Melanogenesis by human uveal melanocytes in vitro

Abstract

To study melanogenesis by cultured human uveal melanocytes, and the relationship between melanin production by uveal melanocytes in vitro with the degree of iris pigmentation in vivo.
Melanin content, melanin production, and tyrosinase activity of cultured uveal melanocytes derived from eyes of various iris color were measured at different stages of cultivation.
Cultured uveal melanocytes maintained a constant level of melanin content, expressed tyrosinase activity, and produced measurable amounts of melanin in vitro. Melanosomes in different stages were seen ultrastructurally. Melanin production correlated directly with the degree of iris pigmentation of the eyes from which the uveal melanocytes were isolated. Tyrosinase activity of cultured uveal melanocytes from black versus white donors was significantly different, but, among white donors, there was no correlation with iris pigmentation or with melanin production in vitro.
Cultured uveal melanocytes can produce melanin in vitro. Cultured uveal melanocytes isolated from eyes of different iris color maintained their inherent capacity for melanogenesis. Therefore, cultured uveal melanocytes are an excellent model system for studying melanogenesis in uveal melanocytes in vitro.

Full text

Melanogenesis by Human Uveal Melanocytes In Vitro
Dan-NingHu*
Steven
A. McCormick*
SethJ.
Orlow,\ Susana Rosemblat,\
Alexander Y. Lin,* and Kevin Wo*
Purpose.
To study melanogenesis by cultured human uveal melanocytes, and the relationship
between melanin production
by uveal
melanocytes
in
vitro with the degree of iris pigmentation
in vivo.
Methods.
Melanin content, melanin production, and tyrosinase activity of cultured uveal mela-
nocytes derived from
eyes
of various iris color
were
measured at different stages of cultivation.
Results.
Cultured uveal melanocytes maintained
a
constant
level
of melanin content, expressed
tyrosinase activity, and produced measurable amounts of melanin in vitro. Melanosomes in
different stages were seen ultrastructurally. Melanin production correlated direcdy with the
degree of iris pigmentation of the eyes from which the uveal melanocytes were isolated.
Tyrosinase activity of cultured uveal melanocytes from black versus white donors was signifi-
cantly different, but, among white donors, there was no correlation with iris pigmentation or
with melanin production in vitro.
Conclusion.
Cultured uveal melanocytes can produce melanin in
vitro.
Cultured uveal melano-
cytes isolated from eyes of different iris color maintained their inherent capacity for malano-
genesis. Therefore, cultured uveal melanocytes are an excellent model system for studying
melanogenesis in uveal melanocytes in vitro. Invest Ophthalmol Vis Sci.
1995;
36:931-938.
.During the past decade, there has been an increasing
interest in the relationship between exposure to visible
and ultraviolet light and the development of certain
eye diseases, including age-related macular degenera-
tion, senile cataract, and uveal melanoma. Ocular pig-
mentation, particularly uveal pigmentation, may play
an important protective role in prevention of these
diseases.
1
"
10
Recently, it has been found that melanin
from the uvea could stimulate experimental autoim-
mune uveitis and may be involved in the pathogenesis
of sympathetic ophthalmia and the Vogt-Koyanagi-
Harada syndrome.""
13
Although many studies of melanogenesis by epi-
dermal melanocytes have been reported and it is well
From the * Department of Pathology and laboratory Medicine and the Department of
Ophthalmology, Tissue Culture
Center,
The Neiu York Eye and Ear Infirmary, New
York,
and jThe Ronald O. Perelman Department of Dermatology and the
Department of
Cell
Biology,
New York University School of
Medicine,
New
York,
Nan
York.
Supported by The New York Eye ami Ear Infirmary Pathology
Research
Fund,
the
Department of
Ophthalmology Research
Fund,
and United States Public Health
Service
grants EY10223 and AR41880 (SJO).
Submitted for publication August 2, 1994; revised
October
14, 1994;
accepted
November 21, 1994.
Proprietary interest
category:
N.
Reprint
requests:
Dan-Ning Hu, Department of
Pathology
and laboratory Medicine,
Tissue Culture Center,
The.
