Human ocular melanocytes and retinal pigment epithelial cells differ in their melanogenic properties in vivo and in vitro.
OBJECTIVE
The vertebrate eye contains both melanocytes and retinal pigment epithelial (RPE) cells. Little is known of the pigmentary behaviour of these embryologically dissimilar cells. The aim of this study was to examine aspects of the pigmentary properties of both cell types in vitro and ex vivo to learn more of the function of these cells.


METHODS
Sections of normal adult human eye were stained for tyrosinase related protein 1(TRP1), and cultures of RPE cells and choroidal melanocytes were examined immunocytochemically for TRP1 and 2 and enzymatically for tyrosinase activity (by assaying dopa oxidase activity).


RESULTS
Over half of the choroidal melanocytes expressed TRP1 ex vivo; in contrast, a very small percentage of RPE cells were TRP1 positive. In vitro, passage 1 to 3 ocular melanocytes expressed TRP1 and TRP2 and had tyrosinase activity, which was influenced by the choice of substrate on which the cells were grown. Tyrosinase activity was highest when cells were grown on fibronectin and plastic, intermediate on laminin and lowest on vitreous extracellular matrix (ECM) containing pigment to which they attached and spread out poorly. In contrast, passage 3 RPE cells (which were unpigmented) showed little evidence of tyrosinase activity in short-term culture, irrespective of the substrate on which they were grown, and failed to express TRP1 and TRP2. When cells were grown on plastic for greater than 3 weeks in culture, a very low percentage of cells (< 0.1%) became TRP1 positive and this percentage was increased threefold if cells were cultured on laminin in the presence of bFGF. A few cells were also seen to contain pigment but cultures failed to show any tyrosinase activity. In contrast, RPE cells (but not melanocytes) showed a marked ability to take up pigment granules in vitro.


CONCLUSIONS
The data suggest that normal human ocular melanocytes retain the capacity to produce pigment throughout adult life, and this can be demonstrated both ex vivo and in vitro. In contrast, we were unable to confirm that the majority of RPE cells play any significant role in active pigment production in the adult.
Smith-Thomas
L
L
University Department of Medicine, Northern General Hospital, Sheffield, UK.
Richardson
P
P
Thody
A J
AJ
Graham
A
A
Palmer
I
I
Flemming
L
L
Parsons
M A
MA
Rennie
I G
IG
MacNeil
S
S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000378:metabolism; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010088:Oxidoreductases; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D014602:Uvea / Q000166:cytology
D008543:Melanins; D008562:Membrane Glycoproteins; D011506:Proteins; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase