Download Effect of TGF-beta on interferon-gamma-induced HLA-DR

Effect of TGF-/5 on Interferon-y-Induced HLA-DR
Expression in Human Retinal Pigment Epithelial Cells
Karine Gabrielian,*^ Roman Osusky*^ Brian D. Sippy,X Stephen J. Ryan*~\
and David R. Hinton%%\\
Purpose. Retinal pigment epithelial (RPE) cells express human leukocyte antigen (HLA)-DR
(class II) antigens when stimulated with interferon gamma (IFN-7) and may be capable of
local antigen presentation. The authors examined the effect of transforming growth factorbeta (TGF-/3), a cytokine normally found in the eye, on the expression of these immunoregulatory molecules in vitro and attempted to determine the mechanism by which this cytokine
acts.
Methods. Human RPE cells were cultured in the presence of IFN-7 and then stained immunohistochemically for HLA-DR antigens. TGF-/?] or TGF-/?2 was added simultaneously with IFN7 or after 3 days of IFN-7 treatment. In parallel experiments, RPE cells were pretreated with
4-phorbol-12 myristate-13 acetate (PMA), staurosporine, or calphostin C before stimulation
with IFN-7 or TGF-/?. Quantitative analysis was performed by fluorescence-activated cell
sorting.
Results. IFN-7 induced HLA-DR expression on RPE cells. Both TGF-/3, and TGF-/32 were able
to inhibit this effect. These inhibitory effects of TGF-/3 were augmented by pretreatment with
either PMA or calphostin C. Pretreatment of the cells with PMA before stimulation with
IFN-7 downregulated HLA-DR expression. Staurosporine pretreatment suppressed HLA-DR
expression by IFN-7-stimulated RPE cells, but this was not additive with TGF-/3.
Conclusions. The authors conclude that TGF-/?i and TGF-/?2 strongly inhibit the IFN-7-induced
upregulation of class II antigens on human RPE cells. The modulation of these IFN-7 and
TGF-/3 effects by calphostin C, staurosporine, and PMA treatment suggests involvement of
the protein kinase C pathway. Invest Ophthalmol Vis Sci. 1994; 35:4253-4259.
Xmmunologic mechanisms are thought to be important in the progression of several retinal diseases,
including uveitis and proliferative vitreoretinopathy.1'2
The human retinal pigment epithelial (RPE) cell may
play a central role in the immunologic microenvironment of the outer retina, based on its ability to synthesize and respond to a large variety of inflammatory
cytokines.3'4 RPE cells are capable of expressing the
From the *Doheny Eye Institute and the Departments off Ophthalmology,
%Palhology, ^Neurological Surgery, and "Neurology, University of Southern
California School of Medicine.
Supported in part by grants EY01545 and EY03040 from the National Eye
Institute, Bethesda, Maryland. The University of Southern California Department of
Ophthalmology is the recipient of an award from Research to Prevent Blindness,
Inc., New York, New York.
Submitted for publication October 7, 1993; revised April 1, 1994; accepted July 18,
1994.
Proprietary interest category: N.
Reprint requests: Stephen J. Ryan, Doheny Eye Institute, 1450 San Pablo Street,
Los Angeles, CA 90033-4681.
major histocompatibility complex (MHC) class II molecule, human leukocyte antigen (HLA)-DR in vivo,2'5
and can present antigens to T cells in vitro.6 Interferon
gamma (IFN-y) induces MHC class II antigens on RPE
cells in vitro and has been identified in the vitreous
of patients with uveitis and proliferative vitreoretinopathy.1'7 Modulation of class II antigen expression
might provide an avenue of therapy for these disorders.
