Introduction

The present in vitro study was undertaken to learn about the effects of leukocytes on tenocytes in respect to complement regulation simulating an inflammatory scenario of the traumatized tissue.

Methods

Human hamstring tendon-derived tenocyte monolayers were co-cultured indirectly with human leukocytes (either Peripheral Blood Mononuclear Cells [PBMCs] or neutrophils) using a transwell system with/without (+ /_wo) 10 ng/ml tumor necrosis factor α (TNFα) for 4 and 24 h. Tenocyte and leukocyte cell survival was assessed by live–dead assay. Tenocyte gene expression of TNFα, the anaphylatoxin receptor C5aR and the cytoprotective complement regulatory proteins (CRP) CD46, CD55 and CD59 was monitored using qPCR. TNFα was detected in the culture supernatants using ELISA.

Results

C5aR gene expression was significantly induced by TNFα after 4 h, but impaired in the presence of leukocytes + TNFα after 24 h. At 4 h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24 h by neutrophils + /_woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4 h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /_woTNFα, probably contributing to complement regulation.

Conclusion

TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.

中文翻译：

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间接共培养模型中白细胞影响下肌腱细胞的补体调节

介绍

本体外研究旨在了解白细胞对肌腱细胞在补体调节方面的影响，模拟创伤组织的炎症情景。

方法

使用带有/不带有 (+ / _wo ) 10 ng/ml 肿瘤坏死因子 α (TNFα)的 transwell 系统，将人腘绳肌腱衍生的肌腱细胞单层与人白细胞（外周血单核细胞 [PBMC] 或中性粒细胞）间接共培养4 和 24 小时。通过活-死试验评估肌腱细胞和白细胞的存活率。使用 qPCR 监测 TNFα、过敏毒素受体 C5aR 和细胞保护性补体调节蛋白 (CRP) CD46、CD55 和 CD59 的肌腱细胞基因表达。使用ELISA在培养物上清液中检测TNFα。

结果

C5aR 基因表达在 4 小时后被 TNFα 显着诱导，但在 24 小时后在白细胞 + TNFα 存在下受损。在 4 小时时，由 TNFα 激活的 PBMC 诱导 CRP CD46 基因表达。然而，CD55 在 24 小时后被中性粒细胞 + / _wo TNFα显着抑制。4 小时后，被 TNFα 激活的白细胞也显着降低了更下游作用的 CRP CD59 的基因表达。TNFα 基因表达和 ELISA 分析显示，在与 PBMC + / _wo TNFα 的肌腱细胞共培养物中，TNFα 表达/释放增加，可能有助于补体调节。

结论

TNFα 可能代表一种关键的可溶性介质，对肌腱细胞补体调节发挥不同的时间依赖性影响。