# Serum Concentration of Interleukin-6 Is Increased Both in Active and Remission Stages of Pemphigus Vulgaris

Authors: Joanna Narbutt; Jolanta Lukamowicz; Jarosław Bogaczewicz; Anna Sysa-Jedrzejowska; Jolanta Dorota Torzecka; Aleksandra Lesiak
Journal: Mediators of Inflammation
 (2008)

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## Abstract

As most studies on pemphigus vulgaris (PV) pathogenesis concern its active stage, we aimed to evaluate the serum concentration of TNF-α, IL-1, and IL-6 in PV patients in clinical remission. The study group consisted of sera from 19 PV patients in active stage and from 24 patients in clinical remission. 19 sera taken from healthy subjects served as the controls. Serum IL-6 concentrations in PV active and PV remission group were significantly higher when compared to the controls (P<.05). In patients in active stage of PV, a significant correlation between serum IL-1 and IL-6 concentrations was found (rP=0.46;P<.05).  We also found a negative correlation between TNF-α level and pemphigus antibodies titer in the patients from the remission group (rS=−0.47303;P<.02). Our data suggest that IL-6 and TNF-α may be involved in maintaining immunological disturbances in remission stage of PV.


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## Body

## 1. Introduction

Pemphigus is an autoimmune
blistering disease, characterised by severe and chronic course. Based on
clinical picture and profile of circulatingpemphigus antibodies, two distinct forms of the disease are distinguished:
pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Transmembrane desmosomal
proteins: desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) are pemphigus target
antigens. In serum of PF patients, anti-Dsg1 antibodies are detected while in
PV patients—antibodies are directed against Dsg3. However, in approximately 50% of the latter
cases, also anti-Dsg1 antibodies may be found. Binding of pemphigus antibodies to the target antigens leads to the failure in
adhesive function of desmogleins, and in consequence to disruption of desmosomes
and thus acantholysis development [1]. Despite numerous investigations performed all
over the world, the exact mechanism of acantholysis has not been fully elucidated.
Recent studies point out at proinflammatory cytokines such as TNF-α, IL-1, or
IL-6 as strong players involved in this process. Moreover, experimental studies
revealed that synergistic cooperation of pemphigus antibodies with Fas-L and TNF-α results in acantholysis. Increased mRNA expression
for TNF-α and enhanced cytokine’s level in serum of PV patients were observed
by many authors [2–4]. Furthermore,
experimental models showed that mice lacking TNF-α receptor are less sensitive
to pemphigus development after passive transfer of pemphigus antibodies [2]. The above data at least in part explain
therapeutic efficacy of anti-TNF-alpha antibodies in pemphigus vulgaris treatment
[5, 6].Despite extensive research, the objective criteria of complete cure in PV
have not been defined so far. Direct immunofluorescence (DIF) test detecting in vivo IgG deposits bound in the
intercellular spaces of the epidermis and indirect immunofluorescence (IIF) test
demonstrating circulating IgG antibodies are routine examinations in pemphigus diagnosis
and their negative findings may indicate treatment cessation. In some cases,
however, despite a long-lasting treatment and lack of clinical symptoms, either
both immunological examinations are still positive, or DIF test is negative,
but circulating antibodies are still detected. It seems controversial,
especially because most dermatologists assume a strong correlation between the
titre ofpemphigus antibodies and the
disease activity. Most studies on PV pathogenesis are focused on immunological
disturbances in active stage of the disease.Thus, the aim of our study was to evaluate the serum
concentration of TNF-α, IL-1, and IL-6 in PV patients both in active stage of
the disease and in clinical remission and assess their potential influence on
disease course. We also assessed the correlation between cytokines’ serum levels
and titer of pemphigu s antibodies.


## 2. Material and Methods

The study group consisted of 19 sera obtained from PV patients
in active stage of the disease and 24 sera obtained from patients in clinical
remission. Sera from active PV patients had been collected before
immunosuppressive treatment; (prednisone+cyclophosphamide) was introduced. 19
sera taken from healthy unrelated subjects, age and sex matched served as
controls. Pemphigus vulgaris diagnosis was based on the clinical picture and results
of histological and immunological examinations. The sera of all the patients
and individuals from the control group were examined by IIF on commercially
available substrate of monkey esophagus (Euroimmune, Lubeka, Germany),
using a standard procedure [7]. The same sera samples were examined by ELISA (MESACUP
Desmoglein Test Dsg1 and Dsg3; MBL Co. Ltd, Nagoya , Japan)
according to the manufacturer’s instruction. ELISA index was assessed as
positive when it was higher than cutoff (for Dsg 1–14; for Dgs 3–7).All the patients’
direct immunofluorescence (DIF) tests revealed in vivo IgG and/or C3 deposits bound in the intercellular spaces
of the epidermis. Active stage of the disease was considered when all the
examinations were positive, blisters and erosions on the mucous membranes
and/or skin were present. Sera samples were taken before introducing
immunosuppressive treatment. Clinical remission was considered when patients
were treated with prednisone (20–40 mg every other
day) and cyclophosphamide (50 mg every other day) and demonstrated lack of
clinical lesions for at least six months, while DIF and IIF still gave positive
results.The same sera
samples were used to measure TNFα, IL-1, and IL-6 levels using commercially
available ELISA kits (R & D, Minneapolis, Minn, USA), according to
manufacturer’s procedure.