Transforming growth factor-beta (TGF-/3), a cytokine found in both normal and pathologic vitreous,8
is capable of inhibiting MHC class II antigen expression on several different cell types and can also suppress autoantigen presentation.9 Some of the effects
of TGF-/3 are antagonistic to IFN-7.10 Although the
mechanism of action of TGF-/? in this form of immunomodulation is unknown, it has been reported that
IFN-y acts through protein kinase C (PKC) to upregu-
Investigative Ophthalmology & Visual Science, December 1994, Vol. 35, No. 13
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology 8e Visual Science, December 1994, Vol. 35, No. 13
late MHC class II antigens1' and that TGF-/? also exerts
some of its actions through this pathway.12 This study
was initiated to determine whether TGF-p could regulate IFN-y-induced MHC class II antigen expression
on human RPE cells and to determine whether this
action is mediated through the PKC pathway.
MATERIALS AND METHODS
Isolation and Culture of Human Retinal
Pigment Epithelial Cells
Retinal pigment epithelial cells were isolated from
adult human eyes after donor death. The tenets of
the Declaration of Helsinki were followed, informed
consent was obtained, and institutional human experimentation committee approval was granted. After the
eyes were cut circumferentially through the sclera approximately 2 mm posterior to the ora serrata, the
vitreous was aspirated, and the retina was gently separated from the RPE layer. The eyecup was washed
with Eagle's minimal essential medium (EMEM; Irvine
Scientific, Santa Ana, CA) and incubated at 37°C with
0.05% trypsin and 0.02% ethylenediaminetetraacetic
acid (Tryp-EDTA; Irvine Scientific). After 1 hour, the
Tryp-EDTA solution was aspirated from the eyecup
and replaced with standard medium (EMEM) containing 10% fetal bovine serum (FBS; Gemini Bioproducts, Calabasas, CA), penicillin (100 U/ml), streptomycin (100 fj,g/m\) (Irvine Scientific), and amphotericin B (2.5 /xg/ml) (Irvine Scientific). The RPE cells,
released by gentle pipetting, were cultured at 37°C in
a humidified incubator saturated with 5% CO2 and
95% air. After 4 to 5 weeks, when the cells had reached
confluence, they were trypsinized and passaged. Fifty
percent confluent cells (passages 4 to 8) were used
for all experiments. Cells were grown either in EMEM
with 10% FBS or in defined media with 0.5% bovine
serum albumin. Cells were grown on 4-well or 8-well
chamber slides (Fisher Scientific, Pittsburgh, PA) and
screened immunohistochemically for contaminating
cells. More than 95% of the cells were positive for
pancytokeratin, indicating epithelial origin. No cells
positive for factor VIII (endothelial cells) oranti-CDll
(macrophages) were found.
IFN-y and TGF-/3 Studies
Retinal pigment epithelial cells were treated with IFNy (Collaborative Biochemicals, Bedford, MA) alone
for 24 or 72 hours; IFN-y plus TGF-/?! (Becton Dickinson, Bedford, MA) for 72 hours; IFN-y plus TGF-/?2
(Genzyme, Cambridge, MA) for 72 hours; IFN-y for
72 hours, plus TGF-/?! for the next 48 hours; IFN-y
for 72 hours, plus TGF-/?2 for the next 48 hours; TGF-
/?! alone for 24, 48, or 72 hours; TGF-/32 alone for 24,
48, or 72 hours.
Protein Kinase C Studies
The PKC pathway was tested by pretreating the RPE
cells with 4-phorbol-12 myristate-13 acetate (PMA;
Sigma, St. Louis, MO), staurosporine (BoehringerMannheim, Indianapolis, IN), and calphostin C (L.
C. Service, Woburn, MA). PMA pretreatment was for
15, 30, 60, or 120 minutes, or for 12 hours. Calphostin
C and staurosporine pretreatment was for 1 hour.
After each pretreatment, the cells were washed three
times with phosphate-buffered saline and then were
exposed to IFN-y and TGF-/5.
MHC Class II Antigen Immunocytochemistry
Immunoperoxidase staining was performed on adherent RPE cells grown on multiwell chamber slides by
the avidin-biotin complex (ABC) method, as described by the manufacturer (Vector, Carpenteria,
CA),13 using primary mouse monoclonal antibody
against HLA-DR (Becton-Dickinson). The percentage of positively stained RPE cells was calculated. An
irrelevant isotypic (IgG2a) primary antibody control
(anti-/?l, Coulter Immunology, Hialeah, FL) showed
no background staining.
