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Department of Internal Medicine, Section of Internal Medicine, Immunoallergology and Respiratory Diseases, University of Florence, Florence, Italy;
Division of Allergology and Clinical Immunology, San Giovanni di Dio Hospital, Florence, Italy; and
Allergy Laboratory, Carlos Haya Hospital, Malaga, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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with poor or no IL-4, whereas Der p 1-specific T cells exhibited
IFN- 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-, 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-. These data suggest that cytokines produced by Th2 cells
play an important role in all ß-L-induced ADR, even when late
clinical manifestations occur and an IgE-mediated mechanism is
apparently indemonstrable. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is in vitro evidence that T cells participate in any type of ADR
to ß-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 ß-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-, 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 ß-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 ß-L. Only a proportion of subjects with
history of ADR to ß-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
ß-L-specific IgE Abs. As expected, SK-specific T cells from
all groups of subjects showed intracellular expression of IFN- with
poor or no IL-4, while Der p 1-specific T cells exhibited prevalent
IFN- 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 ß-L showed high production of IL-4, IL-5, and
IL-13, but poor or no expression of IFN-, 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
ß-L are generally characterized by the activation of Th2 cells
irrespective of the type of ADR induced by these drugs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- (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).
Subjects
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 ß-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 ß-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 ß-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 ß-L exhibited serum IgE Abs specific for pen G, amp, or amox, alone or in combination.
Detection of specific IgE Abs
IgE Abs specific for ß-L (penicilloyl G, penicilloyl V, ampicilloyl, amoxicilloyl), inhaled or food allergens were measured in the serum by commercial kits (CAP System; Pharmacia, Uppsala, Sweden).
Lymphocyte transformation test
PBMC from subjects with ADR and nonatopic donors were
isolated by Ficoll-Hypaque density gradient (Pharmacia), and 2 x
105 cells were cultured in triplicate for 6 days
in antibiotic-free RPMI 1640 medium (Seromed, Berlin, Germany),
supplemented with 2 x 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 ß-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.
Generation of short-term T cell lines and T cell clones
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 x
104 T cell blasts were incubated in triplicate
for 48 h in 0.2 ml volume with 5 x 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 cytokine production
Intracytofluorometric analysis of IL-4, IL-5, IL-13, and
IFN- 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.
Quantitation of cytokine content in T cell clone supernatants
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-, 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.
Statistical analysis
For statistical analysis t and
2 tests were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
ß-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 ß-L also showed positive LTT.
Cross-reactivity in the LTT to at least two of the three ß-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).
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). It is of
note that 10 of 12 subjects showing positive LTT to ß-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.
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To evaluate the cytokine profile of ß-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 ß-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- 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- (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 ß-L, the great majority of SK- or Der p 1-specific T
cells synthesized IFN- (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-),
and only a very few of them produced IFN- alone. In these subjects,
there was a highly statistically significant difference in the number
of IL-4- and IFN--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.
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. 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 ß-L-sensitive patients, apparently lacking serum
drug-specific IgE Abs, displayed a similarly Th2-polarized pattern of
intracellular cytokine expression (data not shown).
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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
ß-L. IL-4 and IFN- 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- alone, thus showing a
clear-cut Th1-like profile; 4 of them (21%) produced both IL-4 and
IFN-, whereas none of them produced IL-4 alone. By contrast, none of
the pen G-specific T cell clones produced IFN- alone, 9 of them
produced both IFN- and IL-4 (53%), and 7 produced IL-4 alone
(41%), thus showing a clearly polarized Th0/Th2 profile.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of our study largely confirm previous results showing that PBMC from a proportion of patients with ADR to ß-L proliferate in vitro in response to one or more of these drugs. Our findings are also consistent with the recent demonstration that some ß-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 ß-L-specific serum IgE Abs.
The reason why PBMC from a proportion of patients with ADR to ß-L apparently do not proliferate in response to these drugs, despite the presence of ß-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. ß-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 ß-L. First, pen G-specific T cell lines could be
generated only from PBMC of patients showing positive LTT to pen G or
other ß-L, but neither from healthy donors nor patients with ADR, but
negative LTT, to ß-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 ß-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-, 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 ß-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 ß-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 ß-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 ß-L.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sergio Romagnani, Dipartimento di Medicina Interna, Sezione di Medicina Interna ed Immunoallergologia, Viale Morgagni 85, 50134 Florence, Italy. E-mail address:
3 Abbreviations used in this paper: ß-L, ß-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.
Received for publication February 1, 1999. Accepted for publication May 6, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß T lymphocytes. J. Clin. Invest. 102:1591.[Medline]
and IL-4 on the in vitro development of human Th1 or Th2 clones. J. Immunol. 148:2142.[Abstract]
and interleukin-4 expressing cells. Eur. J. Immunol. 24:1097.[Medline]
ß+ T cell clones. Int. Immunol. 10:507.This article has been cited by other articles:
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  B. M. Brooks, C. A. Hart, and J. W. Coleman Differential effects of {beta}-lactams on human IFN-{gamma} activity J. Antimicrob. Chemother., December 1, 2005; 56(6): 1122 - 1125. [Abstract] [Full Text] [PDF]
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 W. J. Pichler Delayed Drug Hypersensitivity Reactions Ann Intern Med, October 21, 2003; 139(8): 683 - 693. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 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-