days of culture, T cell lines were assessed for specificity in an MHC-restricted fashion.

Pen G-specific T cell lines could be obtained from 8 of the 12 subjects with ADR and positive LTT to β-L and only from 1 of 17 subjects with ADR, but negative LTT. No pen G-specific T cell line was obtained from any of 17 controls showing no history of ADR. By contrast, Der p 1-specific and SK-specific T cell lines were obtained from all 29 subjects with ADR, 17 healthy nonatopic controls, and 7 atopic Der p-sensitive donors. Pen G-, SK- and Der p 1-specific T cell lines were then assessed for the expression at single cell level of intracellular IL-4 and IFN-γ synthesis by flow cytometry, following 4-h stimulation with PMA plus ionomycin. The results of these experiments are summarized in Fig. 1. In nonatopic subjects without a history of ADR, the great majority of both SK- and Der p 1-specific T cells synthesized IFN-γ (alone or in association with IL-4) and only a very few of them synthesized IL-4 alone. In atopic Der p-sensitive donors, SK-specific T cells showed a similar pattern of intracellular cytokine synthesis, but the proportions of Der p 1-specific, IL-4-producing T cells was significantly higher than in nonatopic subjects (p < 0.01). In patients with ADR to β-L, the great majority of SK- or Der p 1-specific T cells synthesized IFN-γ (alone or in association with IL-4), whereas only a minority of them synthesized IL-4 alone, thus showing a pathway similar to T cell lines generated from nonatopic healthy controls. However, the great majority of pen G-specific T cells showed intracellular synthesis of IL-4 (alone or in association with IFN-γ), and only a very few of them produced IFN-γ alone. In these subjects, there was a highly statistically significant difference in the number of IL-4- and IFN-γ-producing T cells between pen G-specific T cell lines and Der p 1-specific (p < 0.01 and <0.0001, respectively) or SK-specific (p < 0.005 and <0.0001, respectively) T cell lines.

FIGURE 1.

Intracellular IL-4 and IFN-γ expression by pen G-, Der p 1-, and SK-specific T cell lines. Pen G-specific T cell lines were obtained from 9 subjects suffering from ADR (A). Der p 1- (B) and SK-specific (C) T cell lines were derived from 7 subjects suffering from ADR with negative history for atopy, 7 healthy donors (randomly selected from 17), and 7 atopic Der p-sensitive patients with negative history for ADR. T cell blasts were stimulated with PMA plus ionomycin, as described in Materials and Methods, and intracellular cytokine synthesis was evaluated by cytofluorometric analysis. The columns represent the mean values (± SE) of cells expressing IL-4 alone (white columns), IFN-γ alone (black columns), or both IL-4 and IFN-γ (gray columns). Comparison between pen G- and Der p 1- or SK-specific T cell lines derived from drug-sensitive patients is shown. ∗, p < 0.01; ∗∗, p < 0.005; ∗∗∗, p < 0.0001).

FIGURE 1.

Intracellular IL-4 and IFN-γ expression by pen G-, Der p 1-, and SK-specific T cell lines. Pen G-specific T cell lines were obtained from 9 subjects suffering from ADR (A). Der p 1- (B) and SK-specific (C) T cell lines were derived from 7 subjects suffering from ADR with negative history for atopy, 7 healthy donors (randomly selected from 17), and 7 atopic Der p-sensitive patients with negative history for ADR. T cell blasts were stimulated with PMA plus ionomycin, as described in Materials and Methods, and intracellular cytokine synthesis was evaluated by cytofluorometric analysis. The columns represent the mean values (± SE) of cells expressing IL-4 alone (white columns), IFN-γ alone (black columns), or both IL-4 and IFN-γ (gray columns). Comparison between pen G- and Der p 1- or SK-specific T cell lines derived from drug-sensitive patients is shown. ∗, p < 0.01; ∗∗, p < 0.005; ∗∗∗, p < 0.0001).

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The majority of pen G-specific T cells derived from these subjects, as well as those specific for amox or amp, consistently produced IL-5 and IL-13 in addition to IL-4, thus showing a classic Th2-skewed cytokine profile. A representative experiment is shown in Fig. 2. It is of note that pen G-specific T cell lines derived from two pen G-sensitive subjects, who were also atopic and sensitive to Der p, showed a Th2 skewing in the expression of intracellular cytokines, which was even more pronounced than in Der p 1-specific T cells (Fig. 3). Interestingly, no significant difference in the profile of cytokine production was observed when pen G-specific T cell lines were obtained from patients showing immediate or late clinical manifestations (Table III). Likewise, pen G-specific T cell lines obtained from β-L-sensitive patients, apparently lacking serum drug-specific IgE Abs, displayed a similarly Th2-polarized pattern of intracellular cytokine expression (data not shown).

