Download Prostaglandin production by human trabecular cells: in vitro

December 1963
Vol. 24/12
Investigative Ophthalmology
& Visual Science
A Journal of Dosic and Clinical Research
Articles
Prostaglandin Production by Human Trabecular Cells:
In Vitro Inhibition by Dexamethasone
Robert N. Weinreb, Murray D. Mitchell, and Jon R. Polansky
In addition to the well-known ability of prostaglandins (PGs) to raise intraocular pressure (IOP), it
recently has been reported that moderate and low doses of PGE2 and PGF2a significantly reduce IOP
in a variety of experimental animals. These studies suggested to us that PGs might serve as endogenous
regulators of outflow facility in the meshwork if these autacoids were produced and secreted by human
trabecular cells. To examine this possibility, media from well-defined trabecular cell material were
assayed using specific radioimmunoassays. Morphologically differentiated human trabecular cells produced high levels of PGE 2 , and somewhat lower levels of PGF2o and 6KF la in the presence and absence
of serum. In a typical experiment, the following PG levels were detected in the cell culture media after
24 hours: PGE2; 225; PGF 2a , 33.5; 6KF la , 12.7 ng/ml with the presence of 10% fetal calf serum; and
PGE 2 , 30.0; PGF 2a , 4.8; 6KF la , 3.6 ng/ml in serum-free media. Since glucocorticoids are known to
inhibit PG pathways in other tissues, this effect was examined in the cultured trabecular cells. Moderate
concentrations of dexamethasone (DEX) produced a marked inhibition in the levels of all three PGs.
For PGE2 production, 10~8 M DEX inhibited approximately 75%, and 10"7 M DEX inhibited approximately 90%. More detailed dose-response studies revealed that the I so for inhibition of PG production
by dexamethasone was less than 10 nM, thus indicating that the steroid effect probably involved high
affinity glucocorticoid receptors. These findings emphasize the possibility that physiologic levels of
glucocorticoids may regulate PG production within the meshwork, and suggest that studies of endogenous
PG production by trabecular cells could provide new clues to the pathogenesis of a number of glaucoma
syndromes, including primary open-angle glaucoma and steroid glaucoma. Invest Ophthalmol Vis Sci
24:1541-1545, 1983
Prostaglandins (PGs) generally are considered to be
primary mediators of ocular inflammation,1 and also
appear to be putative physiologic regulators in many
different tissues throughout the body.2-3 Although PGs
are reported to raise intraocular pressure (IOP) after
administration of relatively high doses,4"7 recent evidence indicates that PGE2 and PGF 2a significantly
lower IOP when employed in lower and perhaps more
physiologic doses in several species of experimental
animals.8"11 Previously, there has been interest in the
measurement of PGs found in the anterior segment
and aqueous humor during ocular inflammation.12"14
In addition, the production of PGs has been examined
in cell cultures derived from corneal and lens tissues15
and in cell-free microsomal systems of other ocular
tissues.16 However, the important possibility that PGs
might be produced by trabecular cells has not been
evaluated previously. If active PGs were produced by
these cells, then their presence could play a major role
in the physiologic regulation of IOP, and perhaps in
understanding certain drug effects on outflow facility.
To investigate trabecular cell PGs, specific radioimmunoassays for PGE2, PGF 2a , and 6KFi a were employed to measure the amount of these substances released by differentiated cultures of human trabecular
From the Department of Ophthalmology, University of California
Medical Center, San Francisco, California, and Departments of
Ophthalmology, Biochemistry and OB/GYN, University of Texas,
Health Science Center, Dallas, Texas. MDM is an investigator at
Cecil and Ida Green Center for Reproductive Biology, SW Medical
School, Dallas, Texas.
Supported by NIH grants EY 0833 (RNW), EY 02477 (JRP), and
HD 13234 (MDM).
Submitted for publication: March 15, 1983.
Reprint requests: Robert Weinreb, MD, UTHSCD-Ophthalmology, 5323 Harry Hines Boulevard, Dallas, TX 75235.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1983
Fig. 1. Cultured human trabecular cells; phase contrast micrograph
of fourth passage cells (plated at 20>000 cells/35-mm dish according
to Methods) one week after confluency. The cells could be maintained
in a stable morphologic appearance as shown for over one month
in vitro.
cells. Additionally, because glucocorticoids were known
to inhibit PG production in other systems,212 the ability
of dexamethasone to block PG production in our trabecular cell system was investigated.