Direct-Fluorescence-Activated Cell Sorting
Analysis
Retinal pigment epithelium cells were detached from
the flask with EDTA (Ca2+- and Mg2+-free) and were
counted with a Coulter counter (Coulter Immunology). Cells were washed once with Hanks' balanced
salt solution containing 0.2% bovine serum albumin
and 0.01% sodium azide. They were incubated with
20 fi\ of 3% goat serum in phosphate-buffered saline
(Gibco BRL, Gaithersburg, MD) for 15 minutes and
then with 20 /A of mouse monoclonal anti-HLA-DRfluorescein isothiocyanate (FITC) (GenTrack, Plymouth, PA) for 30 minutes on ice. The cells were washed
with buffer, fixed in 2% paraformaldehyde, and analyzed by fluorescence-activated cell sorting analysis
(FACStar Plus; Becton Dickinson, Mountain View,
CA). An irrelevant mouse monoclonal FITC-labeled
isotypic (IgGi) control (FITC-CD4, Dako, Carpinteria,
CA) was also used and showed no difference from a
no primary control in both unstimulated and IFN-7
stimulated cells (results not shown).
RESULTS
Modulation of HLA-DR Expression
Control unstimulated RPE cells maintained in either
10% FBS or defined media were negative for HLA-DR
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Effect of TGF-/3 on HIA-DR Expression in RPE Cells
4255
f
FIGURE l. Immunoperoxidase staining for HLA-DR. (a) Control RPE cells do not express
HLA-DR antigens, (b) RPE cells stimulated with 500 U/ml of IFN-y for 3 days show intense
staining, (c) TGF-^i (50 ng/ml) added for 48 hours after 3 days of IFN-y stimulation
show a significant decrease in the amount and intensity of HLA-DR-positive cells, (d) PMA
pretreatment for 1 hour was additive with the effect of TGF-/?i, decreasing significantly the
amount and intensity of positively stained cells. HLA-DR = human leukocyte antigen-DR;
RPE = retinal pigment epithelial; IFN — interferon; TGF = transforming growth factor;
PMA = 4-phorbol-12 myristate-13 acetate.
surface antigens, as were cells treated with TGF-/3, and
TGF-/32 alone and cells pretreated with PMA, calphostin
C, or staurosporine. Treatment of RPE cells with various
concentrations of IFN-y for 1 day resulted in weak HLADR staining of only a few cells, which is consistent with
other published data.14 We, as well as others, have found
maximal HLA-DR expression after 3 days of incubation
with IFN-y and followed this protocol for all experiments.6 The maximal inhibitory effect for TGF-/3 in modulating histocompatibility antigens on other cell types
was reported to be after 48 hours,15 which is why we
choose 48 hours as the minimal length of incubation.
Treatment of RPE cells with IFN-y for 3 days in
either 10% FBS or in defined media resulted in the
upregulation of HLA-DR antigens in a dose-dependent
manner, with an increase in both the intensity of staining
and the percentage of positive cells. Fifty units per milliliter of IFN-y induced expression of HLA-DR antigens in
the presence of 10% FBS on 91% ± 2.9% of cells; 500
U/ml, 95% ± 1.6%; and 1,000 U/ml, 98% ± 1.0%.
After combined stimulation of the cells with IFN-y plus
TGF-/?] or TGF-/?2> the number and intensity of positively stained cells was dramatically decreased in a dosedependent manner (Fig. 1), with the more prominent
effect induced by TGF-/?2. Table 1 shows the results of
stimulation with 50 U/ml of IFN-y plus TGF-/?, or TGF/?2 (10, 50, and 100 ng/mt each) for 3 days.
