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wileyonlinelibrary.com/journal/exd Experimental Dermatology. 2017;26:1248–1252.
© 2017 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd
Accepted: 8 May 2017
DOI: 10.1111/exd.13382
ORIGINAL ARTICLE
Analysis of serum markers of cellular immune activation in
patients with bullous pemphigoid
Katja Bieber1 | Anna Lara Ernst1 | Stefan Tukaj2 | Maike M. Holtsche3 |
Enno Schmidt1,3 | Detlef Zillikens1,3 | Ralf J. Ludwig1,3 | Michael Kasperkiewicz3
1Lübeck Institute of Experimental
Dermatology, University of Lübeck, Lübeck,
Germany
2Department of Molecular Biology, University
of Gdańsk, Gdańsk, Poland
3Department of Dermatology, University of
Lübeck, Lübeck, Germany
Correspondence
Michael Kasperkiewicz, Department of
Dermatology, University of Lübeck, Lübeck,
Germany.
Email: Michael.Kasperkiewicz@uk-sh.de
Funding information
Deutsche Forschungsgemeinschaft
Abstract
Experimental models of bullous pemphigoid (BP), the most frequent subepidermal
autoimmune bullous disease, revealed that the immune response leading to blister
formation represents an incompletely understood complex process involving different
inflammatory cells. In contrast to previous reports commonly focusing on limited
molecular and cellular phenotypes of the disease, the aim of this study was to investi-
gate a broad spectrum of markers of cellular immune activation in patients with BP.
We found that serum levels of soluble CD4, myeloperoxidase, S100A12, eosinophil
cationic protein and soluble P- selectin were significantly elevated in patients with
active BP compared with normal controls. Mast cell tryptase and neopterin serum
levels significantly decreased at the time of clinical remission of the patients.
Additionally, serum concentrations of soluble IL- 2 receptor, mast cell tryptase and
soluble P- selectin were significantly associated with levels of circulating anti- BP180
autoantibodies. Our findings confirm and extend previous reports suggesting some
concomitant involvement of a panel of molecules representative for a wide spectrum
of cellular players (T cells, mast cells, neutrophils, eosinophils, macrophages and
platelets) orchestrating the inflammatory reaction in BP. These data may favour the
employment of broad- spectrum or combined immunosuppressants, potentially
together with an anticoagulant treatment, over cell- or molecule- specific targeted
therapy in patients with this disorder.
KEYWORDS
autoantibody, inflammation, pemphigoid
1 | INTRODUCTION
Bullous pemphigoid (BP) is the most frequent autoimmune subepider-
mal blistering dermatosis, characterized by autoantibodies directed
against the dermal–epidermal junction proteins BP180/BP230 typi-
cally causing pruritic bullous eruptions.[1] While it is well known that
autoantibodies against the 16th non- collagenous (NC16A) epitope
of the BP180 ectodomain are directly pathogenic and serologically
parallel the clinical course of disease,[2] knowledge on other biological
indicators of BP activity is incomplete.
The immune response leading to blister formation in BP rep-
resents a complex process involving different inflammatory cells and
molecules, including NC16A- reactive CD4 T cells, B cells, comple-
ment factors, mast cells, neutrophils, eosinophils, macrophages and
granulocyte- derived basal membrane zone- degrading proteases and
reactive oxygen species.[1,3,4] Previous studies demonstrated that
symptoms of patients with BP are associated with a plethora of raised
cytokines and chemokines in sera and blister fluids.[1,5,6] Moreover, the
Katja Bieber and Anna Lara Ernst are contributed equally to this work
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activation of the coagulation cascade seems to be involved in the dis-
ease pathophysiology.[7-9]
It is conceivable that the effect of a single mediator cannot
produce all the aspects of the clinically expressed lesion, but it is
rather the interplay of different factors orchestrating the inflamma-
tory reaction which may better explain the clinical outcome in BP.
Therefore, and in contrast to previous reports mainly focusing on
single or restricted molecular and cellular phenotypes of this dis-
ease,[10-18] the aim of the study was to compare the degree of as-
sociations between a broad spectrum of markers of cellular immune
activation and disease activity in patients with BP. We investigated
serum parameters related to activation of T cells (soluble CD4 [sCD4]
and soluble IL- 2 receptor [sIL- 2R]), B cells (IgD), mast cells (mast cell
tryptase [MCT]), neutrophils (myeloperoxidase [MPO], S100A12 and
soluble L- selectin [sL- selectin]), eosinophils (eosinophil cationic pro-
tein [ECP]), macrophages (neopterin) and platelets (soluble P- selectin
[sP- selectin]).
