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From the Department of Ophthalmology,*
W. K.
Kellogg Eye Center, and the Department of
Pathology,
University of Michigan, Ann
Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) antibody, but not by anti-IL-1ß
antibody. TNF- was detected in cell lysates of monocytes detached
from HRPE cells after co-culture stimulation with thrombin. HRPE cells
mainly produced these chemokines. However, thrombin generally
potentiated exogenous IL-1ß- and TNF--induced chemokine production
by HRPE cells, monocytes, and co-cultures.
Interferon-
potentiated chemokine secretion by co-cultures with or
without thrombin. Our results indicate that thrombin may cause
leukocyte recruitment by inducing HRPE cell and monocyte chemokine and
by enhancing HRPE cell/monocyte interactions, in part because
of monocyte TNF- induction, suggesting important mechanisms
for ocular inflammation during blood-retina barrier breakdown and
intra-ocular hemorrhage.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thrombin has numerous activities. In its hemostatic roles, it converts fibrinogen to fibrin, and activates platelet adhesion, aggregation, and secretion.9 Thrombin also elicits mitogenic responses in retinal pigment epithelial cells, retinal glial cells, vascular endothelial cells, vascular smooth muscle cells, and fibroblasts.7,10,11 Thrombin has been tried as a therapeutic agent during intra-ocular surgery for diabetic retinopathy and ocular trauma to control bleeding and for macular holes as a mitogen of retinal cells, frequently resulting in severe ocular inflammation.12,13 Therefore, it seems that thrombin may be a key factor in the clotting cascade that can also induce significant inflammation.
Interleukin (IL)-8, which is chemotactic for neutrophils and eosinophils,14 and monocyte chemoattractant protein-1 (MCP-1), which directly attracts monocytes15 and lymphocytes,16 are the two chemokines responsible for the majority of HRPE-derived leukocyte chemotactic activity.17-19 We reported that IL-8 and MCP-1 are present in eyes from patients with proliferative vitreoretinopathy and proliferative diabetic retinopathy20,21 and it has been shown that MCP-1 was detected in HRPE cells and monocytes in the eyes with age-related macular degeneration.22 In addition, we found that direct interactions between HRPE cells and monocytes could result in chemokine induction.23 Because monocytes are intimately associated with HRPE cells in the epiretinal membranes of proliferative vitreoretinopathy and in the subretinal neovascular membranes of age-related macular degeneration,24,25 HRPE cell/monocyte interactions could induce further leukocyte accumulation and lead to the progression of these diseases by enhancing expression of IL-8 and MCP-1. Previous reports demonstrated that thrombin induces monocyte IL-8 and MCP-1,26,27 which indicates that thrombin might play a role in inflammatory process by inducing chemokines. However, the effects of thrombin on chemokine production by ocular cells are unknown.
In this study we examined the effects of thrombin on chemokine
secretion by HRPE cells and HRPE cell/monocyte co-cultures. In
addition, we tested synergy between thrombin and the pro-inflammatory
cytokines, IL-1ß and tumor necrosis factor- (TNF-), on
chemokine production, and the effects of interferon (IFN)- on
chemokine secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Falcon Primaria flasks were purchased from Becton-Dickinson Inc.
(Lincoln Park, NJ). Transwell chambers were purchased from Costar
(Kennebunkport, ME). Human thrombin was purchased from ICN
Pharmaceuticals, Inc. (Costa Mesa, CA). Recombinant human (rh) IL-8,
rhMCP-1, rhIL-6, rhIL-1ß, rhTNF-, rhIFN-, monoclonal anti-IL-8
antibody (Ab), monoclonal anti-MCP-1 Ab, monoclonal anti-IL-6 Ab,
monoclonal anti-IL-1ß Ab, monoclonal anti-TNF- Ab, biotinylated
anti-IL-8 Ab, biotinylated anti-MCP-1 Ab, biotinylated anti-IL-6 Ab,
and biotinylated anti-TNF- Ab were purchased from R & D Systems
(Minneapolis, MN). Dulbecco’s modified essential medium,
Ca2+, Mg2+-free
phosphate-buffered saline (PBS), fetal bovine serum, Leech recombinant
hirudin produced by cDNA expressed in Saccharomyces
cerevisiae, Triton X-100, sodium chloride, glycerol, magnesium
chloride, ethylenediaminetetraacetic acid, sodium orthovanadate, sodium
pyrophosphate, 4-(2-aminoethyl)-benzenesulfonyl fluoride, sodium
fluoride, and aprotinin were purchased from Sigma Chemical Co. (St.
Louis, MO), and leupeptin was purchased from Roche Molecular
Biochemicals (Indianapolis, IN). Penicillin G, streptomycin sulfate,
amphotericin B, and Moloney murine leukemia virus reverse transcriptase
were purchased from Life Technologies, Inc. (Gaithersburg, MD), and
Ampli Taq DNA polymerase was purchased from Perkin-Elmer
(Norwalk, CT). Ficoll-Paque Plus was purchased from Amersham Pharmacia
Biotechnologies (Uppsala, Sweden), and Fico-Lite monocytes was
purchased from Atlanta Biologics (Atlanta, GA). Diff-Quick was
purchased from Baxter (McGaw, IL), and the limulus amoebocyte lysate
assay was purchased from BioWhittaker (Walkersville, MD).
HRPE Cell Culture
HRPE cells were isolated from donors who had healthy eyes within 24 hours of death as previously described, in accordance with the Helsinki agreement.17 In brief, the sensory retina was separated gently from the HRPE monolayer, and the HRPE cells were removed from Bruch’s membrane using a 1 hour incubation with papain (5 µg/ml). Isolated HRPE cells were seeded into Falcon Primaria flasks in Dulbecco’s modified essential medium containing 15% fetal bovine serum, penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml), and amphotericin B (0.25 µg/ml). The HRPE monolayers exhibited uniform immunohistochemical staining for fibronectin, laminin, and type IV collagen in a chicken wire distribution, characteristic for these epithelial cells. Cells, grown in culture up to six passages, were used for all experiments.
Monocyte Culture
Human monocytes were isolated as previously described with modification, in accordance with the Helsinki agreement.23,28,29 Peripheral blood was drawn into a heparinized syringe from healthy volunteers, diluted 1:1 in normal saline and mononuclear cells separated by density gradient centrifugation (Ficoll-Paque Plus). The cells were washed and then layered onto density gradient (1.068 g/ml) for the enrichment of monocytes (Fico-Lite monocytes). The isolated cells were then washed, cytospun onto a glass slide, stained with Diff-Quick, and differentially counted.
Cell Stimulation
Before experiments, HRPE cells were incubated in serum-free medium
for 24 hours. HRPE cells and monocytes were incubated in control
medium, Dulbecco’s modified essential medium, or in the same medium
also containing thrombin. They were also incubated in the medium
containing rhIL-1ß (0.2 ng/ml), rhTNF- (2 ng/ml), or rhIFN-
(1000 U/ml), either alone or in combination with thrombin. For
enzyme-linked immunosorbent assay (ELISA) of supernatants of HRPE
cell/monocyte co-cultures, enriched monocyte populations (4 x
105/well) were layered onto HRPE monolayers
(2 x 105/well) grown to confluency in a
total of 400 µl of media in 12-well plates. In experiments in which
monoclonal antibodies directed against IL-1ß and TNF- were used,
they were incubated with HRPE cells and monocytes for 1 hour before
addition of monocytes and maintained during stimulation. To detect
whether cell contact was obligatory for chemokine production, HRPE
cells and monocytes were co-incubated in the same cultures, but
separated by porous polycarbonate filters. After experimental
incubations, culture media were collected and then centrifuged to
remove particulates. Cold Ca2+,
Mg2+-free PBS was used to separate monocytes from
HRPE cells as previously described.28
The isolated cells
were cytospun onto a glass slide, stained with Diff-Quick, and counted.
The purity of the cells was >97%. To detect cell-associated
cytokines, HRPE cells and monocytes in 60-mm culture dishes were lysed
with 150 µl of lysing buffer containing 50 mmol/L Hepes (pH 7.4), 1%
Triton X-100, 0.15 mol/L sodium chloride, 10% glycerol, 1.5 mmol/L
magnesium chloride, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L
sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 1 mmol/L
4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 mmol/L sodium fluoride,
10 µg/ml aprotinin, and 10 µg/ml leupeptin. Lysates were then
incubated on ice for 15 minutes with shaking. Then, the extracts were
centrifuged at 14,000 rpm for 10 minutes at 4°C. Supernatants were
decanted into new tubes. Culture media and cell extracts were stored at
-70°C until ELISA was performed. The ELISA values of chemokines were
normalized so that they may be compared. Cytokines and reagents were
negative for endotoxin contamination as determined by the limulus
amoebocyte lysate assay method (<0.05 EU/ml).
ELISA
ELISA was performed on serial dilutions of HRPE cell, monocyte,
and HRPE cell/monocyte co-culture supernatants and cell extracts.
Antigenic IL-8, MCP-1, IL-6, and TNF- were quantitated using a
double-ligand ELISA method as described previously.30
Standards included 0.5 log dilutions of rhIL-8, rhMCP-1, rhIL-6, or
rhTNF- from 5 pg to 100 ng/well. This ELISA method detected MCP-1,
IL-6, and TNF- concentrations of >15 pg/ml and IL-8 concentrations
of >30 pg/ml.
Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Synthetic oligonucleotide primers based on the cDNA sequences of human IL-8, MCP-1, and ß-actin were prepared: IL-8, 5'-AAGCTGGCCGTGGCTCTCTTG-3' and 5'-AGCCCTCTTCAAAAACTTCTC-3'; MCP-1, 5'-GCTCATAGCAGCCACCTTCATTC-3' and 5'-GTCTTCGGAGTTTGGGTTTGC-3'; and ß -actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGA TTTC-3'. PCR was performed in a semiquantitative manner, essentially as previously described.21 First-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase. One µg RNA was denatured at 65°C for 10 minutes and added to the reverse transcription mixture, as indicated by the manufacturer. After incubation at 37°C for 1 hour, cDNA from the reverse-transcription mixture was then subjected to PCR in a 10-µl volume containing 5 pmol of the primer pair and 0.5 U of Ampli Taq DNA polymerase using a DNA thermal cycler. Linearity range of the reaction was determined running 15 to 35 cycles. DNA was denatured for 5 minutes at 94°C, followed by 28, 26, and 20 PCR cycles for IL-8, MCP-1, and ß-actin, respectively. Each cycle included a 1-minute denaturation at 94°C, a 1-minute primer annealing at 65°C, and a 2-minute polymerization at 72°C. Each RT-PCR reaction mixture was analyzed by electrophoresis on a 2% agarose gel and stained with ethidium bromide. The intensity of the ethidium bromide luminescence was measured by an image sensor with a computer-controlled display.
Statistical Analysis
Individual experiments were performed in triplicate three times on three different HRPE cell lines and monocytes isolated from the blood of three different donors on separate days. Each cell line displayed similar fold-increases or decreases over control levels. Data are expressed as mean ± SEM. Various assay conditions were evaluated using analysis of variance test with a post hoc analysis (Scheff multiple comparison test); P values <0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of thrombin on HRPE chemokine secretion were
investigated. Thrombin caused concentration-dependent IL-8 and MCP-1
release from HRPE cells reaching statistical significance at 1 U/ml
(Figure 1, A
and B). Hirudin (20 U/ml), a potent thrombin
antagonist, binds to the anion-binding exosite and the enzymatic
cleavage pocket of thrombin and prevents thrombin from binding and
cleaving its receptor. This antagonist nearly completely blocked the
effects of thrombin on chemokine induction. Higher doses of hirudin (50
and 100 U/ml) did not show more pronounced effects (data not shown).
Thrombin-induced IL-8 and MCP-1 secretion was time-dependent,
increasing for 24 hours (Figure 1, C and D)
. IL-8 and MCP-1 mRNA levels
were increased in thrombin-activated HRPE cells after 1 hour of
stimulation, reaching a maximum after 4 hours (Figure 2, A and B)
.