Results of FACS analysis of one of the experiments
are shown in Figure 2. After 3 days of incubation in
50 U/ml of IFN-y, cells were incubated for an additional 2 days in media with TGF-/?,; mean fluorescence
of stimulated RPE cells was reduced in a dose-dependent manner after the addition of TGF-/?.
Modulation of PKC Pathway
Pretreatment of the RPE cells with PMA, with subsequent incubation with IFN-y, resulted in a time-dependent downregulation of HLA-DR antigen, with the
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Investigative Ophthalmology & Visual Science, December 1994, Vol. 35, No. 13
4256
TABLE l.
Percentage of HLA-DR-Positive Cells After Combined
Treatment of RPE with IFN-y 50 U/ml and Different
Concentrations of TGF-/?i and TGF-/32
Concentration of TGF-fi
0 (IFN-y Alone)
10 ng/ml
50 ng/ml
100 ng/ml
91 ± 2.9
91 ± 2.9
65 ± 7.9*
56 ± 4.3f
18.4 ± 2.1f
3.6 ± 1.5f
8.1 ± 2.1f
1.5 ± 1.6f
TGF-/?,
TGF-/?2
The percentage of HLA-DR positive cells after simultaneous incubation with IFN-y, TGF-/?1? and
TGF-/?2 was compared with the percentage of positive cells after incubation with IFN-y alone (chisquare test).
* P < 0.005; -\P< 0.0001.
most prominent effect seen after 1 to 2 hours of PMA
incubation (Fig. 3).
The addition of PMA to the RPE cells treated for
3 days with IFN-y did not alter significantly the intensity or number of positively stained cells, compared to
the controls. Pretreatment of RPE cells with 0.1 fjM
PMA for 1 hour and further incubation of these cells
with IFN-y (50 U/ml) plus TGF-^i and/or TGF-&
(50 ng/ml) resulted in a significant decrease in inten-
a
b
c
d
A
1
240
480
720
960
A
240
480
720
sity and in the number of positively stained cells compared with the cells incubated with TGF-/?, and/or
TGF-& alone (PMA + IFN-7 + TGF-/?!, 8.6% ± 2.3%,
P < 0.01; PMA + IFN-7 + TGF-/?2, 0.8% ± 0.3%, P
< 0.0002) (Figs. Id, 3).
Table 2 shows that pretreatment of cells with staurosporine (10 nM or 100 nM) for 1 hour and further
incubation with IFN-7 alone (50 U/ml) decreased significantly the number of HLA-DR-positive cells; the
intensity of staining was also significantly decreased.
Subsequent treatment with TGF-/?! (50 ng/ml) did
not enhance the effect of staurosporine. Pretreatment
of IFN-7-treated (50 U/ml) cells with calphostin C
also caused significant inhibition of HLA-DR antigens
compared to the controls. Calphostin C in concentrations of 1 nM and 10 nM appeared to have an additive
effect with that of TGF-/3 in downregulating HLA-DR
antigens.
DISCUSSION
Many pathologic processes in the eye are influenced
by cytokines and growth factors produced by immune
960
FIGURE 2. FACS analysis for HLA-DR of RPE cells incubated
with 50 U/ml IFN-7 and TGF-/5, (50 ng/ml, 100 ng/ml)
(a) Negative control cells without stimulation. A similar profile was obtained using an irrelevant (anti-CD4) isotypic antibody control for HLA-DR on unstimulated and IFN-y-stimulated cells, (b) Positive control cells incubated with IFN-7
for 3 days, (c) Cells incubated with IFN-7 for 3 days and/
or TGF-/3 (50 ng/ml) for 4 days show moderate reduction
in HLA-DR staining from positive control, (d) Cells incubated with IFN-7 for 3 days and then with TGF-/3 (100 ng/
ml) for 48 hours show marked reduction in HLA-DR staining. FACS = fluorescence activated cell sorting; RPE = retinal pigment epithelial; IFN = interferon; TGF = transforming growth factor; HLA-DR = human leukocyte antigen-DR.