New
York
Eye and Ear Infirmary, 310 East 14th Street,
New
York,
NY 10003.
established that epidermal melanocytes do produce
melanin in vivo and in vitro,
14
"
26
little is known about
melanogenesis by uveal melanocytes. Whether uveal
melanocytes produce melanin in vivo during adult-
hood remains controversial.
27
"
32
Little work has been
performed on melanogenesis by uveal melanocytes in
vitro.33'34
Investigation of melanogenesis by uveal mel-
anocytes in vitro has been hampered by an inability
to obtain a sufficient number of pure uveal melano-
cytes for study.
We have developed methods for isolation and cul-
ture of human uveal melanocytes.
35
'
36
This culture sys-
tem is capable of generating large numbers of human
uveal melanocytes cells in pure culture. We have now
established many cell strains from the iris, ciliary body,
and choroid from donors of different races with vari-
ous degree of iris stromal pigmentation (iris color).
The purpose of the present study was to investigate
melanogenesis by uveal melanocytes in vitro. We
wanted to explore specifically whether uveal melano-
cytes produce melanin in vitro, whether melanin con-
tent and melanin production by uveal melanocytes in
vitro correlate with iris pigmentation, whether cul-
tured uveal melanocytes express tyrosinase activity,
and whether tyrosinase activity of uveal melanocytes
Investigative Ophthalmology
&
Visual Sc
Copyright © Association for Research ine, April
1995,
Vol. 36, No. 5
on and Ophthalmology931

932Investigative Ophthalmology
&
Visual Science, April 1995, Vol. 36, No. 5
correlates with iris pigmentation, melanin content and
melanin production. We also wanted to study factors
influencing melanin content and production, as well
as the tyrosinase activity of uveal melanocytes in vitro.
METHODS
Cell Culture
Uveal melanocytes were isolated and cultured from
adult donor eyes as described previously.
35
Briefly, a
circumferential scleral incision was made at the ora
serrata, separating the globe into anterior and poste-
rior portions. The iris
was
excised and placed in a dish
with the posterior surface upward. The iris pigment
epithelium was separated from the stroma after im-
mersion in 0.25% trypsin solution (Gibco, Grand Is-
land, NY) at 37°C for
1
to 2 hours. The remaining iris
stroma was placed in 0.25% trypsin solution at 4°C for
18 hours, followed by incubation at 37°C for 1 hour.
The isolated cells were collected. The trypsin solution
was replaced by collagenase solution (400 U/ml, in F-
12 medium, Sigma, St. Louis, MO) and incubated at
37°C.
The collagenase solution was replaced, and the
cells were collected, centrifuged, resuspended, and
plated each hour for 3 hours. The ciliary body was
separated from the sclera and placed in a culture dish
with its inner surface upward. The ciliary epithelium
was separated from the ciliary body after immersion
with trypsin solution for 2 to 3 hours. Uveal melano-
cytes in the remaining ciliary body were isolated ac-
cording to the method used for the iris. The vitreous
and retina were excised, The retinal pigment epithe-
lium was separated from the choroid after immersion
with 0.05% trypsin-0.02% ethylenediaminetetraacetic
acid solution (EDTA, Gibco) for
1
hour. The choroid
was separated from the sclera, and the uveal melano-
cytes were isolated using the same method.
The isolated uveal melanocytes were cultured in
Falcon culture dishes (Becton Dickinson, Oxnard,
CA) with FIC medium, which consisted of F-12 me-
dium supplemented with 10% fetal bovine serum, 2
mM glutamine (all from Gibco), 10 ng/ml cholera
toxin, 0.1 mM isobutylmethylxanthine, 50 pig/nA gen-
tamicin (all from Sigma), and 20 ng/ml basic fibro-
blast growth factor (Promega, Madison, WI). The cul-
ture dishes were incubated in a humidified 5% CO2
atmosphere. The medium was changed three times a
week. Geneticin (Sigma), a cytotoxic agent, was added
(100 ng/m\) for 3 to 7 days when necessary to elimi-
nate contaminating cells.
