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

Authors


Correspondence

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

E-mail: ts23@cornell.edu

Abstract

Background

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.

Objectives

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).

Methods

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.

Results

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.

Conclusions

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.

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