Abstract
Purpose:
Deficiency in mice of thrombospondin-1 (TSP-1), a major TGFβ activator, results in ocular surface inflammation and goblet cell (GC) dysfunction. Goblet cells are known to participate in ocular surface protection via mucin secretion, although their contribution to the immune response remains largely unknown. To evaluate a potential participation of GCs in the regulation of conjunctival immune response, we assessed whether conjunctival GCs can express and activate TGFβ in response to inflammatory stimuli, and the role of TSP-1 in this activation.
Methods:
Conjunctival tissues were collected from WT (C57BL/6) and TSP-1null mice. Primary cultures of mouse GCs were grown from conjunctival explants. To simulate inflammatory conditions, cultured GCs were treated with lipopolysaccharide (LPS) for 24h. Conjunctival tissues (WT and TSP-1null) and cultured GCs (untreated and LPS-treated) were analyzed by RT-PCR, immunofluorescence and flow cytometry for the expression of TGFβ1, TGFβ2, TSP-1 and CD36. Active TGFβ in the supernatant was assessed using a TGFβ reporter assay (MFB-F11 cells).
Results:
Conjunctiva and GC cultures from WT mice expressed both TGFβ isoforms, with a predominant expression of TGFβ2. In response to inflammatory stimulus (LPS), while the expression of TGFβ1 remained unaltered that of TGFβ2 was significantly increased compared to untreated GCs. Supernatants from LPS-treated WT GCs contained significantly elevated levels of active total TGFβ and TGFβ2 compared to untreated controls, indicating the ability of WT GCs to activate their TGFβ. Similarly TSP-1null conjunctiva expressed significantly increased levels of TGFβ2 compared to WT tissue supporting a relevance of TGFβ2 in conjunctival inflammation. Thrombospondin-1 and its receptor involved in TGFβ activation, CD36, both were detectable in WT GCs suggesting a potential use of TSP-1 by GCs in activating TGFβ. Moreover, LPS-treated TSP-1null GCs increased their expression of TGFβ2 compared to controls, however corresponding increase in active total TGFβ and TGFβ2 was not detected. These results support TSP-1 dependence of GCs in activation of their TGFβ.
Conclusions:
In response to inflammatory stimuli conjunctival GCs increase TGFβ expression and activate its latent form in a TSP-1-dependent manner. These results strongly implicate an as yet unknown regulatory role of GCs in conjunctival immune response, and suggest a key role for TSP-1 in this regulation.
Keywords: 557 inflammation •
474 conjunctiva •
555 immunomodulation/immunoregulation