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We studied the effects of thrombin on chemokine secretion
because of HRPE cell/monocyte interaction. The direct overlay of
monocytes onto HRPE cell cultures consistently resulted in increased
IL-8 and MCP-1 production as we found previously (Figure 3, A and B)
.23
When added to
HRPE cell/monocyte co-cultures, 10 U/ml of thrombin enhanced both IL-8
and MCP-1 production by 2.4-fold and 1.5-fold, respectively (Figure 3, A and B)
. Thrombin-induced chemokines were inhibited by hirudin (Figure 3, A and B)
. Although thrombin also induced elevated monocyte IL-8 and
MCP-1 secretion, the synergistic increases in chemokine secretion by
co-cultures exposed to thrombin were more than additive of the effects
on HRPE cells and monocytes alone. Thrombin-induced IL-8 and MCP-1
secretion was time-dependent (Figure 3, C and D)
.
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. Thrombin enhanced IL-6 secretion by
HRPE cells and HRPE cell/monocyte co-cultures, whereas it had no effect
on monocyte IL-6 secretion (Figure 4)
.
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Thrombin induced significant increases in cell-associated IL-8 and
MCP-1 in cultured HRPE cells (Figure 5, a
versus b and i versus j) and IL-8 in isolated
monocytes (Figure 5A, c
versus d). To detect the cells
responsible for chemokine production during exposure of HRPE
cell/monocyte co-cultures to thrombin, monocytes were separated from
HRPE cells after co-incubation and HRPE cell and monocyte
cell-associated chemokines were measured separately. After 4 hours of
co-culture, cell-associated IL-8 and MCP-1 was induced in HRPE cells
(Figure 5, a
versus e and i versus m). More
cell-associated IL-8 and MCP-1 was detected in HRPE cells in
co-cultures than in monocytes (Figure 5
; e versus g,
2.2-fold; and m versus o, 15.4-fold). Thrombin potentiated
cell-associated IL-8 and MCP-1 in HRPE cells and IL-8 in monocytes
after co-culture (Figure 5, e
versus f, m versus
n, and g versus h). Thrombin did not significantly induce
monocyte MCP-1 (Figure 5B, k
versus l) or significantly
potentiate monocyte MCP-1 after culture with HRPE cells (Figure 5B, o
versus p). HRPE cells also showed substantially more
cell-associated IL-8 and MCP-1 than monocytes when co-cultures were
stimulated with thrombin (Figure 5
; f versus h, 2.5-fold; n
versus p, 14.9-fold).
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We examined whether cell contact was obligatory for IL-8, MCP-1,
and IL-6 production. When HRPE cells were co-incubated with
monocytes in the same cultures, but separated by porous polycarbonate
filters, significantly less (P < 0.01) IL-8,
MCP-1, and IL-6 were secreted into the supernatants (Figure 6, a
versus c, e
versus g, and i versus k). Thrombin induced
significantly less (P < 0.01) IL-8, MCP-1, and
IL-6 secretion into the culture supernatants from HRPE cell/monocyte
co-cultures when the cells were separated by the filters than when
monocytes were directly overlayed onto the HRPE monolayers (Figure 6, b
versus d, f versus h, and j versus l).
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, but Not Anti-IL-1ß Ab, Inhibits Thrombin-Induced
IL-8 and MCP-1 Secretion by HRPE Cell/Monocyte Co-Cultures
We examined the effect of neutralizing antibodies against IL-1ß
and TNF- on IL-8 and MCP-1 production. Neither anti-IL-1ß nor
anti-TNF- Ab had significant effects on IL-8 and MCP-1 secreted by
HRPE cell/monocyte co-cultures (Figure 7, A and B)
. In contrast, anti-TNF- Ab, but not anti-IL-1ß Ab,
inhibited IL-8 and MCP-1 secretion by HRPE cell/monocyte co-cultures
exposed to thrombin by 41 and 21%, respectively (Figure 7, A and B)
.
