Effect of recombinant canine interleukin-6 and interleukin-8 on tissue factor procoagulant activity in canine peripheral blood mononuclear cells and purified canine monocytes
Tracy Stokol, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, S1-058 Schurman Hall, Upper Tower Road, Ithaca, NY 14853, USA
Inflammation 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 and IL-8, induce tissue factor (TF) expression on canine monocyte surfaces, which may contribute to DIC initiation.
The objectives of this study were to determine if (1) IL-6 and IL-8 would induce TF activity on canine monocytes, (2) fetal bovine serum or autologous plasma was required for IL-6– or IL-8–induced TF responses in canine monocytes, and (3) these pro-inflammatory cytokines would enhance TF activity on canine monocytes in response to low concentrations of lipopolysaccharide (LPS).
Canine monocytes were isolated from EDTA-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), or HI-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 from PBMC with 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 factor X generation.
Neither rc-IL-6 nor rc-IL-8 consistently stimulated TF procoagulant activity in canine PBMC or purified monocytes after 4 hours. Serum, plasma, or low concentrations of LPS did not enhance the TF response to these cytokines.
IL-6 or IL-8 at evaluated concentrations may not play major roles in coagulation activation by induction of TF expression on monocytes in dogs with inflammation.