title: Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.
abstract: In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kellner
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Nestler
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Leimert
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Bucher
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany; Department of Anesthesiology, Critical Care and Pain Therapy, St. Georg Medical Center, Leipzig, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Czeslick
authors.affiliation: Department of Anesthesiology, Critical Care and Pain Therapy, St. Georg Medical Center, Leipzig, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Sablotzki
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Halle-Wittenberg University, Germany. Electronic address: christoph.raspe@medizin.uni-halle.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Raspè
mesh_terms: D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D000990:Antithrombin III / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D050718:Complement C1 Inhibitor Protein / Q000494:pharmacology*; D005260:Female; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000097:blood* / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D009000:Monocytes / Q000187:drug effects / Q000473:pathology*; D055815:Young Adult
chemical_list: D000925:Anticoagulants; D050718:Complement C1 Inhibitor Protein; D006629:Hirudins; D008070:Lipopolysaccharides; D000990:Antithrombin III
keywords: Antithrombin; C1 esterase inhibitor; Immunomodulation; Monocytes; Sepsis