Increased expression of the NLRP3 inflammasome components in patients with Behçet’s disease

Patients

Blood samples were taken from 15 active, 15 stable BD patients and 15 healthy volunteers
(HC) (Table 1). All patients consisted of BD patients who presented themselves for the first time
or were monitored at the Department of Dermatology, Ajou University Hospital. BD patients
met the Diagnostic criteria of the BD Research Committee of Japan. The active group
patients had at least one of the BD symptoms despite the treatment and inactive group
patients were in well-controlled states. Informed consent was obtained prior to the
study. This study was approved by the Institutional Review Board (IRB no.: AJIRB-GN3-07-098,
AJIRB-GGEN-GEN-10-119).

Table 1. Characteristics of Behçet’s disease (BD) patients and healthy controls

Immunohistochemistry

Six mm punch skin biopsies of erythema nodosum (EN)-like lesions of 25 BD and 25 EN
patients were performed. Formalin-fixed and paraffin-embedded tissues of EN-like lesions
in BD were cut (3-?m thickness) and mounted onto slides. Specimens were deparaffinated
and endogenous peroxidase activity was blocked by 3 % H
₂
O
₂
in methanol for 15 min at room temperature. After rinsing in phosphate-buffered saline
(PBS) for 10 min, the nonspecific binding sites were blocked by blocking solution
for 10 min at room temperature and all specimens were incubated with polyclonal antibodies
against NLRP3 (1:50 dilution, mouse, Alexis Biochemicals, San diego, CA, USA) and
ASC (1:100 dilution, rabbit, Lifespan bioscience, Seattle, WA) for 30 min at room
temperature. Next, HRP polymer (Thermo scientific, Fremont, CA, USA) was applied and
incubated for 30 min at room temperature. After washing in PBS for 10 min, bound antibodies
were visualized by incubation with AEC chromogen system (Thermo scientific, Fremont,
CA, USA). Slides were counterstained with hematoxylin. Negative controls were isotype
matched. The image was analyzed using Image Pro Plus Version 4.5 (Media Cybertics
Co., MD, U.S.A.) (Additional file 1).

Quantitative real time PCR

Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood samples
by Ficoll Hypaque density gradients (Ficoll paque
^TIM
plus, StemCell Technologies, Vancouver, BC, Canada). PBMCs were stimulated for 4 h
with 100 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich). After 4 h, RPMI containing
1 mM adenosine 5-triphosphate (ATP; Sigma-Aldrich) was added to the cells for another
15 min (LPS/ATP). In separate experiments, 20 ?M zYVAD(Ome)-FMK an irreversible caspase-1
inhibitor (CaspI; Enzo life science, PlymouthMeeting, PA) was added. The method for
Quantitative real time PCR is described in Additional file 2.

Western blotting

Briefly, freshly isolated PBMCs and stimulated PBMCs were harvested and lysed in RIPA
buffer (Sigma–Aldrich, St. Louis, MO, USA) containing protease inhibitors. Cell extracts
were run on Bolt™ 4–12 % Bis–Tris Plus Gel (life technologies, Carlsbad, CA, USA)
and transferred onto polyvinylidene difluoride membranes (Merck Milipore, Darmstadt,
Germany). Following transfer, the membrane was blocked overnight at room temperature
with PBS/0.2 % Tween-20/5 % skim milk. Blots were incubated with primary antibodies
anti-NLRP3, anti-apoptosis-associated speck-like protein containing a CARD (ASC),
and anti-caspase-1 antibodies (Abcam, Cambridge, MA). The membranes were incubated
in a solution containing an appropriate secondary Ab (either anti-rabbit IgG or anti-mouse
IgG Ab) linked to horseradish peroxidase (Invitrogen). Bands were visualized with
Immobilon Western Chemiluminescent HRP Substrate (Merck Milipore, Darmstadt, Germany)
(Additional file 2).

ELISA for IL-1?

Total IL-1? and mature IL-1? level in the supernatants was measured with a commercial
ELISA kit from RD Systems according to the manufacturer’s protocols.

Statistics

The data are presented as mean?±?standard deviation (S.D.). Data were analyzed by
one-way analysis of variance followed by the Scheffé test for overall multiple comparisons
among HC, stable BD, active BD. Student’s t-test was used for comparison between two groups. SPSS 17.0 (SPSS Inc., Chicago, IL)
was used. A p-value 0.05 was considered to indicate statistical significance.