Comparison of morphological and functional characteristics of primary-cultured human conjunctival epithelium and of Wong-Kilbourne derivative of Chang conjunctival cell line.
OBJECTIVE
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 11,000 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.
De Saint Jean
M
M
Department of Ophthalmology, Ophthalmology National Hospital of Quinze-Vingts, EA3123, University Paris VI, Paris, France.
Baudouin
C
C
Di Nolfo
M
M
Roman
S
S
Lozato
P
P
Warnet
J M
JM
Brignole
F
F
D000954:Antigens, Surface / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015820:Cadherins / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D048429:Cell Size; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000954:Antigens, Surface; D015820:Cadherins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma