Observational Study on Cytokine Production by Leukocytes of Papillon-Lefèvre Syndrome Patients and Healthy Probands in Whole Blood Cultures

MATERIAL AND METHODS Materials Lipopolysaccharide (LPS; Escherichia coli, serotype R515) was purchased from Alexis (Lausen, Switzerland) and adenosine triphosphate (ATP) from Sigma (Deisenhofen, Germany). Tumor necrosis factor α (TNF-α) was kindly provided by the Knoll AG (Ludwigshafen, Germany). IL-1β was from Invitrogen/Biosource (Karlsruhe, Germany).

Patients and healthy donors Five patients with established diagnose of PLS are under periodontal treatment at the Department of Periodontology, Center for Dental, Oral, and Maxillofacial Medicine (Carolinum) of the Johann Wolfgang Goethe-University Frankfurt am Main. Antiinfective therapy with adjunctive antibiotics has been rendered to all of them and they are under regular and frequent supportive therapy. The Department of Periodontology has contact to additional 5 PLS patients that are edentulous or under periodontal therapy elsewhere. All patients underwent complete oral examinations as well as inspection of the skin of the palms and soles. Each adult patient or parents received clinical and genetic counselling, and signed a consent form, approved by the ethic committees of the Universities of Dresden and Frankfurt/Main. Clinical data and mutations of all patients have been reported before. All these patients were invited to take part in this study. Healthy donors had abstained from taking drugs for two weeks prior to the study. Due to wide spread use of oral contraceptives only male probands were chosen. The study complied with the rules of the Declaration of Helsinki and was approved by the Institutional Review Board for Human Studies of the Medical Faculty of the Johann Wolfgang Goethe-University Frankfurt/Main (Application# 31/05). All participating individuals were informed on risks and benefit as well as the procedures of the study and gave written informed consent.

Whole blood culture Heparinized blood was mixed with an equal volume of culture medium (RPMI 1640 supplemented with 25 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), 100 U/ml penicillin, 100 µg/ml streptomycin) and 1 ml aliquots were transferred into loosely sealed round-bottom polypropylene tubes (Greiner, Germany). Whole blood cultures were kept at 37 oC and 5 % CO2 for the indicated time periods. Thereafter, cell-free plasma/RPMI samples were obtained by centrifugation and stored at -70oC until assessment of cytokine concentrations by enzyme linked immunosorbent essay (ELISA). Experiments were started within 60 min of blood withdrawal. Thus, the whole blood cultures consisted of the whole range of white blood cells as well as erythrocytes. Except for determination of IL-1β release, cultures were either kept as unstimulated control or stimulated with LPS (10 or 100 ng/ml), or with the combination of IL-1β plus TNF-α (50 ng/ml each) for 24h. For determination of IL-1β release, cells were kept as unstimulated control or were stimulated with Toll-like receptor 4 ligand LPS (100 ng/ml) for altogether 5h. For efficient release of IL-1β from activated cultures, LPS was combined with ATP (2 mM) which was added during the last 2h of the 5h stimulation period in order to achieve activation of the purinoreceptor P2X7.

Analysis of cytokine release by ELISA analysis Concentrations of IL-8, IL-6, interferon-inducible protein (IP)-10, interferon (IFN) gamma (Pharmingen/BD Biosciences), and IL-1β, (R&D Systems), in plasma/RPMI samples were determined by ELISA according to the manufacturers' instructions.

Statistics The individual patient or proband was defined as statistical unit. Data are shown as median with interquartile range and are presented as pg/ml (IL-1β, IL-6, IP-10) or as ng/ml (IL-8). Medians were compared between PLS patients and healthy volunteers using the non parametric Mann Whitney U test. Statistical analysis was performed using a computer program (Systat for Windows version 10.0, Systat Inc., Evanston, IL, USA).