Ó 2007 The Authors
Journal compilation Ó 2007 Blackwell Munksgaard
Allergy 2007: 62: 1429–1438
DOI: 10.1111/j.1398-9995.2007.01542.x
Original article
Differential cytokine and transcription factor expression in patients
with allergic reactions to drugs
Background: Allergic drug reactions (ADR) can be either immediate reaction
(IR) (IgE mediated) or delayed reaction (DR) (T-cell mediated). They follow the
Th1/Th2 paradigm, with DR expressing interferon-c (IFN-c) with down-regulation of interleukin-4 (IL-4) and IR expressing IL-4 with down-regulation of
IFN-c. We studied the extension of this polarization in DR and IR by examining
the cytokine and transcription factor profile in T-cell subpopulations during the
acute phase of an ADR.
Methods: Expressions of cytokines [IL-4, IFN-c and tumor necrosis factor-a
(TNF-a)] and transcription factors (c-maf, GATA-3 and T-bet) were analysed
by semi-quantitative real time-polymerase chain reaction in peripheral blood
mononuclear cells and in CD4 and CD8 subpopulations from ADR patients.
Results: In DR, IFN-c, TNF-a and T-bet increased significantly in both CD4
and CD8 subpopulations, depending on the clinical severity. In IR, IL-4, c-Maf
and GATA-3 were increased, but only significantly in CD4. A positive correlation existed between IFN-c and T-bet in DR and between IL-4 and c-Maf and
GATA-3 in IR. In DR, IFN-c, TNF-a and T-bet were increased during the
acute phase in CD4 and CD8. In IR, IL-4, c-Maf and GATA-3 were all
increased in the acute phase, but only in CD4.
Conclusions: These results support the Th1/Th2 paradigm in ADR, confirming
previous findings that include the expression in both CD4 and CD8 T cells, and
extending the observation to the transcription factors involved in the polarization of the immune response. Monitoring the reactions in the cell populations
implicated, could be an important tool for assessing the mechanisms involved in
ADR.
Allergic drug reactions (ADR) are unpredictable,
uncommon, and usually not related to the pharmacological actions of the drug (1). Allergic drug reactions are
classified, depending on the time interval between the
appearance of the symptoms and the drug intake, as
immediate, accelerated or delayed (2). Immediate reactions (IR) correspond to those occurring <1 h after
drug intake, with anaphylaxis and urticaria being the
typical manifestations. Delayed reactions (DR) appear
>24 h after drug intake and have different clinical
manifestations, ranging from delayed urticaria and
maculopapular exanthema (MPE) to more severe reactions, such as Stevens-Johnson syndrome and toxic
Abbreviations: ADR, allergic drug reactions; DPT, drug provocation test; DR, delayed reactions; IFN, interferon; IL, interleukin;
IR, immediate reactions; MPE, maculopapular exanthema; PBGD,
porphobilinogen deaminase; PBMC, peripheral blood mononuclear
cells; SJS/TEN, Stevens-Johnson syndrome/toxic epidermal necrolysis; TNF, tumor necrosis factor.
J. A. Cornejo-Garcia1,
T. D. Fernandez1,
M. J. Torres2, M. Carballo3,
I. Hernan3, C. Antunez1, M. Blanca2,
C. Mayorga1
1
Research Laboratory for Allergic Diseases, Carlos
Haya Hospital-Fundacion IMABIS, Mlaga; 2Allergy
Service, Carlos Haya Hospital, Mlaga; 3Laboratory
Service, Terrassa Hospital, Terrassa, Barcelona,
Spain
Key words: allergy; cytokine; drug; T-cells; transcription
factors.
Cristobalina Mayorga, PhD
Research Laboratory
Hospital Civil
pabelln 5, stano
Malaga 29009
Spain
Accepted for publication 2 August 2007
epidermal necrolysis (SJS/TEN) (3). Accelerated reactions occur between 1 and 24 h after drug intake with
urticaria being the typical clinical manifestation and may
overlap with IR and DR.
