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Soluble Interleukin 2 Receptor and Interleukin 1 in Toxic Epidermal Necrolysis
A Comparative Analysis of Serum and Blister Fluid Samples
Osvaldo Correia, MD;
Luis Delgado, MD, PhD;
Jean-Claude Roujeau, MD, PhD;
Laurence Le Cleach, MD;
José A. Fleming-Torrinha, MD, PhD
Arch Dermatol. 2002;138:29-32.
ABSTRACT
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Background Toxic epidermal necrolysis (TEN) is a rare but severe adverse drug disease,
characterized by extensive skin and mucosal detachment with participation
of different immunoinflammatory pathways, in particular with early participation
of activated CD8+ T lymphocytes.
Objective To further study the potential role of T lymphocytes in the early phase
of keratinocyte necrosis.
Design Prospective study.
Setting University hospitals.
Patients Thirteen patients with clinical and histopathologic criteria of TEN
and 6 patients with second-degree burns.
Main Outcome Measures Measurement of soluble interleukin (IL) 2 receptor (sIL-2R) and IL-1
in serum samples and fluid of recent blisters.
Results In the blister fluid of patients with TEN, we found significantly higher
levels of sIL-2R than in patients with burns, whereas IL-1 levels were
higher in the blister fluid of burned patients. No significant differences
were found in serum samples of patients with TEN and burns, in either sIL-2R
or IL-1. In TEN we also found significantly higher levels of sIL-2R
in the blister fluid compared with serum samples, pointing to a predominantly
local production contrasting with the low concentration of sIL-2R in the blister
fluid of burned patients.
Conclusions Our findings of elevated sIL-2R levels in blister fluid of patients
with TEN are probably related to a local down-regulation of an immunologically
mediated cytotoxic reaction and further support the involvement of activated
T lymphocytes in the early blisters of TEN.
INTRODUCTION
TOXIC EPIDERMAL necrolysis (TEN) is an acute, life-threatening disease,
frequently drug induced, characterized by severe epithelial detachment of
skin and mucosal membranes. Although its pathophysiologic characteristics
are not fully understood, data emphasize immune mediation.1
The rare recurrent cases of TEN usually begin 48 hours after drug rechallenge,
contrasting with the usual delay of 1 to 3 weeks between the initiation of
the treatment and the appearance of the first lesions, suggesting immunologic
antigen priming. Clinical and histologic similarities with the severe forms
of acute graft-vs-host disease, the association with autoimmune diseases,
and the post-TEN Sjögren syndrome1-3
all favor an immune dysregulation. Recent evidence has shown that the massive
destruction of epidermis results from keratinocytes apoptosis,4-5
but the trigger for apoptosis remains unknown.
The involvement of a drug-specific cytotoxic T-cell response against
keratinocytes has recently been emphasized in different adverse drug reactions
of the skin.6-9
In TEN, several studies reported a predominance of CD8+ T lymphocytes
along the dermoepidermal junction, in the epidermis, or in the blister fluid
of early blisters.10-13
Our group and others have shown that these cells have the phenotype of memory
T cells and function as cytotoxic T cells.12-13
To further study the potential role of T lymphocytes in the early phase of
keratinocyte necrosis, when blistering is just appearing, we measured markers
of their activation, soluble interleukin (IL) 2 receptor (sIL-2R) and IL-1,
in the fluid of recent blisters of patients with TEN.
PATIENTS AND METHODS
We studied 13 patients with clinical and histopathologic findings of
TEN, according to established criteria.14 We
collected fluid from recent blisters (evolution <48 hours), early in the
beginning of the disease and before the introduction of any immunosuppresive
agent. In 7 of these patients, blood samples were also obtained at the same
time as blister fluid. As a control, we took blood and blister fluid from
6 patients with second-degree burns. Samples were centrifuged and stored at -20°C
until use. The IL-1 and sIL-2R were measured in duplicate samples by
means of commercially available specific enzyme-linked immunosorbent assays
(Immunotech, Marseille, France), according to the manufacturer's instructions.
Results are presented as medians and ranges. For statistical analysis, we
used the Mann-Whitney test, the Wilcoxon matched-pairs signed rank test, and
the Spearman rank correlation. A level of P<.05
was considered statistically significant.
RESULTS
Table 1 summarizes the main
clinical characteristics of the 13 patients with TEN. Median age was 36 years
(range, 13-53 years); 10 patients were female and 3 were male. Median skin
detachment on the day the samples were collected was 27% of body surface area
(range, 18%-58%), but it increased to a maximum of 40% (20%-95%). In all 13
patients, we collected blister fluid from recent blisters (evolution <48
hours) and early in the course of the disease. At that time, all patients
had fever (median temperature, 39.0°C), without documented associated
infection. Of the 13 patients, 5 (38.5%) died, most of them achieving the
higher percentages of skin detachment. Associated drugs were found in 12 of
the 13 patients, according to the imputability methods used for toxic drug
reactions.15
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Table 1. Relevant Clinical Characteristics of Patients With TEN*
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For the 6 control patients with second-degree burns, median age was
42 years (range, 20-46 years); 5 were female and 1 was male. Median skin detachment
on the day the samples were collected was 15% of body surface area (range,
10%-40%). At that time, the median temperature was 37.4°C (range, 37.0°C-38.2°C).