2 | METHODS
2.1 | Patients
Sera were obtained from 25 BP patients with active skin lesions (mean
age 73±9 years, 15 females, 10 males) and elevated circulating anti-
BP180 NC16A autoantibodies (mean 504±1080 U/mL, range 53-
5543 U/mL) who were admitted to the Department of Dermatology
of the University of Lübeck. In addition, another set of sera was
analysed from 10 of the 25 patients with BP, from whom follow- up
data were available, after a mean follow- up period of 61±40 weeks.
During this time, they received immunosuppressive treatment lead-
ing to a decline in serum anti- BP180 NC16A autoantibodies and a
complete resolution of skin symptoms. Thus, the follow- up serum
marker evaluation was performed on patients free from clinical dis-
ease who still received maintenance immunosuppression (complete
remission on therapy). Control sera were collected from 56 age- and
gender- matched healthy donors (mean age 73±13 years, 34 females,
22 males). The diagnosis of BP in our study patients was based on
typical clinical findings as well as detection of linear deposits of IgG
and/or C3 at the dermal–epidermal junction and serum anti- BP180
NC16A autoantibodies by direct immunofluorescence microscopy
and enzyme- linked immunosorbent assay (ELISA), respectively.
The investigations were conducted under approval from the Ethics
Committee of the University of Lübeck and with written informed
consent.
2.2 | ELISA
ELISA was used to measure serum levels of anti- BP180 NC16A
IgG autoantibodies (Euroimmun, Lübeck, Germany), sCD4
(Novateinbio, Woburn, USA), sIL- 2R (ThermoScientific, Waltham,
USA), IgD (Bethyl Laboratories Inc., Montgomery, USA), MCT
(neoScientific, Boston, USA), MPO (BioLegend, San Diego, USA),
S100A12 (Biorbyt, Cambridge, UK), sL- selectin (eBioscience, San
Diego, USA), ECP (Novateinbio, Woburn, USA), neopterin (IBL
International, Hamburg, Germany) and sP- selectin (eBioscience,
San Diego, USA) according to the respective manufacturer’s
instructions.
2.3 | Statistical analysis
Data were analysed by t test, Mann- Whitney U test, Wilcoxon signed-
rank test or Spearman’s rank correlation test using GraphPad prism
5 (San Diego, CA, USA). A P- value <.05 was considered statistically
significant.
3 | RESULTS
3.1 | Serum soluble markers of immune
activation in patients with active BP vs controls
Serum levels of sCD4, sIL- 2R, IgD, MCT, MPO, S100A12, sL- selectin,
ECP, neopterin and sP- selectin were measured by ELISA in patients
with active BP and age- and gender- matched healthy individuals. In
comparison with controls, sera from patients with BP contained signif-
icantly higher levels of sCD4, MPO, S100A12, ECP and sP- selectin. In
addition, some trend towards higher serum concentrations was found
for MCT (P=.1) and neopterin (P=.06), whereas the remaining markers
were similar between both groups (Figure 1).
FIGURE1 Serum levels of sCD4, sIL- 2R, IgD, mast cell tryptase
(MCT), myeloperoxidase (MPO), S100A12, sL- selectin, eosinophil
cationic protein (ECP), neopterin and sP- selectin measured by
enzyme- linked immunosorbent assay in 25 patients with active
bullous pemphigoid (BP) and 56 normal controls. The dots/squares
and horizontal bars indicate individual and mean values in each
group, respectively. *P<.05, **P<.01, ***P<.001
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3.2 | Immune activation markers during
follow- up of BP patients
We next analysed the serum concentrations of immune activation
markers during the course of the disease in 10 patients with BP. We
observed a significant reduction of MCT and neopterin as well as
a slight tendency towards a decline in sIL- 2R (P=.1) in parallel with
complete healing of skin lesions after a mean follow- up period of
61 weeks, during which the patients received immunosuppressive
therapy (Figure 2). In addition, a concomitant decrease in levels of
circulating anti- BP180 autoantibodies was observed in these patients
with BP (data not shown), and the concentrations of some of the ac-
tivation markers were significantly associated (sIL2- R, MCT and sP-
selectin) or showed some trend towards a relationship (sCD4, MPO,
ECP and neopterin) with the levels of these autoantibodies (Table 1).