Materials and Methods
Human trabecular tissues obtained within 24 hours
postmortem from Eye Bank eyes were employed for
cell culture as we have described previously.17"19 Once
established in tissue culture, the cells were frozen in
sterile vials at —70 C at 106 cells/ml in tissue culture
media containing 10% DMSO. These were thawed as
needed for each experiment and provided a reproducible source of human trabecular cell material for
repeated studies. It is important to note that all cultures
employed in this study were verified previously to be
authentic trabecular cells based on detailed ultrastructural comparisons of the individual cell cultures with
trabecular cells in situ.19 The particular trabecular cell
lines employed in these studies were chosen because
they were known to maintain a differentiated morphology at both the light and electron microscopic
level through multiple passages in vitro.
For the experiments described here, third passage
cells from young patients (14, 19, and 30 years old)
were plated out at approximately 20,000 cells/35 mm
Falcon tissue culture dish which had been coated with
0.1% gelatin. Tissue culture was performed using 1.5
ml/dish of Dulbecco's modified Eagle's (DME) me-
Vol. 24
dium supplemented with 10% fetal calf serum (FCS),
2 mM glutamine, 20 Mg/ml gentamicin, 2.5 Mg/ml fungizone, in a 10% CO2 humidified incubator at 37 C.
During the growing phase (1-2 weeks), media and fibroblast growth factor (FGF, 250 ng/ml) were changed
every three days. Following confluency, the cells were
maintained for 7 to 14 days, with media changes every
three days but without addition of FGF. When grown
under these conditions, cultures of human trabecular
cells formed a stable single layer of cells as shown in
Figure 1.
To assess the production of PGs, the trabecular cells
were changed to either 10%, 1%, or serum-free media
in different experiments. Varying concentrations of
dexamethasone (Sigma, St. Louis, MO) then were
added; control dishes received no added dexamethasone. Following 24-hour periods, media were collected
and radioimmunoassays were performed for selected
PGs. Sensitive and specific methods to perform radioimmunoassays for the prostaglandins PGE2 and
PGF 2 a j as well as 6-keto F l a (the degradation product
of prostacyclin) had been developed previously and
validated by one of the authors.20"22 The lower limit
of sensitivity for the assays was approximately 1-5
picogram/tube, and the intraassay coefficients of variation were less than 10%. Cross reactivities of the antisera with other PGs were less than 1%.
Results
When the capability of human trabecular cells to
produce PGs was assessed, considerable amounts of
PGE 2 , PGF2«, and 6KF ]a were detected in the media
following 24 hours of incubation. Although the absolute amount of the different PGs varied with individual experiments, PGE2 was always present in significantly greater concentrations compared with the
other PGs evaluated. In a typical experiment shown
in Figure 2, control levels of PGE 2 were greater than
200 ng/ml in media containing 10% FCS and greater
than 25 ng/ml in serum-free media. Although present
in lower concentrations, PGF 2a (greater than 33 ng/
ml in 10% FCS, and greater than 4 ng/ml in serumfree media) and 6KF l a (greater than 12 ng/ml in 10%
FCS, and greater than 3 ng/ml in serum-free media)
also were assayed readily. Figure 2 shows the control
values obtained using 10% serum compared with those
in which dexamethasone (DEX) was added to the cultures at the beginning of the incubation period. Marked
inhibition of the production of all three PGs was observed with both 10~8 and 10~7 M DEX, to approximately 75% and 90% control levels, respectively.
PGF 2a and 6KF l a likewise were inhibited 70-80% by
these concentrations of dexamethasone. When this experiment was continued for a 4-day period in which
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No. 12
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PROSTAGLANDIN PRODUCTION DY TRADECULAR CELLS / Weinreb er ol.
PGE,
Fig. 2. (A, B, Q . Prostaglandin
secretion by cultured human
trabecular cells in DME medium
containing 10% FCS. The concentrations of (A) PGE 2 , (B)
P G F 2 a , and (C) 6KF, O were
measured in media following a
24-hour incubation under control (CONT: no added steroid)
and dexamethasone-treated
conditions (DEX 10"8 M and
DEX 10"7 M).
6KF.
PGF.
2a-
200
150
30
15
C7>
c
20
100
|
c
50
.0
c
5
10
rh
CONT
OEX
DEX
IO~8M
IO~ 7 M
media were changed every 24 hours, the results showed
that the inhibition of PG production was sustained,
and during this time period, no recovery from the
inhibitory effect occurred in the presence of these concentrations of dexamethasone. Steroid-induced inhibition of PG production was observed in both the
presence and absence of serum; however, cultures
maintained with serum appeared healthier and capable
of providing a more reproducible environment for the
quantitative measurement of the dexamethesone effect.