The sIL-2R and IL-1 levels in blister fluid and serum of the
patients with TEN and burns are shown in Table 2. In TEN blisters, we found significantly higher levels of
sIL-2R than in burns (P = .005), whereas IL-1
levels were higher in the blister fluid of burned patients (P = .02). No significant differences were found in serum samples of
patients with TEN and burns, in either sIL-2R or IL-1.
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Table 2. Blister Fluid and Serum Levels of sIL-2R and IL-1 in
Patients With TEN and Control Subjects With Burns*
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Table 3 compares the levels
of sIL-2R and IL-1 in the blister fluid and serum for both disorders,
taken at the same time. In TEN we found significantly higher levels of sIL-2R
in the blister fluid (P = .03) and no correlation
between levels of sIL-2R in serum and blister fluid (r
= 0.46, P = .29, Spearman rank correlation). In contrast,
burned patients had a significantly lower concentration of sIL-2R in the blister
fluid, which correlated positively with serum concentrations (r = 0.83, P = .04). We did not find a correlation
between blister and/or serum sIL-2R level and the severity (skin detachment)
or outcome of the patients. No significant differences were found between
serum and blister fluid levels of IL-1 in both patients with TEN and
burned control patients.
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Table 3. Comparison of Blister Fluid and Serum Levels of sIL-2R and
IL-1 in TEN*
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COMMENT
Toxic epidermal necrolysis is rare, with an incidence of 0.4 to 1.2
cases per 1 million people, but frequently fatal, with an estimated rate of
death around 30%.1-2 The patients
included in our series (Table 1)
were all classified according to a recognized classification.14
The most probable culprit drug was found according to an imputability method
used for adverse drug reactions.15 Toxic epidermal
necrolysis is usually associated with drug intake, but the pathophysiologic
characteristics are not fully understood; however, immunologic mediation is
unquestionable.4-5,10-13
Since the blistering eruption is a rapid event all the studies, the main immunologic
effectors and/or triggers in blood, skin, or blister fluid must be investigated
in the first hours after bulla appearance. With this rule in mind, we demonstrated
that CD8+ T lymphocytes with the phenotype of memory and cytotoxic
T cells were the predominant cells in the first blisters and within the 48
hours of their appearance.12-13
Most of the blister fluid T lymphocytes express the skin-homing receptor cutaneous
lymphocyte–associated antigen.16 In addition,
drug-specific CD8+ T-cell clones have been described in skin adverse
drug reactions,6 and T-cell–mediated
cytotoxic effects against keratinocytes seem to be drug specific and mediated
by perforin.8-9 The present study,
with the finding of high levels of sIL-2R in patients with TEN, mainly in
early blisters, further corroborates the participation of activated T lymphocytes
or associated cytokines in the first steps of this disease.
Soluble IL-2 receptor represents the extracellular domain of the 55-kd -chain
of the IL-2 receptor.17-18 In
vitro studies have shown the release of sIL-2R from activated T cells, correlating
with the rate of expression of the membrane-bound receptor and the degree
of activation.19 A similar release, but at
a much lower rate, has also been described in B cells and monocytes,19 and therefore sIL-2R is usually considered a marker
of T-cell activation. Elevated serum sIL-2R concentrations have been reported
in burned patients, as part of the inflammatory response and immune dysfunction
of the postburn period,18, 20 and
in disorders involving the activation of T cells, like autoimmune diseases21 or organ transplant rejection.22
In transplant rejection, serum sIL-2R concentrations correlate with the severity
of the disease,22 and, interestingly, they
are a sensitive indicator for acute graft-vs-host disease in allogeneic bone
marrow transplantation, a clinical and biological disorder that, in severe
cases, mimics TEN.1-2
This soluble protein (sIL-2R) can be measured in biological fluids by
means of an enzyme-linked immunosorbent assay and thus provides an indirect
indicator of activated T cells, which are known to be present in the early
blisters of TEN, where cytotoxic CD8+ lymphocytes predominate,
as recently shown.12-13 A small
subset of natural killer cells may also express the 55-kd -chain of
the IL-2 receptor (the CD16- CD56brightsubset),
representing less than 10% of natural killer cells.23
However, this discrete natural killer subset lacks killer cell immunoglobulin-like
receptor expression,24 in contrast to blister
fluid lymphocytes reported in TEN.13 In the
blister fluid of patients with TEN, we found significantly increased levels
of sIL-2R compared with control patients with second-degree burns (P = .005; Table 2). Moreover,
only in TEN cases, blister levels were significantly higher than levels in
corresponding serum samples taken at the time of blister puncture (P = .03; Table 3). This
finding, and the lack of correlation with serum levels (r = 0.46, P = .29, Spearman rank correlation),
points to a release of sIL-2R early at the site of blister formation in TEN
and supports the presence of activated T cells in lesional skin.16
In contrast, sIL-2R levels in control blisters of burned patients were significantly
lower than levels in corresponding serum samples and positively correlated
with serum concentrations (r = 0.83, P = .04), consistent with a diffusion from the blood compartment. In
2 patients with TEN, we measured sIL-2R level in blisters with less than 48
hours of evolution, but appearing in the subsequent days, and we found a decrease
in sIL-2R level associated with a decrease in the number of CD8+
lymphocytes (data not shown), further reinforcing the importance of knowing
when the different studies were performed.