TCF*
IFN-y
IFN
-Y
3. The percentage of HLA-DR positive cells identified immunohistochemically under different conditions of
IFN-7 stimulation and PKC inhibition. HLA-DR = human
leukocyte antigen-DR; IFN = interferon; PKC = protein kinase C.
FIGURE
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4257
Effect of TGF-/3 on HLA-DR Expression in RPE Cells
TABLE 2.
Percentage of HLA-DR-Positive Cells After Pretreatment for 1 Hour With
Staurosporine or Calphostin C, Followed by Incubation With IFN-y or TGF-P for 3 Days
Staurosporine
+ IFN-y
Calphostin C
+ IFN-y
+ TGF-pl
Calphostin C
+ IFN-y
+ TGF-pl
92.6 ± 3.6*
75.7 ± 27f
24.7 ± 6.11
49.1 ± 7.4J
55.7 ± 7.3§
65 ± 8.8§
24.5 ± 6.86||
13.9 ± 5.3||
32.1 ± 6.91
4.3 ± 1.5H
5.2 ± 2.5H
11.5 ± 3.5||
Staurospine
Dosage
(nM)
0
1
10
100
IFN^y
(control)
IFN^y + TGF-P
(control)
91 ± 2.9
18 ± 4.0
The percentage of HLA-DR positive cells following pretreatment with calphostin C and/or staurosporine and then stimulated with IFNy (50 U/ml) was compared with the percentage HLA-DR positive cells following incubation with IFN-y alone. The data obtained from
cells pretreated with the above mentioned drugs and combined treatment with IFN-y and TGF-/3, (50 ng/ml) was compared with the
percentage of positive cells following simultaneous treatment with IFN-y and TGF-/3, but without pretreatment (chi-square test).
* P = 0.87; t P < 0.009; %P< 0.0001; § P < 0.0006; || P a 0.1; H P < 0.02.
effector and inflammatory cells. Recently, it was proposed that RPE cells may act as immune competent
cells by expressing MHC class II antigens, 116 by their
ability to present antigens to T cells,6 and by synthesizing various immunomodulating factors such as IL-6,3
M-CSF,17 TGF-/?lf and TGF-&. 18 ' 19 The crucial role of
class II antigen expression in inflammatory eye disease
is emphasized by studies showing that administration
of anti-la monoclonal antibodies to rats altered significantly the course of experimental autoimmune
uveitis, resulting in less ocular inflammation, diminution or elimination of local la expression, and a delay
in onset of autoimmune uveitis.20 Recent evidence suggests that IFN-y may be important in regulation of
the ocular immune microenvironment. 5 ' 8 IFN-y modulates MHC class II antigens on RPE cells in vitro6 and
on ocular cells in vivo21 and is present within the eye
during certain inflammatory conditions. 8'22 Our data
are consistent with those of others 5 in that IFN-y treatment of human RPE cells for 3 days induced HLA-DR
expression in a dose-dependent manner. A 24-hour
incubation of RPE cells with IFN-y resulted in weak
staining of occasional cells, but the effect after a 72hour incubation was strong. This suggests that HLADR expression is a long-term event that is regulated
at the transcriptional level.23 The effects of IFN-y cannot be considered in isolation because in vivo IFN-y
is present along with other cytokines and growth factors that can either augment or block its effect. For
example, the vitreous of patients with proliferative vitreoretinopathy contains increased amounts of TGF/?2 along with IFN-y.8 Our results demonstrate that
both TGF-/3, and TGF-/?2 are able to downregulate
IFN-y-induced HLA-DR expression on RPE cells in a
dose-dependent manner; however, TGF-/?2 was more
potent than was TGF-/?!. It has been suggested that
RPE cells are the most likely ocular cells to synthesize
TGF-/?! and TGF-/52, and they also possess binding
proteins for both TGF-/5] and TGF-/?2,18 suggesting
that RPE cells can also be regulated by exogenous
TGF-/?. In fact, RPE cell effects, such as RPE-mediated
gel contraction or proliferation, may be modulated by
incubation with TGF-/?.24'25
There are indications that, in other cell types, IFNy may induce MHC class II antigens through activation of the PKC pathway,23 whereas TGF-/3 may be
acting, in part, by PKC inhibition. 26 It has also been
proposed that transcriptional regulation of HLA-DR
antigen is modulated by PKC.27 Attempts to induce