These various clinical entities, although all mediated by
effector T cells, have a number of pathological differences
that are currently the objective of continued research.
T cells are a key factor in all types of ADR, regulating
both IgE production or effector cells, depending on their
profile of cytokine production (4). Two main T-cell
phenotypes have been identified, type 1 [interferon-c
(IFN-c) secreting] and type 2 [interleukin-4 (IL-4) secreting]. In DR, different T-cell subsets have been implicated
depending on the clinical entities, CD4 in MPE (5) and
CD8 in SJS/TEN; these have been demonstrated in
peripheral blood and skin in both blister fluids and
biopsies (6–8). Monitoring the allergic response during
the acute episode and the resolution period in the affected
tissue and peripheral blood can provide clues concerning
the mechanisms involved.
1429
Cornejo-Garcia et al.
In a previous study, we reported that cytokine production in peripheral blood mononuclear cells (PBMC)
from patients suffering an ADR followed the classical
Th1/Th2 paradigm depending on the type of reaction,
DR or IR (9). However, discrepancies exist between
investigators (9–12). These can be explained by differences in the clinical evaluation, in the cell type analysed,
or in the methodology used.
More detailed studies concerning the mechanism
underlying Th1/Th2 polarization in ADR are not generally available. These responses are the result of signals
through cytokine receptors that culminate in the binding
of specific transcription factors to multiple regulatory
elements in the promoters and subsequent activation of
cytokine genes. It is increasingly apparent that different
transcription factors such as GATA-3, T-bet and c-Maf,
are involved in directing Th1 or Th2 immune responses
(13). Thus, T-bet (T-box expressed in T-cells) is related
with Th1 responses, whereas the proto-oncogenes c-Maf
and GATA-3 are related with Th2 commitment. However, as far as we know, no studies have been carried out
dealing with ADR.
The aim of this work was to determine Th1 and Th2
cytokine expression, as well as T-bet, c-Maf and GATA-3
expression, in PBMC and into two different T-lymphocyte subpopulations, CD4 and CD8, obtained from
patients with DR and IR to drugs.
Material and methods
Subjects
Patients with a confirmed drug allergy after a clinical history, plus
skin testing or drug provocation test (DPT) when required, were
finally included. The clinical entities were established as described
(14). Patients were classified into two groups depending on the time
of occurrence and type of reaction: IR, appearing within 1 h of drug
intake, including urticaria and anaphylaxis; and DR, appearing
24 h or more after drug intake, including urticaria, MPE and SJS/
TEN. Accelerated reactions were not included in this study.
As controls, we selected a group of age- and sex-matched subjects
to the patients with no history of ADR or any cutaneous or
immunological diseases at the time of selection and good tolerance
to the drugs involved in the reactions. This study was approved by
the institutional review board, and informed consent for all the
diagnostic procedures was obtained from patients and controls.
The procedure to establish the diagnosis was as reported elsewhere (14). Basically, in DR intradermal or patch testing was performed depending on the drug solubility. If skin testing was
negative, and the reaction was not severe, a DPT was performed. In
IR, prick and intradermal tests were carried out although these
could only be performed with soluble drugs.
Sample collection
Blood samples were obtained from each patient, both with DR and
IR, within 24 h of the appearance of the clinical symptoms (T1) and
45 days later, when the reaction had cleared (T2). Peripheral blood
mononuclear cells were isolated by density gradient centrifugation
(Nycomed, Oslo, Norway) from 20 ml of heparinized venous blood.