FIGURE 2.

Prevalent intracellular expression of Th2 cytokines by β-L-specific T cell lines. Intracellular IL-4, IL-5, IL-13, and IFN-γ expression was assessed in β-L-specific (pen G-, amp-, and amox-specific) T cell lines derived from patients suffering from ADR in comparison with cytokine expression by T cell lines specific for a bacterial Ag (SK) or allergen (Der p 1) generated from the same donors, following stimulation with PMA plus ionomycin, as described in Materials and Methods. The cytofluorometric pattern from a representative experiment is shown.

FIGURE 2.

Prevalent intracellular expression of Th2 cytokines by β-L-specific T cell lines. Intracellular IL-4, IL-5, IL-13, and IFN-γ expression was assessed in β-L-specific (pen G-, amp-, and amox-specific) T cell lines derived from patients suffering from ADR in comparison with cytokine expression by T cell lines specific for a bacterial Ag (SK) or allergen (Der p 1) generated from the same donors, following stimulation with PMA plus ionomycin, as described in Materials and Methods. The cytofluorometric pattern from a representative experiment is shown.

Close modal
FIGURE 3.

Higher percentage of IL-4-expressing cells in pen G-specific than in Der p 1-specific T cell lines generated from the same donors. Pen G- and Der p 1-specific T cell lines were derived from two atopic Der p-sensitive patients suffering from ADR and from seven drug-sensitive nonatopic donors. Intracellular expression of IL-4 and IFN-γ was evaluated in CD3+ CD4+ gated cells, as described. One representative experiment from each of the two groups of subjects is reported. A, Shows the results obtained in a drug-sensitive atopic (Der p) donor and B in a drug-sensitive nonatopic donor.

FIGURE 3.

Higher percentage of IL-4-expressing cells in pen G-specific than in Der p 1-specific T cell lines generated from the same donors. Pen G- and Der p 1-specific T cell lines were derived from two atopic Der p-sensitive patients suffering from ADR and from seven drug-sensitive nonatopic donors. Intracellular expression of IL-4 and IFN-γ was evaluated in CD3+ CD4+ gated cells, as described. One representative experiment from each of the two groups of subjects is reported. A, Shows the results obtained in a drug-sensitive atopic (Der p) donor and B in a drug-sensitive nonatopic donor.

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Table III.

Th2-polarized intracellular cytokine expression by pen G-specific T cell lines from β-L-sensitive subjects divided according to the latency of ADR after drug administration

Type of ADR in Cell DonoraNo. of pen G-Specific LinesCD4+ T Cells (%) Showing Intracellular Expression of the Indicated Cytokine (mean values ± SE)b
IL-4IL-4 and IFN-γIFN-γ
Immediate 26.3 ± 8.9 8.2 ± 2.4 14.6 ± 2.2 
Late 38.2 ± 12.0 8.9 ± 1.4 14.7 ± 3.2 
Type of ADR in Cell DonoraNo. of pen G-Specific LinesCD4+ T Cells (%) Showing Intracellular Expression of the Indicated Cytokine (mean values ± SE)b
IL-4IL-4 and IFN-γIFN-γ
Immediate 26.3 ± 8.9 8.2 ± 2.4 14.6 ± 2.2 
Late 38.2 ± 12.0 8.9 ± 1.4 14.7 ± 3.2 
a

Immediate and late ADR were defined as those occurring within 30 min or at least 1 h after drug intake, respectively.

b

Intracellular cytokine expression by pen G-specific T cell blasts was evaluated by flow cytometry after stimulation with PMA plus ionomycin, as described in Materials and Methods. No statistical difference was found in the percentage of cytokine-producing cells between the two groups of subjects.

To provide additional evidence for the Th2-polarized cytokine profile of pen G-specific T cells, pen G- or SK-specific T cell clones were generated from pen G- and SK-specific short-term T cell lines, both derived from two subjects with history of ADR to β-L. IL-4 and IFN-γ concentrations released in the supernatants by T cell clones after 36-h stimulation with PMA and anti-CD3 mAb were then measured. The results obtained in a total number of 19 SK-specific and 17 pen G-specific CD4+ T cell clones are shown in Fig. 4. As expected, 13 of 19 (68%) SK-specific T cell clones produced IFN-γ alone, thus showing a clear-cut Th1-like profile; 4 of them (21%) produced both IL-4 and IFN-γ, whereas none of them produced IL-4 alone. By contrast, none of the pen G-specific T cell clones produced IFN-γ alone, 9 of them produced both IFN-γ and IL-4 (53%), and 7 produced IL-4 alone (41%), thus showing a clearly polarized Th0/Th2 profile.

FIGURE 4.