4 | DISCUSSION
Most data regarding the pathogenic importance of cellular autoim-
munity against BP180 were generated in animal models in which either
injection of rabbit antimurine BP180 antibodies or transfer of human
BP autoantibodies into BP180- knockout mice rescued by the human
ortholog duplicate major characteristics of the human disease.[19]
Accordingly, it is believed that following interaction of autoreactive T
and B cells with consecutive production and binding of autoantibodies
to the dermal–epidermal junction, a sequence of events involving dif-
ferent effector cells is initiated in BP. These pathophysiological steps
include release of interleukin 6 (IL- 6) and IL- 8 from basal keratinocytes,
(ii) complement activation, (iii) mast cell degranulation, (iv) infiltration
of inflammatory cells (neutrophils, eosinophils, macrophages and T
cells) into the upper dermis along with secretion of inflammatory me-
diators and (v) release of proteases and reactive oxygen species from
granulocytes that ultimately induce dermal–epidermal splitting.[1,3,4]
In addition, a Th1/Th2/Th17 mixed cytokine and chemokine profile
including interleukin 1β, interleukin 5, interleukin 6, interleukin 8, in-
terleukin 10, interleukin 15, interleukin 17, tumor necrosis factor α,
chemokine ligand 2 (CCL2), CCL5, CCL11, CCL13, CCL18 and CXCL10
can be found in serum samples and blister fluids of patients with BP,
partially paralleling patients’ disease activity.[1,5,6] The data reported
here confirm and extend previous studies suggesting a broad and si-
multaneous immune cell activation in patients with BP.
In this study, we used various serum soluble markers known to
reflect the activation state of different cell types: sCD4 and sIL- 2R, cell
surface molecules released by T cells upon their stimulation[20]; IgD, an
immunoglobulin associated with B- cell development/maturation and
class- switched autoreactive B cells[21]; MCT, MPO, and ECP, granule
proteins liberated from activated mast cells, neutrophils and eosino-
phils, respectively[22-24]; S100A12, a calcium- binding protein secreted
by activated neutrophils[25]; neopterin, a pyrazino- pyrimidine com-
pound synthesized from activated macrophages[26]; and sL- selectin
and sP- selectin, adhesion molecules indicative of neutrophil and plate-
let activation, respectively.[27]
We could show that the serum levels of sCD4, MPO, S100A12,
ECP and sP- selectin were significantly elevated in patients with ac-
tive BP compared with normal controls. Some trend towards higher
serum concentrations was also found for MCT and neopterin during
ongoing disease, both of which significantly decreased along with
a slight decline tendency for sIL- 2R at the time of clinical remission
of the patients. Additionally, some activation markers were signifi-
cantly associated (sIL2- R, MCT and sP- selectin) or showed some
trend towards a relationship (sCD4, MPO, ECP and neopterin) with
anti- BP180 autoantibody titres. These results are in agreement with
previous studies which reported increased concentrations of sCD4,
sIL- 2R, MCT, MPO, and ECP in BP blister fluids and/or serum, partly
FIGURE2 Individual enzyme- linked immunosorbent assay-
based serum levels of sCD4, sIL- 2R, IgD, mast cell tryptase
(MCT), myeloperoxidase (MPO), S100A12, sL- selectin, eosinophil
cationic protein (ECP), neopterin and sP- selectin at time of
cutaneous symptoms and during remission of skin lesions in 10
bullous pemphigoid patients after a mean period of 61 weeks of
immunosuppressive therapy. Horizontal black bars indicate the mean.
*P<.05, **P<.01
TABLE1 Correlation coefficients between serum levels of cell activation markers and anti- BP180 autoantibodies in BP patients
sCD4 sIL- 2R IgD MCT MPO S100A12 sL- selectin ECP Neopterin sP- selectin
Anti- BP180
r.416 .512 −.024 .722 .401 .066 −.109 .392 .404 .572
P.067 .021 .917 <.001 .079 .781 .647 .087 .077 .008
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correlating with cytokines, autoantibodies and clinical disease sever-
ity in patients with BP.[10-18] The reported mean blister fluid concen-
trations for sCD4 (42.4 U/mL), sIL- 2R (2070 U/mL), MCT (4.35 U/L),
MPO (3498- 16 863 μg/L) and ECP (282- 1559 μg/L) in these patients
were found to be significantly higher compared with those in their sera
and/or suction blisters of healthy controls.[10-12,14-16,18]
The significantly increased serum concentrations of these acti-
vation markers in our study underscore the previously described key
contribution of the respective T cells, neutrophils and eosinophils to
the disease pathophysiology. Results from the BP180- humanised
mouse model revealed the importance of NC16A- reactive CD4 T cells
and accorded with evidence from previous in vitro studies with human
cells that demonstrated a restriction of NC16A- reactive CD4 T cells to
the BP susceptibility gene HLA- DQB1*0301.[1] In addition, a variety
of BP models suggest that autoantibody- dependent neutrophil and
eosinophil degranulation with the liberation of proteinases or cyto-
toxic agents is essential, damaging the basal membrane zone directly
and causing dermo- epidermal junction separation.[1,6] Interestingly, in
addition to classical inflammatory cells, platelets seem to play a role in
the disease as sP- selectin was observed to be significantly increased in
patients’ sera and to correlate with circulating autoantibodies. In fact,
although the role of the blood coagulation system including platelets
has not yet been investigated in experimental models of BP, some ev-
idence exists on the relationship between inflammation and coagula-
tion. It has been reported that patients with BP show elevated levels of
tissue factor, D- dimer, prothrombin fragment F1+2 and mean platelet
volume and that they have an increased risk for thrombotic events.[7-9]
It is interesting to note that, with the exception of MCT and ne-
opterin, no other immune cell activation marker was found to be sig-
nificantly decreased at follow- up. This suggests some latent persistent
molecular inflammatory process during both complete remission on
therapy and normalization of circulating anti- BP180 autoantibodies
of the patients. However, one has to take into account the different
limitations of this study as mentioned below.
