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Cytokine production in thromboangiitis obliterans patients: New evidence for an immune-mediated inflammatory disorder

E.S. Slavov, S.A. Stanilova, D.P. Petkov1, Z.G. Dobreva

Department of Molecular Biology and Immunology, 1Department of Vascular Surgery, Medical Faculty, Thracian University, Stara Zagora, Bulgaria.

ABSTRACT
Objective
To study IL-6, IL-10, IL-12 levels and circulating immune complexes (CIC) containing IgG, IgM or IgA in sera of 14 TAO patients and 12 healthy blood donors. To evaluates the ability of TAO PBMC to produce IL-6, IL-10, IL-12, as well as to detect PBMC apoptosis after stimulation with different stimuli.

Methods
In vitro stimulation of PBMC with lypopolysaccharide (LPS), phytohemagglutinin (PHA), C3 binding glycoprotein from Cuscuta europea (C3bgp), pokeweed mitogen (PWM), and dexamethasone (DM) were performed. The quantities of the secreted cytokines in sera and in culture supernatants, as well as CIC were detected by ELISA. The apoptosis was assessed according to nuclear morphology, after acridine orange staining, by fluorescence microscopy.

Results
Significantly higher IL-6 levels in the patients’, than in the controls’ sera was found. An increased production of IL-6 and IL-12 in TAO PBMC supernatants was detected, regardless of the stimuli used. A hyporeactivity of TAO PBMC toward IL-10 production was found after C3bgp, LPS, PHA and PWM stimulation, compared to the controls’ PBMC. The spontaneous and induced apoptosis was significantly higher in TAO compared to the control group. Increased CIC quantities were detected in 75% of the patients tested. According to the CIC isotype, the IgG CIC positives (75%) prevailed over IgA CIC positives (50 %).

Conclusion
The altered production of IL-6, IL-12 and IL-10, the increased apoptosis as well as the elevated levels of CIC could be a reason for the persisting immune inflammation in TAO.

Key words
TAO, PBMC, IL-6, IL-12, IL-10, apoptosis, immune complex.

Please address correspondence to: Emil Slavov, MD, PhD, Department of Molecular Biology and Immunology, Medical Faculty, Thracian University, 11 Armeiska St., 6000 Stara Zagora, Bulgaria.
E-mail address: e_slav67@mf.uni-sz.bg

Abbreviations:
TAO: thromboangiitis obliterans;
LPS: lypopolysaccharide;
PHA: phytohemagglutinin;
C3bgp: C3 binding glycoprotein from Cuscuta europea;
PWM: pokeweed mitogen;
DM: dexamethasone;
IgG CIC: circulating immune complexes; containing IgG;
IgA CIC: circulating immune complexes; containing IgA;
IgM CIC: circulating immune complexes; containing IgM;
PBMC: peripheral blood mononuclear cells.