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Effects of calcitriol on apoptosis, toll-like receptor 4 expression, and cytokine production of endotoxin-primed canine leukocytes

Jared A. Jaffey DVM, MS1, Juliana Amorim DVM2, and Amy E. DeClue DVM, MS3
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  • 1 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 2 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 3 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

Abstract

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs.

SAMPLE Blood samples from 6 healthy adult dogs.

PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10−7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry.

RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes.

CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

Contributor Notes

Address correspondence to Dr. DeClue (decluea@missouri.edu).