Production of IL-4 and IFN-γ by CD4+ T cell clones generated from pen G- and SK-specific T cell lines. Pen G- (○) and SK-specific (•) T cell clones were generated from T cell lines derived from two randomly selected drug-sensitive nonatopic donors. Clonal T blasts (106/ml) were stimulated for 36 h with PMA plus anti-CD3 mAb, and cell-free culture supernatants were assessed for their IFN-γ and IL-4 content, as described in Materials and Methods. Lines represent the mean values ± 5 SD cytokine concentrations produced by irradiated feeder cells alone in response to PMA plus anti-CD3 mAb.

FIGURE 4.

Production of IL-4 and IFN-γ by CD4+ T cell clones generated from pen G- and SK-specific T cell lines. Pen G- (○) and SK-specific (•) T cell clones were generated from T cell lines derived from two randomly selected drug-sensitive nonatopic donors. Clonal T blasts (106/ml) were stimulated for 36 h with PMA plus anti-CD3 mAb, and cell-free culture supernatants were assessed for their IFN-γ and IL-4 content, as described in Materials and Methods. Lines represent the mean values ± 5 SD cytokine concentrations produced by irradiated feeder cells alone in response to PMA plus anti-CD3 mAb.

Close modal

Adverse reactions to antibiotics can be responsible for serious problems in practical medicine. β-L are the antibiotics that most frequently induce adverse reactions mediated by specific immunological mechanisms (1, 2), and several conceptual and experimental data support the involvement of T cell responses in ADR. First, IgE-mediated allergic reactions to β-L occurring immediately (usually within 10–20 min after drug administration) are responsible for urticaria and/or anaphylactic reactions in a proportion of patients. This suggests the pathogenic role of Th2 cells able to collaborate with B cells for the IgE isotype switching via the production of IL-4 and IL-13 (19). However, a series of different reactions can occur after an interval varying from several hours to some days after drug intake, and IgE Abs are not necessarily detected, thus suggesting different, even if similarly T cell-mediated, effector mechanisms. For example, delayed type hypersensitivity reactions to β-L have been demonstrated in contact dermatitis (20). Accordingly, PBMC from many patients with adverse reactions to β-L can proliferate in vitro in response to these drugs, independently of either the type of clinical manifestation or the presence of β-L-specific serum IgE Abs (21). Finally, drug-specific T cell lines and clones have been recently generated from the blood and tissues of patients with different types of ADR to β-L (10, 22).

The results of our study largely confirm previous results showing that PBMC from a proportion of patients with ADR to β-L proliferate in vitro in response to one or more of these drugs. Our findings are also consistent with the recent demonstration that some β-L-specific T cells exhibit a rather restricted specificity, whereas other T cells show a broader reactivity (23). Moreover, the positivity of LTT was found to be independent of both the type of clinical manifestation (immediate or late) and the presence of β-L-specific serum IgE Abs.

The reason why PBMC from a proportion of patients with ADR to β-L apparently do not proliferate in response to these drugs, despite the presence of β-L-specific serum IgE Abs, is unclear. It could be due to the difficulty to reproduce in vitro the same conditions that allow hapten presentation to Th cells in vivo. β-L are indeed nonpeptide Ags with low m.w. that, per se, are unable to evoke an adequate immune response. For many years, the occurrence of effector T cell responses to haptens, such as penicillin, has been explained by the hapten-carrier model (24). More recently, a series of nonconventional pathways for nonpeptide Ag presentation to T cells has been suggested, since haptenated peptides can be generated by modification of MHC-embedded peptides or haptens can directly alter the MHC molecule itself. Accordingly, it has been shown that TCR recognition of drugs can be MHC restricted, dose dependent, and APC dependent, but it may involve processing-independent presentation, since glutaraldehyde-fixed APC are able to present pen G to drug-specific T cell clones (11, 13, 25). Moreover, other studies have reported an HLA-DR allele-independent recognition of lidocaine by specific T cell clones (26).