Figure 3 shows results from another series of experiments in which the influence of varying concentrations of dexamethasone (from 10~9M through 10"8
M) on PGE2 secretion was measured in cell cultures
maintained in either 10% or 1% serum. These results
show a clear dose-dependent effect of dexamethasone,
with inhibition noted by concentrations as low as 1
CONT
DEX
IO~8M
CONT
DEX
IO~ 7 M
DEX
OEX
IO~ 8 M
IO~ 7 M
nM DEX. The half maximal inhibition (I50) was less
than 10 nM for both serum concentrations and on
four consecutive days in which PGE2 was assayed.
(Thefirstand third days of media collection are shown
in Fig. 3.) The fact that the trabecular cells demonstrated high affinity glucocorticoid receptors with a KD
in each case of less than 10 nM23 suggests that the PG
effect probably is mediated by these receptors. The
sensitivity of the PG production in human trabecular
cells to dexamethasone suggests the possibility that
physiologic concentrations of steroid could exert an
influence on PGs produced by trabecular meshwork
cells in vivo.
Discussion
We have shown that human trabecular cells synthesize a variety of prostaglandins, including significant
B I % SERUM
800
Fig. 3. (A and B). Dose-response
data for dexamethasone inhibition
of PGE production in cultured
human trabecular cells obtained on
days 1 and 3 of steroid treatment.
The media contained either (A)
10% fetal calf serum or (B) 1% fetal
calf serum, and media were collected 24 hours following each addition of dexamethasone.
IO~9M
IO~ 8 M
IO~7M
IO" 6 M
[DEXAMETHASONE]
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IO" 9 M
IO~8M
IO~7M
[DEXAMETHASONE]
IO" 6 M
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / December 1983
levels of both PGE 2 and PGF 2 a . The production of
these PGs in particular may be important in light of
the proposal that administration of low doses of PGE 2
and PGF 2a exert a lowering of IOP by an increase in
outflow facility.9 The relatively high amounts of PGE 2
and PGF 2a produced by trabecular cells increase the
likelihood that these PGs could serve as local regulatory
hormones or "autacoids" within the meshwork. Prostaglandins could influence outflow facility by regulation
of cellular cyclic-AMP levels, since prior studies of
primates have implicated cyclic-AMP as "second messenger" for hormone effects on outflow facility.2425
However, many other PG actions also are possible and
must be considered in future experimental studies of
human trabecular cells.
The marked inhibition of PG secretion observed
following dexamethasone treatment of human trabecular cells was of particular interest because of the potency of this steroid to produce elevations in IOP. 26
The mechanism(s) for dexamethasone inhibition of
PG levels in trabecular cells may be similar to that
described in other systems in which active corticosteroids induce the synthesis of proteins that inhibit the
action of cell membrane phospholipases27"29 by binding
to glucocorticoid receptors. The net effect is a reduction
of arachidonic acid available from the cell membrane
to participate in PG synthesis, and a diminution of
the production of all PGs produced by the cell. Our
findings are compatible with this model of action since
dexamethasone treatment of human trabecular cells
results in a decrease of the three different PGs assayed.
However, considerably more investigation will be necessary to evaluate this question and to determine
whether specific inhibitors of arachidonic acid release
are induced by dexamethasone in human trabecular
cells.
Irrespective of the mechanism(s) for the observed
steroid effect, the measurement of PGE 2 and other PG
products will provide a valuable means to quantitate
the potency of different ophthalmic steroids based on
their direct action on human trabecular cells. Using
this method, agonist, partial agonist, and antagonist
properties of a steroid can be evaluated directly, which
cannot be readily determined by receptor assays. We
have reported previously the presence of specific highaffinity glucocorticoid receptors in trabecular cells, and
have found that low doses of glucocorticoid inhibit
3H-thymidine uptake in these cells. 1823 The present
findings may have greater importance than our prior
observations since the individual PG products produced by trabecular cells may be involved in the regulation of outflow facility.
The suppression of PG secretion by dexamethasone
may provide a useful means to quantitatively measure
the glucocorticoid "sensitivity" of trabecular cell cul-
Vol. 24
tures and to investigate factors that may alter the steroid
responsiveness of trabecular cells. This may help in
the evaluation of trabecular cells from steroid-sensitive
individuals and from patients with primary open-angle
glaucoma, if differentiated cultures from these sources
become available. Further studies may help to clarify
whether changes in trabecular cell PGs play a role in
the etiology of primary open-angle glaucoma (POAG)
and other glaucoma syndromes, or in certain drug
effects on aqueous outflow facility. In subsequent studies, a monitoring of the effects of donor age and source
of trabecular tissue on trabecular cell PGs may provide
additional information in evaluating these possibilities.
Key words: prostaglandins, cell culture, trabecular meshwork,
glaucoma, dexamethasone, glucocorticoids
Acknowledgments
The authors would like to thank Douglas Craig for technical
assistance, and Dr. Denis Gospodarowicz for providing FGF.
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