Abstract
Abstract: :
Purpose: To develop a new method of isolation and purification of human intraepithelial lymphocytes (IEL) and to establish the phenotype of IEL in human conjunctiva with multicolor fluorescence flow cytometry. Methods: 25 conjunctival biopsies (1cm2, inferior bulbar conjunctiva) were obtained from Cornea Bank of St. Antoine Hospital. The isolation procedure included mechanical disruption of the mucosal layer, treatment with reducing agent (dithioerythritol) and Percoll gradient centrifugation. Finally, leucocytes were removed from the epithelial fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Flow cytometric multiparametrical analysis of CD45, CD45RO, CD45RA, CD3, CD4, CD8, CD80, CD86, CD103, CD56, CD14, TCRαß and HLA DR was performed. Dendritic intraepithelial cells were detected with multiparametrical analysis of CD45, CD83, CD1a and of intracellular Langerhin (Birbeck granules). Results: The isolation procedure yielded the mean of 14,220 intraepithelial leucocytes per sample (ranging from 6016 to 28416 cells). Most of the isolated cells (90%) were of the T lineage (CD3+) and expressed a phenotype of cytotoxic-suppressor T cells (CD8+) for 73% and of T helper cells (CD4+) for 16%. 48% of CD45+ positive cells expressed the memory T cell marker CD45RO, whereas 29% were CD45RA+ (native T cell marker). Almost all CD45+ cells (91%) were CD103+ (alphaEbeta7 integrin), which confirmed their intraepithelial character. There were about 4% of macrophages (CD14+). Most of T cells (90%) expressed the αßTCR . There was less than 1% of B lymphocytes and of CD45+ cells expressing NK marker, CD56. HLADR, CD83, CD1a were present in respectively 16%, 9% and 17% of CD45+ cells. Co-stimulatory molecules CD80 and CD86 were expressed by respectively 10% and 18% of CD45+ cells. After permeabilization, intracellular Langerhin was detected in 22% of CD45+ large size cells (as determined with the forward scatter parameter). Conclusions: Conjunctival intraepithelial leukocyte population constitutes a lymphoid diffuse tissue presenting some unique characteristics only in part similar to other barrier mucosal systems. We developed a reproducible procedure which allowed to enrich leucocyte fraction isolated from conjunctival epithelium. This method could be applied in analysis of inflammatory cell population isolated from conjunctival biopsies in different ocular surface disorders.
Keywords: conjunctiva • clinical laboratory testing • flow cytometry