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SHORT COMMUNICATION
Increase in FoxP3-positive Cells and Their Contacts with Mast Cells in Köbner-negative Patients
with Psoriasis after Tape-stripping
Mireille-Maria SUTTLE, Riikka-Liisa RITANEN and Ilkka T. HARVIMA
Department of Dermatology, University of Eastern Finland and Kuopio University Hospital, POB 1777, FIN-70211 Kuopio, Finland. E-mail
[email protected]
Accepted Aug 29, 2018; Epub ahead of print Sep 3, 2018
In psoriasis, regulatory T cells (Tregs) can modulate the
function of effector Th cells and mast cells (MCs) (1,
2). Defined by their expression of CD4, CD25, and the
transcription factor forkhead box P3 (FoxP3), Tregs are
central in protecting an individual from autoimmune
diseases. FoxP3 is a nuclear protein that is considered
to be a master regulator in the development and function
of Tregs (3, 4). There have been few studies on Tregs in
psoriasis; however, it is known that the function of Tregs
is impaired in psoriasis, in the lesional skin and in the
peripheral blood, with respect to their ability to suppress
effector T cells. Treatment of psoriasis may increase the
numbers and activity of Tregs (5–7).
MC activation is one of the earliest morphological
changes in the developing psoriatic lesion (2), but it is
unknown to what extent pro-inflammatory MC cytokines
are involved in the development of psoriatic lesions. The
interaction between Tregs and MCs has been shown to
be essential for immunosuppression in a variety of ex-
perimental models, such as hepatocarcinoma, colorectal
carcinoma, and skin allografts (8–10).
The purpose of this study was therefore to investigate
Tregs and their interaction with MCs in the early deve-
loping lesion in psoriasis. To this end, the tape-stripping
technique was used to induce the Köbner reaction, and
a series of skin biopsies was obtained at 0 days, 2 h, 1
day, 3 days and 7 days (11, 12). Patients who did not
develop the Köbner reaction were the focus of this study,
because we hypothesized that suppressive factors pre-
vent the development of lesions compared with patients
with positive Köbner reactions. These biopsies were
analysed for expression of CD25/IL-2Rα and FoxP3 and
for apparent morphological contacts (AMCs) between
tryptase-positive MCs and FoxP3 + cells by immunohisto-
chemistry and double-staining. FoxP3 has been described
as the marker of Tregs, but it may also be expressed by
other cell types. This aspect must be remembered when
interpreting the results.
MATERIALS AND METHODS
Mouse monoclonal anti-human FoxP3 (clone 236A/E7) was
purchased from Abcam (Cambridge, UK). Mouse monoclonal
anti-human CD25/IL-2Rα (clone 24204) was obtained from R&D
Systems Europe Ltd (Oxon, UK). The substrate of tryptase, Z-Gly-
Pro-Arg-4-methoxy-2-naphthylamide (Z-Gly-Pro-Arg-MNA),
was obtained from Bachem (Bubendorf, Switzerland), and the
chromogen, Fast Black K, was acquired from Sigma (St Louis,
MO, USA). Reagents for immunohistochemistry were purchased
from Vector Laboratories (Burlingame, CA, USA).
As described in our previous studies (11, 12), the Köbner reac-
tion was studied by the tape-stripping technique on non-lesional
arm skin in 18 patients with psoriasis (4 females and 14 males,
age range 24–77 years). Four-mm punch biopsies were taken at 0
days, 2 h, and 3 days or at 0 days, 1 day, and 7 days. The Köbner
phenomenon was evaluated at the follow-up visit 2–2.5 weeks
later. Eight of 18 subjects showed an initial developing lesion
(Köbner-positive group). The patients had not received any ef-
fective systemic or local treatment in the preceding month. The
skin biopsies were embedded immediately in optimal cutting
temperature (OCT) compound (Miles Scientific, Naperville, IL,
USA) and frozen to prepare 5-μm thick cryosections (11, 12).
The methods were approved by the ethics committee of Kuopio
University Hospital, Kuopio, Finland.
For staining CD25 and FoxP3, the skin cryosections were fixed
in cold acetone for 10 min and blocked with 1.5% normal horse
serum in phosphate-buffered saline (PBS). The bound anti-FoxP3
(10 µg/ml) and anti-CD25 mAbs (5 µg/ml) were visualized with
the avidin-biotin-peroxidase (ABC) technique using the Vectastain
Elite ABC kit, 0.05% 3,3’-diaminobenzidine tetrahydrochloride,
0.04% nickel chloride, and 0.03% hydrogen peroxide. The num-
bers of CD25 + and FoxP3 + cells were counted in a blinded fashion
on separate cryosections on a 0.2×0.2 mm ocular grid (Ella Gra-
ticules, Tonbridge, Kent, UK) in an area 0.4 mm (depth)×1.0 mm
(width) immediately beneath the papillary dermis. The results are
expressed as cells/mm 2 .
The technique of double-staining tryptase + MCs and FoxP3 +
cells has been developed to analyse the morphological relation
ships between tryptase + MCs and sensory nerves (13). The 5-µm
thick skin cryosections were fixed in cold acetone for 10 min and
blocked with diluted normal horse serum in PBS. The sections
were first treated with anti-FoxP3 and then with biotin-conjugated
secondary Ab. Next, tryptase + MCs were identified histochemically
using 1 mM Z-Gly-Pro-Arg-MNA and Fast Black K, resulting in
dark-blue to violet MCs. Finally, immunolabelled FoxP3 + cells
were visualized with the ABC technique, generating a black-
stained product. AMCs were identified in an area of 0.4 mm (depth)
× 1.0 mm (width) immediately beneath the papillary dermis, and
the results are expressed as the percentage of tryptase + MCs in
AMCs with at least one FoxP3 + cell. Only clearly stained tryptase +
MCs, FoxP3 + cells, and AMCs were counted. The cryosections
were blinded before the cells were counted.
Statistical analysis
The results were analysed by paired or unpaired 2-tailed t-test,
and p < 0.05 was considered statistically significant.
RESULTS
In the Köbner-negative group, the number of FoxP3 +
cells increased significantly in the 3–7-day and 7-day
biopsies (Table SI 1 , Fig. S1b 1 ) compared with day 0. No
This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta
Journal Compilation © 2019 Acta Dermato-Venereologica.
doi: 10.2340/00015555-3023
Acta Derm Venereol 2019; 99: 103–104