14
Fibroblasts and pigment
epithelial cells are much more sensitive to geneticin
than are uveal melanocytes.
35
At confluence, the uveal
melanocytes were detached by trypsin-EDTA solution,
diluted 1:3 to 1:4, and subcultured.
The 10 cell strains of uveal melanocytes used in
the present study were isolated from donors with dif-
ferent iris color. Iris pigmentation was classified into
three categories: light pigmentation
(1
+
),
blue to
light yellow-green, four cell strains (two from the iris
and two from the choroid); moderate pigmentation
(2+)> green or brown, five cell strains (three from
the iris, one from the ciliary body, and one from the
choroid); dense pigmentation (3+), dark brown, one
cell strain from the iris of
a
black donor. The research
was conducted in accordance with the tenets of the
Declaration of
Helsinki,
and was approval was granted
by institutional human experimentation committee.
Melanin Measurement
Cultured uveal melanocytes were detached by trypsin-
EDTA solution and counted in a hemocytometer, the
cell suspensions were centrifuged, and the pellet was
dissolved in 1 N NaOH. Melanin concentration was
determined by measurement of optic density at 475
nm and compared with a standard curve obtained
using synthetic melanin (Sigma).
19
'
21
Melanin content
was expressed as ng/cell.
Calculation of Melanin Production
Melanin production was calculated by determining
the melanin content and the cell counts at the begin-
ning and the end of each generation by the following
formula:
C
p
= C,P - C
O
/1.3D(P - 1)
where C
o
and C, represent the melanin content per
cell at times O and time t, respectively; P is the popula-
tion increase during time t, D is the doubling time of
the uveal melanocytes; and C
p
is melanin production
per cell per day during time t.
DOPA Reaction
Uveal melanocytes cultured in chamber slides were
fixed with 5% formalin in phosphate buffer (pH 7.0)
at 4°C for 30 minutes, rinsed with distilled water, incu-
bated with 0.1% L-DOPA (3,4-dihydroxyphenylala-
nine,
Sigma) in phosphate buffer at 37°C for 3.5 hours
with one change of solution, then fixed with 10% for-
malin in phosphate buffer at 25°C for 1 hour, air-
dried, coverslipped, and examined by light micros-
copy.
17
'
23
Two cell strains of retinal pigment epithelial
cells isolated from adult human eyes (5th and 10th
generation) and two cell strains of fibroblasts isolated
from human sclera (5th generation and 7th genera-
tion) were tested as controls (both cell types were
cultured with F12 medium supplemented with 10%
fetal bovine serum).
Tyrosinase Activity
Tyrosinase activity was evaluated in nine cell strains
using an adaptation
37
of the Pomerantz method,
38
*/•
u

Melanogenesis by Human Uveal Melanocytes933
1.0 :
0.1 :
0.01 :
Melani
Comsnt (ng/c
——~"
all)
FIGURE 1. Melanin content (ng/cell) of two cell strains of
cultured human uveal melanocytes (UM) at various stages
during cultivation. Uveal melanocytes from light green iris
(solid
line)
and from brown iris
(dashed
line).
which is based on the measurement of
3
H
2
O released
by the enzymatic hydroxylation of tyrosine.
Ultrastructure
Cultures of uveal melanocytes in various stages were
fixed, embedded in Epon, stained with lead citrate-
uranyl acetate, and examined by routine transmission
electron microscopy.
RESULTS
Pure cultures of human uveal melanocytes were estab-
lished from the iris, ciliary body, and choroid using
the described methods. Most of the uveal melanocytes
attached and spread within 1 to 3 days after plating.
After
6
to 12 days, most of the spread cells had divided.
The dividing cells showed a gradual dilution of pig-
ment. Each cell developed two or more dendritic pro-
cesses. The uveal melanocytes grew quickly upon sub-
culture, and the pigment content became stable dur-
ing the active growth stage. These cell strains have
been passaged for 15 to 30 generations over 3 to 7
months, with 30 to 45 divisions. The doubling time
was 2 to 3 days during the active growth stage. When
the cultures became senescent, the cytoplasm of the
uveal melanocytes spread to form round or polygonal
platelike configurations, and the pigment content
gradually increased.