1430
Table 1. Clinical data of patients with immediate and delayed allergic reaction to
drugs. Results of the allergological work-up for the drug involved in the reaction
Patient
Sex
Age
Reaction
type
Drug
involved
Clinical
symptoms
Skin
test
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
M
F
M
F
M
F
F
F
M
M
M
M
M
F
M
F
F
M
F
F
F
F
F
M
F
F
M
F
F
F
F
F
M
F
F
F
F
F
F
F
F
19
21
49
68
39
21
35
35
57
54
24
35
31
43
26
43
52
25
21
46
31
62
58
55
38
52
62
35
28
39
59
30
37
30
20
77
29
50
55
52
38
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
IR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
DR
AX
MET
MET
CEF
AX
AX
CEF
AX
AX
AX
AX
AX
CEF
AX
AX
AX
AX
CEPH
AX
CEPH
AX
SPI
MET
MET
AX
AX
AX
PHE
AX
AX
AX
PHE
CAR
AX
PAR
DIP
ALO
DIP
ALO
ALO
DIP
URT
URT
URT
URT
URT
URT
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
ANAPH
URT
URT
URT
URT
URT
URT
URT
URT
MPE
MPE
MPE
MPE
MPE
MPE
MPE
MPE
MPE
MPE
MPE
SJS/TEN
SJS/TEN
SJS/TEN
SJS/TEN
SJS/TEN
SJS/TEN
)
+
+
+
)
+
+
+
+
+
+
)
)
)
+
+
+
)
AX
)
)
)
)
)
)
+
)
)
)
ND
+
)
)
)
)
ND
ND
ND
ND
ND
ND
DPT
+
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
+
+
+
ND
ND
+
ND
+
+
+
+
+
+
ND
+
+
+
ND
ND
+
+
+
+
ND
ND
ND
ND
ND
ND
IR, immediate reactions; DR, delayed reactions; AX, amoxicillin; CEF, cefuroxime; MET,
metamizol; PAR, paracetamol; ALO, alopurinol; CEPH, cephalexin; DIP: diphenylidantoine; CAR, carbamazepine; PHE, phenobarbital; SPI, spiramicin; URT, urticaria;
ANAPH, anaphylaxis; SJS/TEN, Stevens-Johnson syndrome/toxic epidermal necrolysis; MPE, maculopapular exanthema; DPT, drug provocation test; ND, not done.
T-cell subpopulation isolation
CD4 and CD8 T-cell subpopulations were obtained from PBMC by
negative selection using magnetic beads (Miltenyi Biotech GmbH,
Bergisch Gladbach, Germany), according to the manufacturerÕs
instructions. The purity of the subpopulations was checked by flow
cytometry, using CD3- and CD14-PerCP, CD16-FITC, CD4-APC
and CD8- and CD19-PE monoclonal antibodies (Becton-Dickinson,
San José, CA, USA) on a Facscalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using Cell Quest software as described
(15).
Transcription factor in allergic reaction to drugs
Semi-quantification of cytokine and transcription factor mRNA
expression
Total RNA was extracted in TriPure (Roche, Indianapolis, IN, USA)
according to the manufacturerÕs instructions. One microgram of total
RNA was reverse-transcribed with random hexamers and moloney
murine leukaemia virus-reverse transcriptase at a final volume of
20 ll. Specific cDNA amplification of human cytokines IL-4, IFN-c,
tumor necrosis factor-a (TNF-a) and transcription factors T-bet,
c-Maf and GATA-3 was carried out with 2 ll of cDNA by using
FastStart DNA Master SYBR Green I (Roche). The specific primers
for cytokines, transcription factors and the housekeeping gene
[porphobilinogen deaminase (PBGD)] used were: 5¢-CAGATGTAGCGGATAATGGA-3¢ and 5¢-ACCTTGAAACAGCATCTGAC-3¢
for IFN-c; 5¢-CCCAGGCAGTCAGATCAT-3¢ and 5¢-GATGGTGTGGGTGAGGAG-3¢ for TNF-a; 5¢-TGCTGCCTCCAAGAACAG-3¢ and 5¢-TCACAGGACAGGAATTCAAG-3¢ for IL-4; 5¢CCCATTCCTGTCATTTACTG-3¢ and 5¢-GGCTCTCCGTCG-
TTCACCTC-3¢ for T-bet; 5¢-GGGACGCGTACAAGGAGAAA3¢and 5¢-TCAGGGGTAGGTGGTTCTCC-3¢ for c-Maf; 5¢-ACAAAATGAACGGACAGAAC-3¢ and 5¢-CTTTTTGGATTTGCTAGACA-3¢ for GATA-3 and 5-TCCAAGCGGAGCCATGTCTG3¢ and 5¢-AGAATCTTGTCCCCTGTGGTGGA-3¢ for PBGD. The
concentration was obtained by crossing-point extrapolation into a
standard curve with known cDNA concentrations by using Light
Cycler system software (Roche, Mannheim, Germany) and results
were expressed as relative units that represent the ratio of concentration between the specific mRNA and the housekeeping mRNA.
