Comparison of morphological and functional characteristics of primary-cultured human conjunctival epithelium and of Wong-Kilbourne derivative of Chang conjunctival cell line

Purpose. To analyze the relevance of a human conjunctival cell line in a study of conjunctival epithelium. We investigated and compared the effects of IFNgamma and TNFalpha in a primary culture of human conjunctiva and in a human conjunctival cell line. Methods. A primary-cultured human conjunctival epithelium and a human conjunctival cell line (Chang cells) were treated for 72 hr with 20, 200,400 and 600 U ml(-1) IFNgamma or with 1100 and 11000 U ml(-1) TNFalpha. Then, the expression of HLA DR, CD40, CD44, CD63, CD80, CD86, Fas receptor, E-cadherin, ICAM-1, MUC1, cytokeratins and vimentin were investigated by flow cytometry. Cell morphology was studied with phalloidin staining. Apoptosis was detected by flow cytometry with Annexin V and via cell cycle analysis. Results. The primary culture of human conjunctival epithelium expressed cytokeratin K4, non-keratinized squamous epithelial marker. Chang cells presented a more dedifferentiated phenotype and were cytokeratin K4 negative. In primary-cultured cells, IFNgamma (600 U ml(-1)) induced only a low level of apoptosis and a significant upregulation of most tested proteins such as HLA DR, Fas, ICAM-1, CD40 and CD63. In the Chang cell line, IFNgamma induced a significant level of apoptosis at concentrations of 200, 400 and 600 U ml(-1). HLA DR and CD63 were induced at lower levels than in primary-cultured cells. Other proteins were modified in a similar manner after IFNgamma treatment in both systems. In the primary-cultured cells, TNFalpha induced an important upregulation of ICAM-1, Fas and CD40 whereas CD44 and CD63 were significantly decreased. Conversely, only a very weak alteration of CD63 and ICAM-1 was observed in the Chang cell line after TNFalpha treatment. Conclusions. A primary culture of a human conjunctival epithelium demonstrated well-defined epithelial features. TNFalpha and IFNgamma, two inflammatory cytokines, induced different effects in both cellular systems, in a primary-cultured conjunctival epithelium and a human conjunctival cell line. Inflammation-related molecules were highly upregulated in the primary culture and, to a lesser extent, in the Chang cell line. Thus, the Chang cell line differs in certain features from a primary culture of human conjunctival epithelium, a fact which emphasizes the complexity of interpretation of in vitro data and this should be taken into consideration in in vitro studies of human conjunctival epithelium. (C) 2003 Elsevier Ltd. All rights reserved.