Melanin Contents
Melanin content per cell in cultured uveal melano-
cytes decreased rapidly during early passages and then
stabilized. Despite the dilutional effect of cell division,
the melanin content per cell remained stable in each
cell strain during the active growth stage. The melanin
content increased after the cells became senescent
(Fig. 1).
Melanin content of cultured uveal melanocytes
during active growth stage varied from 0.0118 ng/
cell to 0.102 ng/cell in the 10 cell strains tested. The
melanin content increased after the cells became se-
nescent (Table 1). The difference of melanin content
between actively growing cells and senescent cells was
statistically significant (P < 0.01).
The melanin content of iridal melanocytes from a
black donor during active growth stage and senescent
stage were significantly higher than those from white
donors (P < 0.01).
During the active growth stage and the senescent
stage, the melanin content of uveal melanocytes from
moderately pigmented eyes was significantly greater
than that from lightly pigmented eyes but less than
that from the darkly pigmented eye (Table 1). Correla-
tion tests showed that the melanin content of uveal
melanocytes in the active growth stage or senescent
stage correlated well with iris color (P < 0.01).
Melanin content of uveal melanocytes isolated
from the iris, ciliary body, and choroid did not show
significant differences.
The melanin content of uveal melanocytes at dif-
ferent stages of culture correlated with the growth rate
within a particular cell strain. Correlation tests showed
that melanin content correlated well with the dou-
bling time in four cell strains in which the doubling
time and melanin content were measured in each gen-
eration from primary culture to senescence (P <
0.01).
Melanin Production
Melanin production was 0.0070 ± 0.0043 ng/cell per
day (mean ± SD) in the 10 cell strains tested during
the active growth stage, continued to increase in early
senescence (0.0181 ± 0.0130 ng/cell per day), and
decreased to a slightly lower level (0.0151 ± 0.0124
ng/cell per day) in senescent cells that remained sta-
tionary for a long period (Fig. 2).
Melanin production by iridal melanocytes from a
black donor during active growth and senescent stages
were also significantly higher than those from white
donors (P < 0.01).
Melanin production by uveal melanocytes from
moderately pigmented eyes was significantly greater
than that from lightly pigmented eyes and less than
that from the darkly pigmented eye (Table 1). Correla-
tion tests showed that melanin production of uveal
melanocytes in either the active growth or senescent
stage correlated well with iris color (P < 0.01).
Melanin production also correlated with growth
rate in four cell strains with complete data (P < 0.01).
DOPA Test
The DOPA tests in the four cell strains of cultured
uveal melanocytes tested revealed positive results (Fig.

934
Investigative
Ophthalmology & Visual
Science,
April 1995, Vol. 36, No. 5
TABLE
l. Melanin Content, Melanin Production, and Tyrosinase Activity of Cultured
Melanocytes
From Eyes of
Various
Iris Color
Iris
Color
Light
Moderate
Dark
Melanin
Groiuth
Stage
0.0140
P
< 0.01
0.0249
P<
0.01
0.1020
Content
(ng/cell)
Senescent
Stage
0.0978
P<
0.01
0.2777
P<
0.01
0.6980
Melanin
Production
(ng/cell
per day)
Groivth
Stage
0.0041
P<
0.01
0.0071
P <
0.01
0.0180
Senescent
Stage
0.0100
P
< 0.01
0.0189
P<
0.01
0.0480
Tyrosinase
Activity (units)
Groivth
Stage
51.7
P
> 0.05
37.4
P<
0.01
115.4
Senescent
Stage
121.1
P >
0.05
89.9
P >
0.05
247.9
3),
whereas all cultured retinal pigment cells and fi-
broblasts were negative.
Tyrosinase Activity
The tyrosinase activity varied from 19.2 U to 115.4 U
(a unit of tyrosinase was defined as the activity of en-
zyme that catalyzed the hydroxylation of 1 pmol of
tyrosine/mg protein per hour) in the eight cell strains
tested during the active growth stage, and it increased
after senescence (Table 1).
The tyrosinase activity of iridal melanocytes from
a black donor during the active growth stage and the
senescent stage were also significantly higher than
those from white donors (P < 0.01).
However, in the uveal melanocytes from white do-
nors,
tyrosinase activity of uveal melanocytes from
moderately pigmented eyes did not differ significantly
from lightly pigmented eyes in either the active growth
stage or the senescent stage (Table
1).
The correlation
coefficients between iris pigmentation and tyrosinase
activity of uveal melanocytes from white donors were
—0.4209 (P > 0.05) during active growth stage and
-0.3193 (P > 0.05) in senescence.
The tyrosinase activity of
uveal
melanocytes corre-
Melanin
Production (ng/cell/day)
0.02-
0.015
•
0.01-
0.005 •
10
Generation15
FIGURE
2.
Melanin production (ng/cell per
day)
of
two
cell
strains
of
uveal
melanocytes
at
various
stages during cultiva-
tion.
Uveal
melanocytes
from light
green
iris
(solid
line)
and
from
brown iris
(dashed
lated with melanin content and melanin production
in uveal melanocytes in the active growth stage (P <
0.05) but did not correlate with cultures in the senes-
cent stage (P> 0.05). After excluding the uveal mela-
nocytes from the black donor, the tyrosinase activity
in the uveal melanocytes from white donors did not
correlate with melanin content or melanin production
in either the active growth stage or in the senescent
stage (P > 0.05 in all correlation coefficients).
FIGURE
3.
DOPA test of
cultured
human uveal melanocytes.
(A)
Before
DOPA
test.
Magnification,
X250.
(B) DOPA test
revealed
positive reaction. Magnification,
X200.

Melanogenesis by Human Uveal Melanocytes935
Ultrastructural Study
Transmission electron microscopy revealed premela-
nosomes and all stages of melanosomes (stages I to
IV) in the cytoplasm of cultured uveal melanocytes
(Fig. 4). In actively growing cells, there were fewer
melanosomes, and most were mature melanosomes
(stage IV). In early senescent stage cells, many premel-
anosomes and immature melanosomes (stages I to III)
appeared in the cytoplasm, indicating active melano-
some biogenesis and melanogenesis. In late senes-
cence cells, mature melanosomes again predomi-
nated.
The size of melanosomes in uveal melanocytes
from the black donor (0.35 fim X 1.02 fim) was sig-
nificantly larger than those from white donors (0.25
/itm X 0.71 A*m) (P < 0.01).
DISCUSSION
In the past decade, extensive studies of melanogenesis
by epidermal melanocytes have been presented.
14
"
26
Not only do epidermal melanocytes produce melanin
and transfer it to keratinocytes in vivo, they also ex-
press tyrosinase activity and synthesize melanin in
vitro.
l4
-
a6
Controversy exists concerning the capacity of
uveal melanocytes for melanogenesis in vivo in adult-
hood. The traditional view is that the uveal melano-
cytes produce melanin before and after birth, leading
to increased pigmentation of the iris within the first
several months of life. It was considered that melano-
genesis then ceased and that all melanosomes in the
uveal melanocytes were fully mature by 10 months.
30
However, stage III melanosomes have been found in
iridal melanocytes in the adult monkey.
29
Some au-
thors reported that tyrosinase activity could be demon-
strated in adult human uvea
31
and mature rabbit iris
and choroid.
32
However, others have reported that
tyrosinase and related proteins were synthesized only
early after birth.
25
These various reports leave unre-
solved the question whether the capacity for melanin
production is retained by the uveal melanocytes in
vivo in the adult.
Other than a few reports of positive DOPA reac-
tions and measurable tyrosinase activity in cultured
choroidal melanocytes, little is known about melano-
genesis by uveal melanocytes in vitro.
33
'
34
For accurate
investigation of melanogenes in vitro, it is essential to
generate a large population of uveal melanocytes in
pure culture. Study of melanogenesis by uveal melano-
cytes in vitro has been hampered by their low prolifer-
ative potential and the tendency for contamination
with other cell types under usual culture conditions.
We have developed a method for isolation and cultiva-
tion of pure cultures of human uveal melanocytes.
33
A culture system of human uveal melanocytes has been
developed that can provide large numbers of pure
human uveal melanocyte cells.
35
'
36
Therefore, mea-
surement of melanin content, melanin production,
and tyrosinase activity of uveal melanocytes in vitro
became possible.
The present study revealed that cultured human
uveal melanocytes isolated from iris, ciliary body, or
choroid maintained a constant level of melanin con-
tent even during the active growth stage. They also
produced measurable amounts of melanin in vitro and
showed a positive DOPA reaction. Appreciable levels
of tyrosinase activity also were demonstrated. Melano-
somes in different stages were seen ultrastructurally.
These results indicate that uveal melanocytes, similar
to epidermal melanocytes but in contrast to retinal
pigment epithelium, can produce melanin in vitro.
Melanin content and the production of melanin
by uveal melanocytes in vitro apparently are influ-
enced by racial and genetic factors because uveal mela-
nocytes from a black donor showed significantly
higher melanin content, rate of melanin production,
and tyrosinase activity, as well as larger melanosomal
size,
than seen in uveal melanocytes derived from
white donors.
In white donors, melanin content and melanin
production in cultured uveal melanocytes correlated
well with iris pigmentation in vivo, indicating that cul-
tured uveal melanocytes retain their different capacit-
ies for melanin production ability in vivo well. There-
fore,
cultured uveal melanocytes provide an excellent
model system for studying melanogenesis in uveal mel-
anocytes.
The content of melanin per cell in cultured uveal
melanocytes is not only determined by the production
of melanin but also by growth rate. In stationary cells,
the melanin produced accumulates within the cell and
results in a rapid increase of melanin content per cell.
In growing cells, the melanin is diluted to daughter
cells during division. If the melanin production rate
equals the rate of dilution, the melanin content per
cell would remain unchanged. In rapidly growing
cells,
if the dilution rate
is
greater than that of melanin
production, melanin content per cell would decrease,
as we observed in the first few generations of cultured
uveal melanocytes.
Melanin production by cultured uveal melano-
cytes also correlated with the growth rate in four cell
strains when compared to the doubling time in each
generation. Presumably, stationary cells arrested in
the Gi stage of the cell cycle devote a greater propor-
tion of cellular metabolism to the production of mela-
nin than do rapidly growing cells.
Melanin production of uveal melanocytes de-
creased to a lower level in senescent cells that re-
mained stationary for long periods. This finding is

936Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
1.0
urn
FIGURE 4. Transmission electron microscopy of cultured
uveal melanocytes (UM) demonstrating premelanosomes
and melanosomes in various stages of maturation (stages I
to IV). (A) Uveal melanocytes from a black donor during
growth stage showing mitochondria (M), endoplasmic retic-
ulum (E), nuclei (N), and melanosomes. Most melanosomes
are stage IV, whereas only a few are stage II or stage III
melanosomes. Magnification, X6,000. (B) Uveal melano-
cytes from a white donor during the early senescence stage
showing numerous melanosomes, most of which are stage I
and stage II melanosomes. Relatively few are stage III mela-
nosomes. Magnification,
X
15,000.
(C) Uveal melanocytes
from
a
white donor during the late senescence stage demon-
strated an increased number of stage III and stage IV mela-
nosomes (compare to Fig. 5B). Some stage II melanosomes
also are present. Magnification, X2O,OOO.
• '.
•«**•.,
v"
•*•
1.0 /im,;,
v
•
•.
consistent with results indicating that tyrosinase activ-
ity of cultured epidermal melanocytes was inhibited
by high concentrations of melanin and that virtually
no tyrosinase activity could be identified in fully ma-
ture melanosomes (stage IV).1
Tyrosinase is thought to control the rate limiting
step on the enzymatic production of melanin in epi-
dermal melanocytes. Therefore, tyrosinase activity is
thought to be the major regulatory factor in melano-
genesis. In the present study, the relationship between
tyrosinase activity of uveal melanocytes in vitro with iris
pigmentation in vivo seems more complicated. Uveal
melanocytes from the eye of a black donor contained
higher tyrosinase activity than eyes from white donors.
