Respiratory Treatment and Prevention (Advances in Experimental Medicine and Biology)

2.1 Patients

The study group consisted of 22 patients
(18 men; mean age 46.59 years) suffering
from sarcoidosis diagnosed according to the clin-
ical and pathological guidelines of Costabel and
Hunninghake ( 1999 ). The disease was at the sec-
ond stage, with bilateral hilar lymphadenopathy
and pulmonary infiltrations, and was histologi-
cally confirmed. The control group consisted of
15 healthy volunteers (13 men, mean age
49.7 years) without any inflammatory conditions.
The following functional tests were performed:
spirometry, plethysmography, and diffusing lung
capacity for carbon monoxide (DLCO)
according to the guidelines of the American Tho-
racic Society ( 1995 ). Basic diagnostic biochemi-
cal work-up, gasometry, and ECG were
conducted in each patient. In addition, bronchofi-
beroscopy (Pentax FB 18 V, Pentax Corporation,
Tokyo, Japan) with BALF collection were
performed in all patients and control subjects.
We used premedication with intramuscular atro-
pine and midazolam, and subsequent local anaes-
thesia with lidocaine before bronchoscopy. The
bronchofiberoscopy was inserted and wedged in
the middle right lobe, and three 50 ml aliquots of
sterile saline solution, warmed to 37C, were
instilled and aspirated from the subsegmental
bronchus. The recovered fluid was filtered
through sterile gauze and centrifuged at
1500 rpm for 15 min at 4C. Supernatant was
stored at 70 C until use. Samples of BALF
were assayed for total and differential cell
counts, including CD4+, and CD8+
lymphocytes, using fluorescence-based flow
cytometry (Becton Dickinson; Mountain View,
CA). Cell differentials were made in smears
stained with the Grünwald-Giemza method by
counting at least 400 cells under a light micro-
scope (magnification 1 k). The number of CD
and CD8 cells was counted a percentage of posi-
tively stained cells. The cell suspension of BALF
was incubated with fluorescein isothiocyanate-
labeled anti-CD8 antibody and phycoerythrin-
labeled anti-CD4 antibody (Becton Dickinson)
for 20 min, then washed with PBS twice.

2.2 Concentrations of OPG, sRANKL,

and IL-18 in BALF

We measured the level of OPG (Biomedica

Medizinprodukte; Vienna, Austria), sRANK

(BioVendor – Laboratornı ́medicı ́na a.s.; Brno,

Czech Republic), and IL-18 (MBL International

Corp.; Woburn, MA) in BALF supernatant using

commercially available enzyme-linked immuno-

sorbent assay (ELISA) kits. The minimum

detectable levels of OPG, sRANKL and IL-

were 0.14 pmol/l, 0.1 pmol/l, and 1.5 pg/ml,

respectively.

2.3 Statistical Analysis

Data distribution was analyzed with the Shapiro-

Wilk test. Normally distributed data were

presented as meansSE. Data with skewed

distribution were presented as medians and

minimum-maximum ranges. Differences

between the mean values were assessed using a

t-test for normally distributed data and the Mann-

Whitney U or Wilcoxon tests for skewed distri-

bution. Correlations between variables were

investigated with Spearman’s rank test.

Receiver-operating characteristic (ROC) curves

were used to find out the cut-off levels for

sRANKL. A p-value<0.05 defined statistically

significant differences. Statistical elaboration

was performed using Statistica 12.0 software

(StatSoft Inc., Tulsa, CA).

3 Results

There were no significant differences in age or

gender between the patients and healthy subjects.

With respect to pulmonary function, BBS

patients had a significantly reduced vital capacity

(VC) – 86.215.2 vs. 97.23.1 %pred.,

p¼0.02 and lung diffusion for carbon monox-

ide (DLCO) – 81.2 25 vs. 91.411.2 %

pred., p¼0.01, compared with healthy subjects.

BALF analysis revealed that the BBS patients

had a higher percentage of lymphocytes and a

Osteoprotegerin/sRANKL Signaling System in Pulmonary Sarcoidosis... 3