Effect of recombinant canine interleukin‐6 and interleukin‐8 on tissue factor procoagulant activity in canine peripheral blood mononuclear cells and purified canine monocytes

Abstract

BackgroundInflammation is a major cause of disseminated intravascular coagulation (DIC) in dogs, but underlying mechanisms for its initiation are unknown. We hypothesized that pro‐inflammatory cytokines, interleukin (IL)‐6 andIL‐8, induce tissue factor (TF) expression on canine monocyte surfaces, which may contribute toDICinitiation.ObjectivesThe objectives of this study were to determine if (1)IL‐6 andIL‐8 would induceTFactivity on canine monocytes, (2) fetal bovine serum or autologous plasma was required forIL‐6– orIL‐8–inducedTFresponses in canine monocytes, and (3) these pro‐inflammatory cytokines would enhanceTFactivity on canine monocytes in response to low concentrations of lipopolysaccharide (LPS).MethodsCanine monocytes were isolated fromEDTA‐anticoagulated blood as peripheral blood mononuclear cells (PBMC) by double‐density gradient centrifugation and adhesion to plastic. Adherent cells were stimulated for 4 hours with recombinant canine (rc)‐IL‐6 or rc‐IL‐8 (10–5000 pg/mL) with or without 10% heat‐inactivated (HI) fetal bovine serum, untreated autologous canine plasma (ACP), orHI‐ACP. Lipopolysaccharide (100 ng/mL) served as a positive control. Cells were also costimulated with either cytokine (100 pg/mL) or low concentrations of LPS (0.1 and 1 ng/mL). Monocytes immunopurified fromPBMCwith anti‐CD14 antibodies were also stimulated with both cytokines (100 and 5000 pg/mL). TF activity on cell surfaces was measured by a 2‐stage amidolytic assay, based on activated factorXgeneration.ResultsNeither rc‐IL‐6 nor rc‐IL‐8 consistently stimulatedTFprocoagulant activity in caninePBMCor purified monocytes after 4 hours. Serum, plasma, or low concentrations ofLPSdid not enhance theTFresponse to these cytokines.ConclusionsIL‐6 orIL‐8 at evaluated concentrations may not play major roles in coagulation activation by induction ofTFexpression on monocytes in dogs with inflammation.