Defective Efferocytosis of Ultraviolet B-induced Apoptotic Fibroblasts by M1 Macrophage in Pterygium

초록

Purpose : The phagocytic clearance of apoptotic cells, termed efferocytosis, is a critical immunological mechanism in the maintenance of tissue homeostasis. Given that chronic exposure to ultraviolet (UV) light is a well-known pathogenic factor of pterygium, we investigated whether efferocytosis mechanism is compromised in pterygium compared to normal conjunctiva using an in vitro efferocytosis model. Methods : Pterygium fibroblasts (PFs) and conjunctival fibroblasts (CJFs) were primarily cultured from the surgically obtained pterygium body stroma and normal conjunctival stroma, respectively. For efferocytosis modeling, apoptosis of CJFs and PFs was induced by UVB exposure (312 nm, 1 J/cm2), then they were co-incubated with M1 macrophages derived from THP-1 monocyte. Apoptosis was confirmed by Annexin V staining and apoptotic signal expression change of Bcl2 and Bax The proportion of efferocytosis of M1 was compared between apoptotic CJFs and PFs and efferocytosis receptors (Mertk [Mer Tyrosine Kinase, MerTK], Tyro3, Axl) and ligands (Mfge8 [Milk fat globulin protein E8], Gas6, Pros1 [Protein S], Itgb3 [Integrin subunit beta 3]) were analyzed to assess activation of efferocytosis. Results : Apoptosis was clearly induced in both CJFs and PFs by UVB exposure. Efferocytic receptor Mertk and ligand Itgb3 were activated in CJFs and PFs by UVB. Unexpectedly, receptor Axl and ligand Mfge8 and Gas6 was downregulated by UVB in both cells. After UVB exposure, the proportion of the engulfed cells by M1 macrophages which indicates the phagocytosis efficiency was significantly increased in both CJFs and PFs, however, such an increase was less in PFs. Unlike the efferocytosis efficiency maintained in CJFs before and after UVB exposure, the efferocytosis of PFs by M1 was significantly decreased after the UVB exposure (56.1±11.4 %) compared to before UVB exposure (84.7±10.7 %, p=0.028). Conclusions : Efferocytosis is induced by M1 macrophage with efferocytic synapse activation in both apoptotic CJFs and PFs, however, the efferocytosis efficiency of PFs become defective when exposed to UVB.