class II expression on RPE cells by drug modulation
of PKC expression in the absence of IFN-y failed when
the protocols used were those that had induced HLADR on other cell types.14
Treatment of RPE cells with PMA and the relatively specific PKC inhibitor calphostin C before stimulation with IFN-y reduced significantly the number of
HLA-DR-positive cells and was additive with the effect
of TGF-/3] and TGF-/32. Staurosporine's effect was not
additive with TGF-/?, most likely because staurosporine
is not specific for PKC, but it can influence other
signal transduction pathways, such as cAMP,28 which
may also modulate PKC activity.29'30 PMA can act as
either an inhibitor or an activator of the PKC cascade,
depending on its dose, incubation period, and target
cells.31 The minimal dose of phorbol ester that can
cause downregulation of PKC varies from cell type to
cell type and has not been studied in RPE cells. In
this study, PMA (0.1 fjM, 1 fjM) probably inhibited the
PKC pathway in IFN-y stimulated RPE cells because
its effect was similar to that of calphostin C and staurosporine. We suggest that the TGF-/? effect in this study
may be due to involvement of the PKC pathway, but
we cannot rule out that TGF-/3 may act by a parallel
mechanism synergistic with the effect of the PKC pathway. TGF-P is known to inhibit certain PKC-mediated
events, such as PKC-mediated DNA synthesis in some
cells.30 It is also known that phorbol esters regulate
TGF-/0 gene expression in appropriate target cells32
and that they modulate some of the actions of TGF^
33-36
I f
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Investigative Ophthalmology 8c Visual Science, December 1994, Vol. 35, No. 13
the possible mechanisms of the additive effect of PMA
and TGF-P in downregulation of HLA-DR expression.
Although it has been demonstrated that TGF-/? stimulates phosphoinositol metabolism and translocation of
PKC from cytosol to membrane in different cells,36 it
has been reported that translocation of PKC does not
necessarily indicate activation.35 A major function of
PKC is related to immediate negative feedback control.31 TGF-/3 may prolong the activated state of PKC
and induce production of endogenous TGF-/3.37 PMA
could prolong the TGF-/? effect by upregulating either
endogenous TGF-/? or independently inhibiting PKC,
resulting in an additive effect with TGF-/3 in downregulation of HLA-DR antigens.38 There are at least seven
isoforms of PKC, each of which has its own specificity.3940 It is not well known, though, how IFN-y, TGFP, or HLA-DR relates specifically to each isoform. Possible specificity of each cytokine for specific isozymes
might explain this complex interaction. These reports
show that TGF-/3 may have differing actions, depending on its concentration and type of target cells,31
and may act differently in vivo than in vitro.
In summary, our results show that TGF-/5i and
TGF-/?2 act to inhibit IFN-y-induced upregulation of
HLA-DR expression by human RPE cells. The complex interaction between TGF-/3 and IFN-y may directly involve the PKC cascade, although it is not clear
whether the TGF-/3 effect was due solely to inhibition
of this pathway or due, in part, to additional mechanisms. Detailed studies of the role of PKC and its different isoforms in RPE cells, the relationship with
their target proteins, and the alteration by different
immunomodulating and inflammatory cytokines may
be of vital importance in understanding the biochemical basis and potential therapy of inflammatory ocular
disease.
Key Words
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
histocompatibility complex, cytokine, protein kinase C,
PMA, calphostin C
13.
Acknowledgments
The authors thank Dr. Thomas E. Ogden and Ann Dawson
for their editorial assistance; Verna M. Hailey for her secretarial assistance; and Dr. Rayudu Gopalakrishna for his expert advice and comments.
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