## 3. Statistical Analysis

Statistical analysis was performed using Statistica
v.0.5 and GraphPad Prism v.4.03. As laboratory data did not fit a Gaussian distribution
according to Shapiro-Wilk test, all results were expressed as median (50th) and
interquartile range (25th–75th), and
nonparametric tests were used to test statistical significance. To avoid errors inherent in repeated application of
Mann-Whitney U tests, the Kruskal-Wallis test was performed to make
simultaneous comparison of the assay data from each group, and to determine whether
there was a significant variation in the medians of the groups analysed. If
this achieved 95% significance, Dunn’s multiple comparison posttest was then
used to compare the assay results of one group with the other. For
correlation studies, the Pearson's correlation or Spearman's rank correlation was
used. In all calculations,P<.05 was regarded as statistically
significant.


## 4. Results

Indirect immunofluorescence test revealed circulatingpemphigus antibodies in all PV patients.
In the subjects from active stage of the disease, antibodies titers ranged from
1:80 to 1:2560 (median 1:640) while in the patients from remission group from
1:20 to 1:1280 (median 1:80).Median serum concentrations of IL-1, IL-6, and TNF-α in all the examined patients are presented in
Table 1 and Figures 1–3. IL-1 serum
concentrations in the PV patients from active and remission groups did not significantly
differ from the controls. IL-1 concentration in the PV remission group was
higher than in the PV active group, however, this did not reach statistical
significance. Serum IL-6 concentrations in PV active and PV remission
group were
significantly higher when compared to the controls. IL-6 levels presented
slightly higher values in the active group in comparison to the remission
group, but the difference also did not reach statistical significance. Serum
TNF-α levels did not differ significantly between PV
active group, PV remission group and the control. The obtained values also did
not differ between PV active and PV remission groups.Table 1
Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) concentrations in serum samples from patients
with active stage of pemphigus vulgaris (PV active), remission stage of PV (PV
remission), and in normal controls.
CytokinesPV activeN=19PV remissionN=24ControlsN=19IL-1 (pg/mL)Median0.30.50.3(Interquartile range, 25th–75th)0.1–0.70.3–0.70.1–0.4IL-6 (pg/mL)Median3.382.581.97(Interquartile range, 25th–75th)2.72–4.982.085–3.861.64–2.25TNF-α (pg/mL)Median6.253.053.9(Interquartile range, 25th–75th)4.1–1002.8–1003.4–5.6Figure 1
Interleukin-1 (IL-1) concentration in serum samples
from patients with active stage of pemphigus vulgaris (PV active), remission
stage of PV (PV remission), and in normal controls. Box
plots with upper and lower bars showing the data range, and upper, middle, and
lower lines in the box showing 75th, 50th (median), and 25th centiles,
respectively.Figure 2
Interleukin-6 (IL-6)
concentration in
serum samples from patients with active stage of pemphigus vulgaris (PV
active), remission stage of PV (PV remission), and in normal controls. Box plots with upper and lower
bars showing the data range, and upper, middle, and lower lines in the box
showing 75th, 50th (median), and 25th centiles, respectively. Kruskal-Wallis
test across all three groupsP<.0001. Only significant Dunn’s multiple
comparison posttest p values between groups are shown.Figure 3
Tumor necrosis factor-α (TNF-α) concentration in serum samples from patients
with active stage of pemphigus vulgaris (PV active), remission stage of PV (PV
remission), and in normal controls. Box plots with upper and lower bars showing the data range, and
upper, middle, and lower lines in the box showing 75th, 50th (median), and 25th
centiles, respectively.A significant
correlation between serum IL-1 and IL-6 concentrations in patients presenting active
stage of PV was found(rp=0.46;P<.05) (see Figure 4). No similar relation was observed for the patients
in the remission stage of PV. We also did not find any significant correlations
between serum concentration of IL-1 and TNF-α or IL-6 and TNF-α in all the studied groups.Figure 4
Correlation between serum concentrations of IL-1 and IL-6 in patients
with active stage of PV.When analysing correlations between titer of
circulatingpemphigus antibodies and
serum concentrations of the examined cytokines, we found a negative correlation
between TNF-α level and antibodies titer in the patients
from the remission group (rS=−0.47303;P<.02).


