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Intramyocardial synthesis of pro- and anti-inflammatory cytokines in infants with congenital cardiac defects

Ma Qing, MD^*,*, Kathrin Schumacher, MD^*, Ruth Heise, Michael Wöltje, PhD, Jaime F. Vazquez-Jimenez, MD, Thomas Richter, MD, Monika Arranda-Carrero, MD^||, John Hess, MD^*, G.ötz von Bernuth, MD^|| and Marie-Christine Seghaye, MD^*

^* Department of Pediatric Cardiology and Congenital Heart Diseases, German Heart Center Munich, Technical University Munich, Munich, Germany
Interdisciplinary Center for Clinical Research BIOMAT, Aachen University of Technology, Aachen, Germany
Department of Thoracic and Cardiovascular Surgery, Aachen University of Technology, Aachen, Germany
Institute of Pathology, Technical University Munich, Munich, Germany
^|| Department of Pediatric Cardiology, Aachen University of Technology, Aachen, Germany

View larger version (13K): [in a new window]   Figure 1 Levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, IL-6, and IL-10 measured by enzyme-linked immunosorbent assay in the right ventricular myocardium of infants with tetralogy of Fallot (solid bar; n = 6) or ventricular septal defect (open bar; n = 6). Results are expressed as the mean value ± SEM. *p < 0.02; p < 0.05; and p < 0.01 between groups.

View larger version (13K): [in a new window]   Figure 2 (A) Levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 measured by Western blotting in the right ventricular myocardium of infants with tetralogy of Fallot (TOF) (solid bar; n = 7) or ventricular septal defect (VSD) (open bar; n = 8). Band intensities for TNF-alpha and IL-10 are normalized for bands of beta-actin. Results are expressed as the mean value ± SEM. *p < 0.01 between groups. (B) Exemplary gels obtained after Western blotting representative of seven experiments in infants with TOF and eight experiments in infants with VSD, showing a higher expression of TNF-alpha but not IL-10 in the former group. The upper panel includes positive controls (PC) (human monocytes stimulated with lipopolysaccharide) and negative controls (NC) (nonstimulated human monocytes). M = marker.

View larger version (12K): [in a new window]   Figure 3 Relationship between the right ventricular pressure (RVP)/left ventricular pressure (LVP) ratio and protein levels of tumor necrosis factor (TNF)-alpha measured by Western blotting in all patients (n = 15). Spearman coefficient: 0.73 (p = 0.002).

View larger version (88K): [in a new window]   Figure 4 Immunocytochemistry of the right ventricular myocardium in one infant with tetralogy of Fallot, showing the presence of tumor necrosis factor (TNF)-alpha in cardiomyocytes (My), interleukin (IL)-10 in cardiomyocytes, macrophages (M), and endothelial cells (EC), inducible nitric oxide synthase (iNOS) in cardiomyocytes, and nuclear factor (NF)-kappa-B p50 and p65 subunits in the cytoplasma and nuclei of cardiomyocytes. C = negative control (no primary antibody). Magnification: x400 for control, cytokines, and iNOS and x1,000 for NF-kappa-B p50 and p65 subunits. These stains are representative of those obtained in four patients (two in each group).

View larger version (24K): [in a new window]   Figure 5 (A) Deoxyribonucleic acid (DNA)-binding activity of nuclear factor (NF)-kappa-B measured by EMSA with supershift in the right ventricular myocardium of infants with tetralogy of Fallot (TOF) (solid bar) or ventricular septal defect (VSD) (open bar). Results are expressed as counts (CNT)/mm2. (B) Exemplary gel showing the presence NF-kappa-B (arrow) detected by electrophoretic mobility shift assay and supershift with anti-p65 and anti-p50 in nuclear extracts of the right ventricular myocardium of one patient with TOF and one patient with VSD. This gel is representative of those obtained in three patients from each group. PC = positive control (human HepG2 cells stimulated by TNF-alpha); NS = nonspecific.

View larger version (9K): [in a new window]   Figure 6 (A) Levels of phospho-I-kappa-B-alpha measured by Western blotting in the right ventricular myocardium of infants with tetralogy of Fallot (TOF) (solid bar; n = 7) or ventricular septal defect (VSD) (open bar; n = 8). Band intensities for phospho-I-kappa-B-alpha are normalized for bands of beta-actin. Results are expressed as the mean value ± SEM. (B) Exemplary gel obtained after Western blotting representative of independent experiments in seven patients with TOF and eight with VSD, showing phosphorylation of I-kappa-B-alpha in all patients. The upper panel includes positive controls (PC) (human monocytes stimulated with endotoxin). M = marker.

View larger version (8K): [in a new window]   Figure 7 (A) Levels of phospho-p38 mitogen-activated protein kinase (MAPK) measured by Western blotting in the right ventricular myocardium of infants with tetralogy of Fallot (TOF) (solid bar; n = 5) or ventricular septal defect (VSD) (open bar; n = 5). Results of phospho-p38 MAPK are normalized for the bands of beta-actin and are expressed as the mean value ± SEM. *p < 0.05 between groups. (B) Exemplary gel obtained after Western blotting representative of five experiments in each group, showing higher phosphorylation of p38 MAPK in the right ventricular myocardium of patients with TOF than of those with VSD. The upper panel includes the positive control (PC; human monocytes stimulated with lipopolysacchardide). M = marker.