Statistical analysis
Comparisons for quantitative variables were performed by nonparametrical analysis, Mann–Whitney and Kruskall–Wallis tests for
nonrelated samples. Comparisons at two different times (T1 and T2)
were carried out with the Wilcoxon test for related samples. Correlation studies were performed by using PearsonÕs test. All reported
Figure 1. Box plots of the relative IFN-c, TNF-a and IL-4 mRNA levels in patients during the acute phase of immediate (IR) and
delayed reaction (DR) compared with the control group in peripheral blood mononuclear cells (PBMC), CD4 and CD8 T cells.
Results were normalized to the levels of porphobilinogen deaminase (PBGD) and expressed as arbitrary units.
1431
Cornejo-Garcia et al.
P-values represented two-tailed tests, with values £ 0.05 considered
statistically significant. For the statistical analysis the spss program,
version 11.5 was used.
Results
A total of 41 patients (mean age: 42 ± 14.76 years), 28
female and 13 male, finally diagnosed as allergic to drugs,
were included (25 with DR and 16 with IR). The drugs most
frequently involved in the reactions were betalactams in 12
DR cases and 14 IR cases, nonsteroidal antiinflammatory
drugs in three DR and two IR, anticonvulsant in six DR
cases, alopurinol in three DR cases and macrolide in one
DR case. The percentage of atopy of this group of patients,
tested with a panel of inhalant and food allergens, was 20%.
This was similar to that of the general population. Table 1
shows the clinical characteristics and the results of the
allergological work-up (skin test and DPT) of the patients.
Cytokine expression from patients with DR and IR at the acute
phase of the allergic reaction (T1)
We found a significant increase in IFN-c expression in
PBMC in DR compared with both IR (P = 0.027) and
controls (P = 0.0001). With respect to TNF-a production, we found significant differences with controls
(P = 0.001) and although the median of this was higher
in DR than in IR, this was no significant (Fig. 1). For
Figure 2. Box plots of the relative IFN-c, TNF-a and IL-4 mRNA levels in patients with immediate (IR) and delayed reaction (DR)
with varying degrees of severity of the clinical symptoms (mild, moderate and severe) compared with the control group. Results were
normalized to the levels of porphobilinogen deaminase (PBGD) and expressed as arbitrary units.
1432
Transcription factor in allergic reaction to drugs
IL-4 expression, although an increase was detected in IR
compared with DR and controls, this was not statistically
significant (Fig. 1).
When we analysed these comparisons into two T-cell
subpopulations (CD4 and CD8), we found an increase in
IFN-c in DR compared with IR and controls in CD4
(P = 0.006 and P = 0.008 respectively) and CD8
(P = 0.017 and P = 0.006 respectively). Tumor necrosis
factor-a expression had similar variations in both CD4
(P = 0.02 and P = 0.043 respectively) and CD8
(P = 0.049 and P = 0.003 respectively) (Fig. 1). In IR
we found an increase in IL-4 production compared with
DR and controls only in the case of the CD4 T-cells
(P = 0.001 and P = 0.045 respectively).