Thrombin-induced IL-8 and MCP-1 secretion by cultured HRPE cells or
isolated monocytes alone was not inhibited by either anti-TNF- Ab or
anti-IL-1ß Ab (data not shown)
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Synthesis in Monocytes during HRPE
Cell/Monocyte Co-Cultures
Because anti-TNF- Ab inhibited thrombin-induced chemokine
secretion by HRPE cell/monocyte co-cultures, we tested whether thrombin
could induce TNF- synthesis by co-cultures. Although TNF- was not
detected in supernatants of HRPE cell/monocyte co-cultures stimulated
with thrombin, thrombin did induce substantial levels of
cell-associated TNF- in monocytes after co-culture with HRPE cells
(P < 0.01) (Table 1)
. Only low levels
(P > 0.05) of TNF- were detected in
monocytes co-cultured with HRPE cells in the absence of thrombin. No
detectable TNF- levels were observed in HRPE cells under any of the
experimental conditions and in isolated monocytes with or without
thrombin stimulation.
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-Induced IL-8, MCP-1,
and IL-6 Secretion
Since we previously reported that pro-inflammatory cytokine
(rhIL-1ß and rhTNF-) potentiated IL-8 and MCP-1 secretion by HRPE
cells, monocytes, and HRPE cell/monocyte co-cultures,23
the effects of thrombin on these pro-inflammatory cytokine-induced
chemokine secretion were examined. Co-incubation of cultured HRPE cells
and isolated monocytes with IL-1ß and thrombin potentiated
IL-1ß-induced IL-8 secretion by 1.6-fold and 1.7-fold, respectively,
at the maximum concentration of thrombin (10 U/ml) (Figure 8A)
. However, thrombin substantially
synergized IL-1ß-induced IL-8 secretion by HRPE cell/monocyte
co-cultures 3.8-fold (Figure 8A)
. Thrombin had no effects on
IL-1ß-induced HRPE MCP-1 secretion, but caused small, but significant
increases in MCP-1 production by isolated monocytes exposed to IL-1ß
(Figure 8B)
. As in the case of IL-8, thrombin significantly potentiated
MCP-1 secretion by HRPE cell/monocyte co-cultures exposed to IL-1ß by
1.7-fold (Figure 8B)
.
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-induced IL-8 secretion
by HRPE cells, monocytes, and HRPE cell/monocyte co-cultures at maximum
thrombin concentrations (10 U/ml) (Figure 8C)
. Co-incubation of TNF-
and thrombin potentiated TNF--induced MCP-1 secretion by cultured
HRPE cells and HRPE: monocyte co-cultures whereas it had no effects on
MCP-1 production by isolated monocytes (Figure 8D)
.
We also examined the effects of thrombin on pro-inflammatory cytokine
stimulated IL-6 secretion. Thrombin enhanced IL-1ß- and
TNF--induced IL-6 secretion by HRPE cells and HRPE cell/monocyte
co-cultures and IL-1ß-induced IL-6 secretion by monocytes (Figure 8E)
. Co-incubation of TNF- and thrombin induced monocyte IL-6
secretion whereas TNF- or thrombin alone had no effects on monocyte
IL-6 secretion.
Recombinant hIFN- Modulates IL-8, MCP-1, and IL-6 Secretion
We examined the effects of IFN- on IL-8, MCP-1, and IL-6
secretion by HRPE cells, monocytes, and HRPE cell/monocyte co-cultures.
