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D. J. Keegan, T. M. Holmes, D. M. Wallace, K. P. Kennelly; Investigating the Role of Toll-Like Receptor 4 in Subretinal Graft Cell Loss. Invest. Ophthalmol. Vis. Sci. 2010;51(13):835.
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Subretinal transplants are known to suffer significant graft cell loss. The mechanisms of this loss are currently unknown. Toll-like receptors (TLRs) are innate triggers of the adaptive immune response. Heat shock protein 60 (HSP60) is produced by cells under stress and is known to be an endogenous ligand of TLR4. TLR4 activation has been shown to play a role in pancreatic graft cell loss. This study aimed to determine if TLR4 is involved in graft cell loss in retinal pigment epithelium (RPE) cell subretinal transplants.
DH 01 is an extended-life (SV40 T) mouse RPE cell line currently being used in subretinal allograft studies. We examined the TLR expression profile in DH 01 cells by real-time PCR using gene-specific primers (qPrimerDepot). TLR4 expression by cultured cells was tested by immunohistochemistry with an antibody specific for TLR4. DH 01 cells were transplanted to the subretinal space of allogeneic mice. Eyes were harvested at post-operative day (POD) 1 and 3, snap-frozen and processed for immunohistochemistry. Sections were immunostained with antibodies specific for TLR4 and HSP60.
PCR demonstrated expression of all known mouse TLRs (TLR1-9). Immunohistochemistry showed that cultured DH 01 cells expressed TLR4 on the cell membrane. Immunostaining of subretinal DH 01 allografts illustrated expression of TLR4 and HSP60 throughout the graft cell bolus at POD 1 and 3.
TLR4 was found to be expressed by DH 01 RPE cells both in vitro and in subretinal allografts. Importantly, the endogenous ligand of TLR4, HSP60, was also found in the graft bolus. This raises the possibility that activation of TLR4 plays a role in triggering the immune response against subretinal RPE transplants.
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