Delayed reactions can be classified, according to the
severity of the clinical symptoms, as mild (urticaria),
moderate (MPE) and severe (SJS/TEN). Comparison
showed that IFN-c was increased in all three groups
compared with controls (P = 0.05 for mild, P = 0.004 for
moderate and P = 0.011 for severe reactions). Although
higher in the most severe cases, significant differences were
only obtained when compared with mild reactions
(P = 0.05). Tumor necrosis factor-a analysis showed
statistical increases in moderate (P = 0.003) and severe
reactions (P = 0.05) compared with the controls (Fig. 2).
When we analysed the two T-cell subpopulations we
found that in the IFN-c production for CD4 T cells
(P = 0.011 for mild, P = 0.007 for moderate and
P = 0.006 for severe reactions) whereas for CD8 the
differences were only found for moderate (P = 0.008)
and severe (P = 0.017) reactions compared with controls. In the case of TNF-a, we found greater differences
Figure 3. Box plots of the relative T-bet, c-Maf and GATA-3 mRNA levels in patients during the acute phase of immediate (IR) and
delayed reactions (DR) compared with control group in peripheral blood mononuclear cells (PBMC), CD4 and CD8 T cells. Results
were normalized to the levels of porphobilinogen deaminase (PBGD) and expressed as arbitrary units.
1433
Cornejo-Garcia et al.
in both CD4 T cells (P = 0.007 for urticaria, P = 0.034
for MPE and P = 0.011 for SJS/TEN) and CD8 cells
(P = 0.021 for urticaria, P = 0.011 for MPE and
P = 0.025 for SJS/TEN) (Fig. 2).
Depending on the severity of the clinical symptoms, IR
can be classified as mild (urticaria) or severe (anaphylaxis). Comparisons showed no significant differences for
any of the cytokines in PBMC, CD4 and CD8 subpopulations. However, a tendency for increased IL-4 production was found in CD4 cells from patients with severe
IR reactions (Fig. 2).
Transcription factor expression in patients with DR and IR at the
acute phase of the allergic reaction (T1)
The determination of transcription factors showed that
T-bet was increased in DR compared with IR and
controls in PBMC (P = 0.002 and P = 0.0001 respectively), CD4 (P = 0.0001 for both) and CD8 (P = 0.023
and P = 0.02 respectively). The c-Maf was increased in
IR compared with DR and controls in PBMC
(P = 0.0001 for both) and CD4 (P = 0.0001 and
P = 0.002 respectively) but not in CD8. In GATA-3,
the differences were only found in the CD4 subpopulation
an increase in IR compared with DR and controls
(P = 0.0001 for both) (Fig. 3).
When we analysed the transcription factor expression in
patients suffering a DR according to the severity of the
clinical symptoms, significant differences were found compared with controls in T-bet expression in PBMC
(P = 0.016 for mild, P = 0.0001 for moderate and P =
0.007 for severe), in CD4 (P = 0.005 for mild, P = 0.014
for moderate and P = 0.013 for severe) and in CD8
(P = 0.007 for mild and P = 0.014 for severe). Interest-
Figure 4. Box plots of the relative T-bet, c-Maf and GATA-3 mRNA levels in patients with immediate (IR) and delayed reactions
(DR) with varying degrees of severity of the clinical symptoms (mild, moderate and severe) compared with the control group. Results
were normalized to the levels of porphobilinogen deaminase (PBGD) and expressed as arbitrary units.
1434
Transcription factor in allergic reaction to drugs
ingly, we detected an increased expression of T-bet in
severe, compared with mild and moderate reactions
(P = 0.034 and P = 0.017 respectively) in CD8 (Fig. 4).
The analysis of IR, according to the severity of clinical
symptoms, showed differences for c-Maf in severe and
mild vs controls in PBMC (P = 0.001 and P = 0.01
respectively) and CD4 (P = 0.017 and P = 0.007 respectively) but not in CD8. These differences were stronger
for severe reactions with a significant increase when
compared with mild in CD4 (P = 0.02). For GATA-3, as
occurred before, differences were only observed in CD4
T-cells, with increased expression in severe reactions
compared with mild reactions and controls (P = 0.0001
and P = 0.013 respectively) (Fig. 4).
