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Role of toll-like receptor 4 and caspase-3, -8, and -9 in lipopolysaccharide-induced delay of apoptosis in equine neutrophils

Stacy L. Anderson DVM, MVSC, PhD1, Hugh G.G. Townsend DVM, MSC2, and Baljit Singh BVSc&AH, MVSc, PhD3
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  • 1 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.
  • | 2 Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.
  • | 3 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

Abstract

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro.

SAMPLE Venous blood samples from 40 adult horses.

PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities.

RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis.

CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Abstract

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro.

SAMPLE Venous blood samples from 40 adult horses.

PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities.

RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis.

CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Contributor Notes

Dr. Anderson's present address is College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752.

Dr. Singh's present address is Office of the Dean, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4Z6, Canada.

Address correspondence to Dr. Singh (baljit.singhl@ucalgary.ca).