Jaffey Jared A, Amorim Juliana, DeClue Amy E
Am J Vet Res. 2018 Oct;79(10):1071-1078. doi: 10.2460/ajvr.79.10.1071.
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 × 10M) 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.
目的 确定骨化三醇对健康犬内毒素预致敏血样中免疫系统功能指标的体外作用。样本 来自6只健康成年犬的血样。程序 通过将血样与脂多糖（LPS；内毒素）或PBS溶液（未致敏对照组）孵育1小时来预致敏白细胞。预致敏后，将血样与骨化三醇（2×10M）或乙醇（对照物质）孵育24小时。样本孵育后，用犬特异性多重检测法测量LPS刺激的白细胞产生肿瘤坏死因子（TNF）和白细胞介素-10（IL10）的情况，并通过流式细胞术评估细胞凋亡和Toll样受体4（TLR4）表达。结果 未致敏白细胞经LPS刺激而非内毒素预致敏白细胞经LPS刺激会导致TNF和IL10产生显著增加，证实犬体外存在内毒素耐受。内毒素预致敏显著提高了中性粒细胞活力，对淋巴细胞活力以及中性粒细胞和单核细胞的TLR4表达无影响。骨化三醇处理显著降低了未致敏和内毒素预致敏白细胞经LPS刺激产生的TNF。相反，骨化三醇处理对未致敏白细胞产生IL10无影响，但显著增加了内毒素预致敏白细胞产生IL10的量。骨化三醇对中性粒细胞或淋巴细胞凋亡程度无显著影响，对未致敏或内毒素预致敏白细胞中的中性粒细胞和单核细胞TLR4表达也无影响。结论及临床意义 这些数据表明，骨化三醇在体外可诱导未致敏和内毒素预致敏犬白细胞发生抗炎转变，而不影响中性粒细胞和单核细胞的TLR4表达，也不改变犬血样中中性粒细胞和淋巴细胞的活力。