A fraction of the investigated cell activation markers was not dif-
ferent (sIL- 2R, IgD and sL- selectin) or did not quite reach a statistically
significant level (MCT and neopterin) when compared with healthy
controls. This finding does not exclude the possibility that these fac-
tors or their associated cell types are involved in the disease process,
considering that some of these markers significantly declined during
healing of skin lesions (MCT and neopterin) or paralleled autoantibody
serum levels (sIL2- R and MCT) in our patient cohort. There may be
several mutually non- exclusive influencing variables that could have
accounted for these potentially false- negative and partly incongruent
results: (i) the relatively small population size; (ii) the reported pres-
ence of higher concentrations of activation markers in blister fluids
than in sera of patients with BP[10,11,15,16,18]; (iii) the fact that differ-
ent biomarkers corresponding to the same cell type exist which may
yield different results[20,28-32]; (iv) differences in disease duration and
time intervals from active disease stage (ie initial sample) to reaching
remission (ie follow- up sample); (v) the impact of immunosuppressive
(pre- )treatment on the different cell types; (vi) diverse stability of the
investigated markers in the circulation as well as during procession and
storage of blood samples[33]; and (vii) ELISA- related factors (eg anti-
body quality, kit manufacturer, measurement conditions and technical
issues related to dynamic ranges and dilutions).[33]
It also needs to be mentioned that the serological profile described
in our study may not be specific for BP as the tested markers represent
indicators of inflammation in a variety of other disease conditions.[20-27]
For example, all investigated markers that were significantly increased
in our patient cohort have been also found to be elevated in common
inflammatory dermatoses such as psoriasis and/or atopic dermati-
tis.[34-38] Especially platelets, which in addition to playing a central role
in normal haemostasis, have emerged as important players in various
types of inflammatory and non- inflammatory disorders including cere-
brovascular and neurodegenerative diseases.[27,28,39,40] Regarding the
latter, it is worth noting that the main risk factors for BP include neuro-
logic diseases comprising stroke and dementia,[41] both of which were
documented in the medical history of four of our study patients. Thus,
unspecific serum immune marker alterations reflecting rather general
aspects of inflammation or comorbidities cannot be fully excluded in our
study.
In summary, our findings confirm and extend previous reports sug-
gesting some concomitant involvement of a panel of molecules rep-
resentative for a wide spectrum of cellular players (T cells, mast cells,
neutrophils, eosinophils, macrophages and platelets) orchestrating the
inflammatory reaction in BP. These data may favour the employment
of broad- spectrum or combined immunosuppressants, potentially to-
gether with an anticoagulant treatment, over cell- or molecule- specific
targeted therapy in patients with this disorder.
ACKNOWLEDGEMENTS
This work was supported by Deutsche Forschungsgemeinschaft
(DFG) Excellence Cluster “Inflammation at Interfaces” (EXC 306/2)
and the Clinical Research Unit “Pemphigoid Diseases” (KFO 303/1).
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTION
M.K. and R.J.L. conceived and designed the experiments; K.B. and
A.L.E. performed the experiments; K.B., A.L.E., S.T., E.S., M.M.H., D.Z.,
R.J.L. and M.K. analysed and interpreted the data. M.K. wrote the
manuscript. All authors reviewed the manuscript.
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How to cite this article: Bieber K, Ernst AL, Tukaj S, et al.
Analysis of serum markers of cellular immune activation in
patients with bullous pemphigoid. Exp Dermatol.
2017;26:1248–1252. https://doi.org/10.1111/exd.13382
