Expression of hematopoietic cell markers by retinal pigment epithelial cells.
OBJECTIVE
We investigated the expression of various isoforms of the hematopoietic cell marker CD45 on retinal pigment epithelial cells in relation to their expression of CD68 and the cytokine-reactive intercellular adhesion molecule-1 (ICAM-1). We also determined the effect of the pro-inflammatory cytokines IL-1 beta, TNF alpha and IFN gamma on the expression of these molecules by RPE cells in culture.


METHODS
Monolayers of RPE cells between 3rd and 7th passages were cultured in the presence or absence of cytokines, followed by immunohistochemical staining for CD45 (170-220 kD), CD45RA (205 and 220 kD), CD45RO (180 kD), CD68 and ICAM-1, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. Total (membrane and cytoplasmic) expression of each of the three CD45 isoforms was determined by enzyme-linked immunoassays (ELISA).


RESULTS
The majority of RPE cells expressed all isoforms of CD45 on their membranes and the pattern of expression of these molecules was not modified by culture. The greatest intensity of membrane staining was consistently observed with antibodies to CD45RA (205 + 220 kD), while CD45 (170-220 kD) showed to be the predominant isoform within the whole cell, as judged by ELISA assays. Unlike the membrane expression of CD45, only 20% of RPE cells stained for the macrophage surface molecule CD68 following 4 h of culture, but progressive increase in the proportion of CD68 positive cells was observed by extending the culture to 24 and 48 h. Neither the expression of CD68 nor the various isoforms of CD45 were modified by incubation with pro-inflammatory cytokines. Staining for ICAM-1 was observed in 21-25% of RPE cells throughout the 48 h culture. However, incubation with 50 pg/ml of IL-1 beta, TNF alpha and IFN gamma caused a marked increase in the RPE cell expression of ICAM-1 following 4, 24 and 48 h culture.


CONCLUSIONS
The observations suggest that hematopoietic cell markers are constitutively expressed on RPE cells and that functions governed by these molecules are not influenced by pro-inflammatory signals. Expression of hematopoietic molecules by RPE cells may influence the macrophage-like properties of these cells and may also aid in the identification of RPE cells during pathological processes, particularly in the proliferative retinopathies, where these cells undergo phenotypic and functional changes.
Limb
G A
GA
Department of Ophthalmology, St. Thomas' Hospital, UMDS, London, UK.
Cole
C J
CJ
Earley
O
O
Hollifield
R D
RD
Russell
W
W
Stanford
M R
MR
D015703:Antigens, CD / Q000096:biosynthesis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis*; D015415:Biomarkers; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D017493:Leukocyte Common Antigens / Q000096:biosynthesis*; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D016207:Cytokines; D018799:Intercellular Adhesion Molecule-1; D017493:Leukocyte Common Antigens; C067980:CD68 antigen, human; D000097382:CD68 Molecule