Complement regulation in tenocytes under the influence of leukocytes in an indirect co-culture model.

Abstract

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. 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) 10ng/ml tumor necrosis factor α (TNFα) for 4 and 24h. 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. C5aR gene expression was significantly induced by TNFα after 4h, but impaired in the presence of leukocytes + TNFα after 24h. At 4h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24h by neutrophils + /woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /woTNFα, probably contributing to complement regulation. TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.