The most interesting findings of this study came, however, from the experiments performed on T cell lines generated from PBMC of patients with ADR to β-L. First, pen G-specific T cell lines could be generated only from PBMC of patients showing positive LTT to pen G or other β-L, but neither from healthy donors nor patients with ADR, but negative LTT, to β-L (except 1 of 17). Regarding this latter group of patients, this may be due to the difficulty to reproduce in vitro the conditions occurring in vivo during presentation of β-L to T cells, as mentioned above, or may be related to the very low numbers of drug-reactive circulating T cells in some patients, since specific cells are mainly localized to lymph nodes or other tissues. Moreover, even if the possible role of cells other than T lymphocytes in some ADR cannot be excluded, no evidence for such a possibility has been reported so far. More importantly, the cytokine profile of pen G-, as well as of amox- and amp-, specific T cell lines, as detected by the expression of intracellular cytokine synthesis at the single cell level, was highly and consistently polarized toward the Th2 profile (production of IL-4, IL-5, and IL-13) in all pen G-reactive patients. In two of them, the prevalent Th2 profile of pen G-specific T cells was confirmed by measuring levels of soluble cytokines produced by a panel of pen G-specific T cell clones. These data are apparently at variance with those recently reported by Brander et al. (14) and Zanni et al. (15), showing that penicillin-specific T cell clones mainly produce high amounts of IFN-γ, and rather variable levels of IL-4 and IL-5. The reason for this discrepancy is presently unclear, but it may rest on differences in patient selection, culture conditions, or detection techniques. In a different study, T cell clones generated from the skin of patients with penicillin-induced bullous exanthems exhibited a CD8+ Th1-like phenotype, whereas T cell lines derived from peripheral blood of patients with penicillin-induced urticarial exanthems were predominantly CD4+ and displayed a Th2-like cytokine pattern. This suggests heterogeneity of effector responses depending on the type of clinical manifestations (10, 27).

Notably in our study, 10 of the 12 patients with ADR and positive LTT to β-L had no history of atopy, and, accordingly to that, not only bacterial Ag (SK)-specific but also Der p 1-specific T cell lines showed a Th1-polarized profile. It is also of note that Der p 1-specific T cell lines generated from atopic Der p-sensitive donors had a Th0/Th2-polarized cytokine profile at the intracellular level, which is consistent with the results obtained by using other techniques, such as the measurement of in vitro cytokine produced by specific T cell clones (28) and detection of cytokine mRNA by in situ hybridization (29) or PCR (30). Accordingly, in the two patients with ADR to β-L, who were atopic and Der p sensitive, Der p 1-specific T cells showed a Th2-skewed pattern of cytokine production. However, the cytokine profile of pen G-specific T cells, derived from either nonatopic donors or the two donors showing both types of sensitization, was even more Th2-polarized than the cytokine profile of Der p 1-specific T cells from atopic donors.

Taken together, these findings strongly suggest that mechanisms involved in the Th2 polarization of β-L-specific T cells are genetically different from those responsible for Th2 polarization of T cells specific for common environmental allergens in the so-called atopic donors. A different possibility, even if not mutually exclusive, is that environmental factors (site of Ag penetration, physiochemical structure and/or dose of the immunogen, etc.) related to drug sensitization may favor a more pronounced Th2 response than common environmental allergen. Both genetic and environmental factors contribute indeed in a variable, even if not yet clarified, way to the selective polarization of Th cell response toward the Th1 or the Th2 pathway (31). A second important remark comes from the observation that the cytokine profile of pen G-specific T cells was equally highly Th2 polarized in patients apparently lacking pen G-specific IgE Abs in their serum and/or those with clinical manifestations not reconducible to IgE-mediated immune reactions. The absence of pen G-specific IgE Abs in some patients with strongly Th2-polarized pen G-specific T cells may be simply explained by the longer persistence of circulating drug-specific T cells in comparison with the duration of drug-specific Abs in the serum. However, even patients with clinical manifestations that could not be easily explained by IgE-mediated reactions do actually show expansion of highly polarized pen G-specific T cells, suggesting that Th2 effector responses may be involved in different immunopathological reactions, including those that do not necessarily require IgE Ab production. These reactions may result from the dermal infiltration of Th2 cells, the recruitment of eosinophils by IL-5 produced by Th2 lymphocytes, and/or IL-4 and IL-13-mediated up-regulation of adhesion molecules, as recently described in late phase cutaneous reactions following allergen challenge (32, 33). On the other hand, the presence of both IL-4 and IL-5 mRNA and proteins has been recently demonstrated in bronchial biopsies from patients with either atopic or nonatopic asthma (34, 35), a finding that provides evidence against the concept that “intrinsic” and atopic asthma represent distinct immunopathological entities. Likewise, on the basis of our findings, it is reasonable to suggest that T cell effectors producing Th2 cytokines may account not only for ADR sustained by classic IgE-mediated reactions, but also for clinically distinct forms of ADR to β-L.

1

This work was supported by grants from European Community Biotech Project (Bi04.CT96.0246), Istituto Superiore di Sanitá (ISS; AIDS Projects 1998), and Associazione Italiana per la Ricerca sul Cancro (AIRC).

3

Abbreviations used in this paper: β-L, β-lactam; ADR, adverse drug reaction; pen G, penicillin G; amp, ampicillin; amox, amoxicillin; SK, streptokinase; Der p 1, Dermatophagoides pteronyssinus group 1; LTT, lymphocyte transformation test; MI, mitogenic index.

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