## 5. Discussion

Pemphigus vulgaris is a life-threatening disease of
autoimmune background. During last decades, its pathogenesis has been widely
investigated and new insight into mechanisms triggering disease development and production of antibodies has been recognised.
Despite the fact of that detection of circulating and bound in vivo IgGpemphigus antibodies is that main immunological feature, current
knowledge point out at involvement not only humoral but also cellular
immunological response in pemphigus development. It was shown that
Dsg3-specific lymphocytes T proliferate under desmoglein 3 stimulation release Th2-dependent
cytokines such as IL-4 and IL-10 and demonstrate ability to modulate activity
of B cells [8, 9]. Although it is obvious that autoimmune response in PV is T
cell-mediated, the exact role of those cells in acantholysis development has
not been elucidated yet. IL-2 is one of the main activators of T lymphocytes.
Increased values of soluble receptor for IL-2 (sIL-2R) were detected in sera of
patients with pemphigus, and these values correlated with activity of the
disease. However, their concentrations were significantly higher in blister
fluid thus suggesting presence of activated T cells in PV skin lesions [10]. It
was also proven that isolated T lymphocytes from PV patients are required for
induction of histological and immunological phenomena in the animal models of
pemphigus [11]. Passive transfer of pemphigus antibodies into hairless mice genetically thymus deficient caused their binding
in intercellular spaces of the epidermis, however blister formation over the
basement layer of epidermis was observed in less than 20% of cases. It is
another proof for an important role of lymphocytes T in acantholysis development
[12].Keratinocytes stimulated with serum
obtained from PV patients presented higher expression of mRNA for IL-1α, TNF-α, and urokinase plasminogen activator (uPA) and
developed acantholysis. When antibodies against these proteins were used in
these experiments, the authors observed a significant inhibition of
acantholysis [2, 13]. Alecu et al. [14] found increased levels of ICAM-1, TNF-α, and IL-6 in sera and blister fluid obtained from PV
patients. The above results seem to at least in part confirm the role of these
proteins in PV development. Similar results were obtained by D’Auria et al. and
López-Robles et al. [3, 4] who showed increased serum levels and in situ expression of IL-6 and TNF-α. In experimental models, it was also demonstrated
that IL-1α and TNF-α were able to activate C3 mRNA in keratinocytes culture
after stimulation with pemphigus antibodies obtained from PV patients. This phenomenon is strongly inhibited
after subsequent incubation of keratinocytes with anti-TNF-α and anti-IL1α antibodies [15].D’Auria et al. [3] also showed a positive
correlation between serum concentration of these cytokines and the number of
skin lesions. In our study, we attempted to assess TNF-α, IL-1, and IL-6 sera
concentrations in PV patients being either in the active stage of the disease
or in clinical remission. In the patients presenting the active stage of the
disease, only IL-6 serum concentration was significantly higher when compared
to the control group. IL-1 and TNF-α serum levels did not differ between
patients in the active stage and the controls. In the patients who were in
clinical remission, we found similar results. Only IL-6 serum level was higher
while the rest of the examined cytokines presented similar values to the
controls. Decreased levels of IL-6 in remission stage of PV when compared to
the active patients (statistically insignificant) may result from the
immunosuppressive treatment administration. In many reports, it is noted that
prednisone and cyclophosphamide influence proinflammatory cytokines levels, including
IL-6 levels [16, 17]. Our results confirm the role of IL-6 in PV pathogenesis, not only
in the initial stage of the disease development but also the pathophysiological
process maintenance. In most published studies, the authors found an increased
level of TNF-α in the active stage of pemphigus [4]. TNF-α is a cytokine involved in the majority of
inflammatory processes, and its increased activity is found in many skin
diseases including psoriasis, SLE, or systemic sclerosis [18–20]. TNF-α is released by cells under various stimuli
including bacterial infections or ultraviolet radiation. It plays a role in
many biological processes, enhances phagocytosis, cytotoxicity, and modulates activity
of other cytokines such as IL-1 and IL-4 [21]. We did not confirm TNF-α increased concentration in PV patients when
compared to the controls. However, its negative correlation with pemphigus antibodies titer and
relatively increased values when compared to the controls (no statistical
significance) may suggest its involvement in maintaining pathological mechanisms.
Treatment with immunosuppressive agents is sufficient to reduce skin lesions
formation and decrease antibodies production, however it probably does not
influence disturbed cellular response. These data also partially explain why
anti-TNF-α treatment is successful in refractory PV patients.There is only a scarcity of data on
cytokines’ levels in sera of PV patients in clinical remission. Bhol et al. [22]
revealed a marked reduction of IL-1α and IL-1β with simultaneously increased
levels of IL-1R in sera of patients in clinical remission. Intravenous
immunoglobulin (IVIG) therapy caused similar effects thus suggesting that its
therapeutic effect could depend on modulation of IL1 isoforms and its receptor levels.
Galle et al. [23] and Keskin et al. [17] revealed also that IVIG exerts
anti-IL-6 properties thus to some extent, explaining mechanisms of its activity
in PV patients. IL-10 concentration in serum was also significantly lower in
patients in clinical remission when compared to the active ones and its values
correlated with pemphigus antibodies titers [24]. To the best of our knowledge,
there is no data on IL-6 serum concentration in the PV patients in clinical
remission. Our data suggest the role of IL-6 in the immunological disturbances maintenance
in remission stage of PV, namely, in the patients in whom, despite lack of
clinical lesions, antibodies are still produced and possess the ability to bind
to keratinocytes surface antigens. We believe that this observation suggests
clinical application of anti-IL-6 antibodies in refractory cases of pemphigus
vulgaris.



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