Tumor necrosis factor-α production in whole blood after cardiopulmonary bypass: Downregulation caused by circulating cytokine-inhibitory activities J.

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Tumor necrosis factor-α production in whole blood after cardiopulmonary bypass: Downregulation caused by circulating cytokine-inhibitory activities J. Börgermann, MD, I. Friedrich, MD, S. Flohé, MD, J. Spillner, MD, M. Majetschak, MD, O. Kuss, A. Sablotzki, MD, T. Feldt, MD, J.C. Reidemeister, MD, F.U. Schade, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 124, Issue 3, Pages 608-617 (September 2002) DOI: 10.1067/mtc.2002.122300 Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 1 A, Ex vivo TNF-α synthesis of endotoxin-stimulated (S friedenau, 100 ng/mL) whole blood normalized to 1 × 106 WBCs. Double asterisk indicates P <.01 versus preoperative values (T1); triple asterisk indicates P <.001; P values are Bonferroni adjusted and calculated as pairwise comparisons within a repeated measurement nonparametric analysis of variance model. Data points represent mean for 15 patients from triplicate cultures for each time point for each patient; error bars represent SD. B, Comparison between WEHI bioassay and enzyme-linked immunosorbent assay (n = 150). High correlation between bioactive TNF and total TNF-α (line) indicates little interference by plasma proteins such as soluble TNF receptors (Spearman correlation coefficient r = 0.492, P <.001). The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 2 Analysis of TNF-α and β-actin mRNA in whole blood after 4 hours of stimulation by endotoxin (S. friedenau, 100 ng/mL). Left, Agarose gel stained with ethidium bromide of 1 typical patient among 6. Right, Results of semiquantitative reverse transcriptase-PCR. In parallel with protein secretion, expression of TNF-α mRNA was significantly reduced after CPB to 21.15% ± 20.51% (range 7.56%-29.55%) of preoperative values and had recovered to approximately 50% on postoperative day 6. Bar heights represent mean for 6 patients; error bars represent SD. Asterisk indicates P <.05 versus preoperative values (T1); double asterisk indicates P <.01; triple asterisk indicates P <.001; P values are Bonferroni adjusted and calculated as pairwise comparisons within a repeated measurement nonparametric analysis of variance model. The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 3 Inhibition of whole blood TNF-α synthesis by patient serum prepared after CPB. Mixtures contained 100 μL (33%) additional serum and were stimulated with endotoxin (S. friedenau, 10 ng/mL). Serum withdrawn at end of CPB caused statistically significant inhibition of TNF-α synthesis in heterologous whole blood from healthy volunteers relative to preoperative serum (preop.) or volunteer serum (control). Bar heights represent mean for 10 independent experiments; error bars represent SD. P values were calculated by signed rank test if data from same patient were compared and by Wilcoxon test for comparison with control. All P values were Bonferroni adjusted. The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 4 Dose-dependent inhibition of whole blood TNF-α synthesis by patient serum prepared after CPB. Heterologous whole blood from healthy volunteers was incubated with endotoxin (S. friedenau, 10 ng/mL) for 4 hours in presence of 1 to 100 μL (0.33%-33%) patient serum taken before operation or at end of CPB and volunteer serum. Control indicates TNF-α synthesis on endotoxin stimulation in presence of 100 μL volunteer serum. For serum taken after CPB, linear regression analysis revealed significant decline of 12.55% (95% confidence interval 11.30-13.82, P <.0001) for every unit of concentration of patient serum on log scale. Serum withdrawn before operation (b = 0.97, 95% confidence interval −2.55 to 4.50, P =.523) or control serum (b = 5.25, 95% confidence interval 1.98-8.52, P =.0077) had no or negligible influence on TNF-α synthesis. Data points represent mean; error bars represent SD. The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 5 No inhibition of TNF-α synthesis by heparin. Whole blood was incubated with endotoxin (10 ng/mL, S. friedenau; LPS) for 4 hours in absence or presence of heparin (0.16-100 U/mL). Doses that exceeded those used for CPB by 10 times did not affect TNF-α production in vitro. Bar heights represent mean for 4 independent experiments; error bars represent SD. The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions

Fig. 6 Immune absorption of interleukin-10 does not remove inhibitory activity from patient serum. A, Inhibitory activity in patient serum after CPB before immune absorption is shown on left. When IL-10 was completely removed by immune absorption (right), serum after CPB still contained potent inhibitory activity (P =.075 vs preoperative serum). B, Result of immune absorption with anti-IL-10 antibodies, HLA-DR antibodies (isotype control), or phosphate-buffered saline solution (PBS, control); n.d., not detectable. Bar heights represent mean for 3 independent experiments; error bars represent SD. The Journal of Thoracic and Cardiovascular Surgery 2002 124, 608-617DOI: (10.1067/mtc.2002.122300) Copyright © 2002 American Association for Thoracic Surgery Terms and Conditions