Cytokine and transcription factor correlations in DR and IR at the
acute phase of the allergic reaction (T1)
Statistical analysis showed that in DR, IFN-c was associated with T-bet in both PBMC (r2 = 0.644, P = 0.018)
and CD4 (r2 = 0.522, P = 0.046) whereas TNF-a expression correlated only in CD4 lymphocytes (r2 = 0.842,
P < 0.005). In IR, IL-4 expression, correlated with the
expression of c-Maf and GATA-3 in both PBMC
(r2 = 0.653, P = 0.029; r2 = 0.895, P = 0.016, respectively) and CD4 (r2 = 0.796, P = 0.01; r2 = 0.771,
P = 0.025 respectively). We also found a statistical
association between the two Th2 transcription factors
GATA-3 and c-Maf (r2 = 0.818, P = 0.047) (Fig. 5).
No correlations were found in the case of the CD8
subpopulation for any of the transcription factors or
cytokines studied in either DR or IR (data not shown in
figures).
Cytokine and transcription factor expression in patients with DR
and IR at the acute phase (T1) and after symptoms subsided (T2)
In DR, we observed an increase in IFN-c expression in
PBMC (P = 0.008), CD4 (P = 0.05) and CD8
(P = 0.043) and in TNF-a in PBMC (P = 0.006) and
CD4 (P = 0.001) at T1. With respect to the transcription
factors, we found that T-bet was increased in PBMC
(P = 0.05), CD4 (P = 0.037) and CD8 (P = 0.012).
Interestingly, the expression of the two Th2 transcription
factors, c-Maf and GATA3, was reduced in T1 compared
with T2 in those patients with DR, though only in CD4
lymphocytes (P = 0.028 and P = 0.012, respectively)
(Table 2).
Figure 5. (A) Correlation scatter plots of IL-4 with c-Maf and GATA-3 expression in both peripheral blood mononuclear cells
(PBMC) and CD4 in immediate reaction (IR). (B) Correlation scatter plots of IFN-c with T-bet in both PBMC and CD4 in delayed
reaction (DR).
1435
(0.00001–0.1)
(0.01–0.23)
(0.18–1.06)
(0.11–0.16)
(0.05–0.21)
(0.028–0.36)
In IR, there was a significant increase of IL-4 in PBMC
and CD4 T cells (P = 0.003 and P = 0.028, respectively)
at T1. The c-Maf expression was increased in PBMC
(P = 0.05) and in CD4 lymphocytes (P = 0.028),
whereas GATA-3 expression was only significantly
increased in CD4 cells (P = 0.028) (Table 2).
(0.13–0.39)
(0.14–1.37)
(0.13–0.37)
(0.3–1.18)
(0.1–0.49)
(0.07–0.26)
(0.17–0.53)
(0.23–1.03)
(0.01–0.45)
(1.11–4.1)
(0.062–0.42)
(0.02–0.16)
0.025
0.09
0.002
0.15
0.3
0.24
(0.04–0.18)
(0.18–0.12)
(0.001–0.009)
(0.02–1.5)
(0.22–0.54)
(0.09–0.53)
Discussion
1436
(0.04–0.29)
(0.13–0.44)
(0.06–0.12)
(0.14–0.65)
(0.002–0.49)
(0.21–3.7)
All data are represented as median and interquartile range (25–75).
PBMC, peripheral blood mononuclear cells, IR, immediate reaction; DR, delayed reactions. IFN, interferon; TNF, tumor necrosis factor; IL, interleukin.
Significant differences (P < 0.05) by Wilcoxon test are shown in bold.