A significant reservoir of preformed sequestered IL-1 exists
in epidermis, mainly in keratinocytes, and may be released on injury. External
stimuli, such as burns, or internal triggers, such as local cytokine production,
can induce the release of IL-1 for local or systemic delivery.25
Fluid taken from blisters on thermally injured skin, early after burn injury,
contains substantial amounts of IL-1, and the source is the injured keratinocytes.26 In fact, human keratinocytes contain predominantly
IL-1, but more than 50% of the IL-1 activity within the epidermis is
confined to the stratum corneum.27 Our data
confirm the rapid and intense aggression to keratinocytes in burned patients,
with consequent significantly higher levels of IL-1 in the early blisters,
suggesting that thermal injury is a major stimulus for the production of this
inflammatory cytokine by keratinocytes. However, different lesional mechanisms
may explain the significant difference we found in blister fluid IL-1
level of burned patients compared with those with TEN—an exogenous thermal
injury that induces keratinocyte necrosis at the epidermal surface (in burned
patients) vs an endogenous immunologic cytotoxic reaction that induces a Fas-ligand–mediated
keratinocyte apoptosis5 at the dermal-epidermal
surface.
The significance of our findings of high levels of sIL-2R in the blister
fluid of patients with TEN is difficult to establish, as the biological consequences
of high concentrations of sIL-2R in several diseases are still poorly understood.
In TEN, CD8+ cytotoxic T lymphocytes directly and/or by induced
cytokines are probably the main effectors to induce blister formation,6, 8-12
and an increased number of lymphocytes with the natural killer phenotype and
cytotoxic function were recently described in the blister fluid of patients
with TEN.13 As IL-2 is especially relevant
for the generation of cytotoxic T cells, natural killer cells, and lymphokine-activated
killer cells,17-18,27-29
it is possible that high levels of sIL-2R in the skin, even resulting from
immune activation, may be involved in a local down-regulation of an immunologically
mediated cytotoxic reaction. In this regard, Jobin et al,18
studying serum samples from burned patients, showed that purified sIL-2R inhibited
natural killer cell activity by 50% and suppressed IL-2–induced interferon-
production by peripheral blood mononuclear cells.
As the recently described expression of the killer cell immunoglobulin-like
receptor in most lymphocytes present in the early blister fluid of patients
with TEN,13 elevated sIL-2R levels in blister
fluid could be another way to down-regulate a massive cytotoxic reaction against
keratinocytes. In fact, recent reports indicate that sIL-2R suppresses both
natural killer cell activity and IL-2–induced interferon-,18 a cytokine required for the efficient specific killing
of keratinocytes by cytotoxic T cells30 and
drug-specific T-cell clones.8
While the cellular sources of the cytokines we measured in the blister
fluid of patients with TEN were not established in our study, their expression
and/or secretion in situ may participate in a cytokine cascade that culminates
in the extensive skin and mucosal injury seen in the disease. A better understanding
of the contribution of these and other immunoregulatory cytokines to the pathogenesis
of TEN may allow the design of more specific and more effective therapy.
AUTHOR INFORMATION
Accepted for publication May 1, 2001.
This study was partially supported by a 1996 grant from the Sociedade
Portuguesa Dermatologia Venereologia, Lisbon.
We thank Fernando Campilho, MD, Instituto Português Oncologia,
Porto, Portugal, for the statistical evaluation.
Corresponding author and reprints: Osvaldo Correia, MD, Department
of Dermatology, Instituto Português Oncologia, 4200 Porto, Portugal
(e-mail: osvaldo.correia{at}netc.pt).
From the Department of Dermatology, Instituto Português Oncologia
(Dr Correia), and Department of Immunology, Faculty of Medicine (Drs Correia,
Delgado, and Fleming-Torrinha), Porto, Portugal; and Department of Dermatology,
Hôpital Henri Mondor, Université Paris XII, Créteil, France
(Drs Roujeau and Le Cleach).
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