This is consistent with previous reports that epidermal
melanocytes derived from black skin expressed higher
tyrosinase activity than those from white skin.19"21
However, when the tyrosinase activity of cultured uveal
melanocytes from eyes with light irides was compared
to that from dark irides, approximately equivalent lev-
els of tyrosinase activity
was
present in the two groups.
Moreover, in uveal melanocytes from white donors,
tyrosinase activity did not correlate with melanin con-
tent and melanin production in vitro, indicating that,
although tyrosinase activity attributed to racial differ-
ence
was
present in cultured uveal melanocytes; within
the same race,
tyrosinase
activity in vitro did not corre-
late with iris pigmentation in vivo. This may be ex-
plained by the complexity of the role of tyrosinase in
melanogenesis.22^"'1
Tyrosinase holds a central position in the biosyn-
thesis of melanin because of its ability to catalyze the
first two rate-limiting reactions, namely the hydroxyla-
tion of tyrosine to DOPA and its subsequent oxidation
to dopaquinone. It was previously thought that the
subsequent steps proceeded more or less spontane-
ously, ending with the format of melanin. However,
more recently, it has been found that many other fac-
tors regulate melanogenesis, such as the activity of
auxiliary enzymes (e.g., dopachrome tautomerase and
peroxidase) and certain metal ions, especially copper
and iron.22"24 An additional level of melanogenic con-
trol exists even before tyrosinase.3940Therefore, tyrosi-
nase activity is important, but it is not the sole factor
for determining the rate of melanin production. Many
other factors may modify the biosynthesis of melanin.
These concepts may explain our findings that differ-
ent levels of melanin production existed in uveal mela-

Melanogenesis by Human Uveal Melanocytes937
nocytes that expressed similar levels of tyrosinase activ-
ities.
Regulation of melanogenesis in uveal melanocytes
may have significance in the pathogenesis of several
eye diseases. Recently, it was found that development
of certain eye diseases may be related to exposure to
visible and ultraviolet light. Uveal pigmentation could
protect intraocular tissues from irradiation and may
play a role in the prevention of these disorders.
1
"
3
For example, a population-based study indicated that
exposure to sunlight may be associated with age-re-
lated macular degeneration.
4
Near-ultraviolet radia-
tion is thought to be one of the factors responsible for
oxidative changes in lens protein in senile cataract.
5
'
7
'
8
Epidemiologic studies have shown a high correlation
between increased ultraviolet-B exposure and human
cortical cataract formation.
6
Sunlight exposure has
been identified as a possible risk factor for the devel-
opment of intraocular melanoma.
9
Persons with blue
irides have a significantly greater risk for ocular mela-
noma than those with brown irides.
10
Recent studies demonstrated that purified uveal
melanin was uveitogenic and could induce severe ex-
perimental autoimmune anterior uveitis.
1
' Ocular im-
mune response to modified melanocytic autoantigens
may play a role in the pathogenesis of sympathetic
ophthalmia and Vogt-Koyanagi-Harada syn-
drome."-'
3
Although uveal pigmentation may play an im-
portant role in the physiology and pathology of the
eye,
little is known about melanogenesis by uveal mela-
nocytes in vivo or in vitro. We have developed a
method for uveal melanocytes culture, and we have
demonstrated that cultured uveal melanocytes can
synthesize melanin in vitro and that melanin content
and melanin production of uveal melanocytes in vitro
correlate with iris pigmentation in vivo. It is now possi-
ble to use this model system to study melanogenesis
by uveal melanocytes and to explore the functions of
melanin in the eye and the role of melanin in the
pathogenesis of various eye diseases.
Key Words
melanogenesis, uveal melanocytes, melanin production,
ty-
rosinase, iris pigmentation
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