0.11
0.22
0.082
0.59
0.19
0.92
(0.12–0.77)
(0.18–0.88)
(0.05–0.14)
(1.26–2.14)
(0.001–0.45)
(0.26–0.5)
(0.027–0.072)
(0.07–0.22)
(0.11–0.53)
(0.17–0.21)
(0.22–0.29)
(0.06–0.27)
(0.014–0.03)
(0.018–0.06)
(0.14–0.36)
(0.19–0.52)
(0.013–0.04)
(0.01–0.2)
(0.04–0.12)
(0.08–0.24)
(0.006–0.1)
(0.34–0.95)
(0.001–0.68)
(0.36–1.1)
0.11
0.21
0.15
0.78
0.9
0.74
IFN-c
TNF-a
IL-4
T-bet
c-Maf
GATA-3
T1
(0.025–0.2)
(0.067–0.33)
(0.06–0.52)
(0.66–1.07)
(0.6–1.23)
(0.33–0.19)
0.084
0.163
0.068
0.48
0.25
0.49
T2
0.073
0.046
0.95
0.42
1.45
1.02
T1
(0.008–0.097)
(0.017–0.21)
(0.72–1.45)
(0.28–0.58)
(0.97–2.94)
(0.62–1.39)
0.015
0.037
0.17
0.33
0.02
0.092
T2
0.02
0.14
0.124
0.195
0.21
0.097
T1
(0.08–0.06)
(0.1–0.32)
(0.11–0.73)
(0.19–0.43)
(0.09–0.35)
(0.05–0.25)
0.04
0.09
0.125
0.177
0.26
0.154
T2
0.28
0.48
0.073
1.65
0.22
0.32
T1
PBMC
CD8
CD4
PBMC
Immediate
Table 2. Cytokine and transcription factor expression in PBMC, CD4 and CD8 T cells from patients with IR and DR at T1 and T2
T2
0.28
0.48
0.19
1.79
0.15
0.055
T1
CD4
Delayed
T2
0.23
0.31
0.24
0.68
0.16
0.168
T1
CD8
0.0001
0.037
0.25
0.064
0.73
0.083
T2
Cornejo-Garcia et al.
The induction of allergic diseases depends on the coordinated regulation of many genes, whose products
determine, among other processes, cytokine synthesis.
The expression of gene products is regulated at multiple
levels, with transcription factors being a key element in
the tightly regulated mechanisms governing Th1 and Th2
cell differentiation and the maintenance of the corresponding immune response (16). T-bet plays a key role
for the development of Th1 cells producing IFN-c (17)
and represses the production of Th2 cytokines, IL-4 and
IL-5 (13). The proto-oncogene c-Maf is mainly expressed
by Th2 cells, but, although c-Maf is specific for IL-4, it is
not sufficient (18). A more important transcription factor
for Th2 differentiation is GATA-3, which inhibits the
production of IFN-c, increases the transactivation of the
IL-4 promoter, and directly regulates IL-5 and IL-13
expression (13, 19).
An imbalance between the Th1-specific T-bet and the
Th2-specific GATA-3 and c-Maf transcription factors
can result in several immunological diseases (20–25).
T-bet is increased in some Th1 diseases such as celiac,
CrohnÕs disease and multiple sclerosis (20–22). In Th2
allergic diseases, GATA-3 is increased in patients with
allergic rhinitis (25), and both GATA-3 and c-Maf in
asthma (23). However, although in ADR both Th1 and
Th2 responses can be involved, we are unaware of any
study analysing the relationships between these transcription factors and cytokines with the exception of contact
dermatitis where T-bet plays an important role (26).
The results of the present study on cytokine expression
in PBMC showed a Th1 pattern with a high expression of
IFN-c and TNF-a in DR. However, the Th2 pattern was
not so well defined in IL-4 expression in IR. Additionally,
analysis of different T-cell subsets showed a Th1 pattern
in both CD4 and CD8 cells for DR but a Th2 pattern,
which did not appear in PBMCs, in just CD4 T cells in
IR. Previous studies developed not only with mRNA gene
expression (9, 27) but also with cytokine protein determination by flow cytometry (8) and by enzyme-linked
immunosorbent assay (9), showed a similar pattern,
although with lower sensitivity. This work supports those
data, with additional information about the T-cell subpopulations involved. These cytokine profiles have not
been so clearly found before because studies have used
T cells without specifically examining subpopulations,
and this study shows that this can be a limitation in the
final analysis of the results (11, 12). On the other hand,
Transcription factor in allergic reaction to drugs
in vitro cell stimulation conditions, ionomicin, phorbol
12-myristate 13-acetate and the time of incubation can
alter cytokine production, even if the analysis is performed in different T-cell subsets (10, 11). The approach
followed in our study was to observe the different T-cell
subpopulations involved by obtaining lymphocytes from
peripheral blood, with no other additional intervention,
to verify in detail the nature of the ongoing process.
Our results confirm the Th1/Th2 paradigm with the
involvement of the corresponding transcription factors.
In DR, a Th1 pattern with T-bet in PBMC and in both
CD4 and CD8 T cells was observed, and in IR, a Th2
pattern with c-Maf in PBMC and CD4 T cells. For
GATA-3, differences were only detected in the CD4
T-cells, although studies examining PBMC have shown
the importance of GATA-3 in the induction of Th2
responses (19, 24, 25). This could be explained by
compensation in PBMC levels with a decrease in CD8
or because the expression of GATA-3 is earlier than the
induction of c-Maf (28) and our samples were collected
after GATA-3 peaked.
This study also confirms previous results showing that
the production of different cytokines is correlated with the
severity of ADR (9). However, in the more severe reactions
we also found a higher expression of T-bet (DR) and of
both c-Maf and GATA-3 (IR). Subpopulation analysis in
IR only showed differences in the CD4 T cells, whereas in
DR these differences depended on the clinical manifestation, with higher levels of T-bet in CD4 for moderate
reactions and in CD8 T-cells for severe reactions. Examination of the relationship between the severity of the
reaction (MPE vs SJS/TEN) showed that CD4 is mainly
involved in MPE (5) and CD8 in SJS/TEN (7). These
differences are more marked for T-bet. Although T-bet is
mainly produced in CD4 T-cells, this transcription factor
is also required for the differentiation of naı̈ve CD8
T lymphocytes into cytotoxic effector cells (29).
We also found significant positive correlations between
cytokines and transcription factors, as reported by others
(13, 30, 31). In IR, we observed a correlation between
IL-4 and c-Maf and GATA-3 expression, and between
GATA-3 and c-Maf, and in DR between both IFN-c and
TNF-a and T-bet. A correlation has previously been
shown between IFN-c and T-bet in PBMC from patients
with rheumatoid arthritis (30) and in CD4 T cells from
allergic patients (31). We are unaware of the explanation
for the positive correlation between TNF-a and T-bet. It
may be as a result of two coexisting pathological
phenomena: one related to the severity (TNF-a) and the
other to the degree of involvement of the Th1 immunological response (T-bet).
Although drugs can affect several parameters of the
immune response, including cytokine levels (32, 33), in the
absence of immunological manifestations, no such effect
was observed in our control group. Nevertheless, the
results were not epiphenomena as cytokines and transcription factors all returned to levels detected in controls during
the resolution phase of the disease in both DR and IR.
This is the first study to demonstrate the potential
involvement of various transcription factors in ADR and
their association with key Th1 and Th2 cytokines. We
also show the relevance of monitoring the reactions as a
tool for assessing the mechanisms involved. These data
emphasize that, as suspected, IR show a homogeneous
pathophysiological mechanism whereas in DR each
clinical entity is a consequence of different mechanisms
with the involvement of a cell subpopulation. This raises
the need to analyse the different T-cell markers in the
particular T-cell populations (CD4 or CD8). Understanding the cytokine and the transcription factors
involved will enable development of immunological
intervention to control the ongoing processes.
Acknowledgments
We thank Ian Johnstone for help with the English language version
of the manuscript. This work has been funded by Spanish Ministry
of Health (FIS 01/0014, FIS 01/3031 and FIS PIO20640) and the
Junta de Andalucı́a (134/01).
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