IFN- had no effects on HRPE IL-8 and IL-6, and monocyte IL-6, and
inhibited monocyte IL-8 whereas IFN- enhanced HRPE and monocyte
MCP-1 (Figure 9)
. IFN-
potentiated thrombin-induced HRPE IL-8, MCP-1, and IL-6, and monocyte
MCP-1, but had no effects on thrombin-stimulated monocyte IL-8 and
IL-6. IFN- potentiated IL-8, MCP-1, and IL-6 secretion by
co-cultures both in the absence and presence of thrombin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we demonstrated that thrombin induced elevated HRPE IL-8 and MCP-1 mRNA and increased corresponding chemokine secretion in a dose- and time-dependent manner. These stimulatory effects of thrombin were significantly inhibited by the specific thrombin inhibitor, hirudin. We have found that cell to cell interaction of HRPE cells and monocytes induces IL-8 and MCP-1 production.23 In this study, we also demonstrated that thrombin enhanced chemokine secretion by HRPE cell/monocyte co-cultures. This effect was also inhibited by hirudin. Thrombin has been shown to be directly chemotactic for monocytes and polymorphonuclear cells.33,34 Our findings indicate that thrombin has also indirect chemotactic activity for monocytes and polymorphonuclear by inducing MCP-1 and IL-8 by HRPE cells, monocytes, and interacting HRPE cells and monocytes, revealing an important mechanism in the positive amplification of leukocyte recruitment. Thrombin effects on IL-6 secretion were generally similar to those on chemokines, which suggests that thrombin might also be involved in lymphoid immunomodulation by producing IL-6.35
Thrombin action on IL-8, MCP-1, and IL-6 production seems to be
enhanced by cell-to-cell contact, because co-incubation of HRPE cells
and monocytes in the same cultures with thrombin, but separated by
porous polycarbonate filters, did not induce the secretion of the high
levels of these chemokines measured after direct overlay of human
monocytes onto HRPE cells in the presence of thrombin. Therefore,
HRPE-monocyte adhesion and/or ambient mediators subsequently produced
seem to be involved in the chemokine production by HRPE cell/monocyte
co-cultures stimulated by thrombin. Because we have found that both
IL-8 and MCP-1 can be induced by stimulation of HRPE cells, monocytes,
and HRPE cell/monocyte co-cultures with the proinflammatory cytokines,
IL-1ß or TNF-,23
we examined whether thrombin
stimulation of chemokine production was mediated by these cytokines.
Thrombin-induced secretion of IL-8 and MCP-1 by HRPE cell/monocyte
co-cultures were inhibited by anti-TNF- Ab, but not by anti-IL-1ß
Ab. Neither of these neutralizing antibodies had significant effects on
thrombin-induced chemokine secretion by cultured HRPE cells or isolated
monocytes alone. Thrombin did not induce the release of measurable
levels of TNF- in supernatants of cultured HRPE cells, isolated
monocytes, or HRPE cell/monocyte co-cultures. However, TNF- was
detected in cell lysates of monocytes detached from HRPE cells after
the co-cultures were stimulated with thrombin, but not in lysates of
isolated monocytes or cultured HRPE cells exposed to thrombin
independently. Because HRPE cells and monocytes were closely associated
in our culture system, cell-associated TNF- in monocytes could have
effects on HRPE cells. Our results thus indicate that both thrombin
treatment and interaction with HRPE cells might be required to induce
monocyte TNF-, and that the cell-associated TNF- could, at least
in part, play an important role in thrombin-induced HRPE-monocyte
chemokine production. Our findings of cell-associated chemokines
suggest that HRPE cells in co-cultures produce more IL-8 and MCP-1 than
monocytes do. However, monocytes are likely to be responsible for HRPE
cell activation during HRPE cell/monocyte interactions in the presence
of thrombin. HRPE IL-8 and MCP-1 seem to be efficiently secreted as we
previously reported,19
because the levels of these
chemokines in culture supernatants are much higher than those in cell
lysates. Recent studies have shown that the direct adhesion of cells
may be regulated by various adhesion molecules such as ICAM-1, VCAM-1,
and integrin family receptors on the surfaces of interacting
cells.36-38
These reports, taken together with our
findings, suggest that cell-to-cell interaction through adhesion
molecules might induce chemokine production and work together with
thrombin to induce TNF-, which results in further chemokine
production because of paracrine effects.
Pro-inflammatory cytokines such as IL-1ß and TNF- are considered
to be important in the pathophysiology of the inflammatory component of
numerous ocular disorders. They have been identified on epiretinal
membranes removed surgically from patients with proliferative
vitreoretinopathy and on epiretinal membranes produced
experimentally.39,40
In this study, thrombin generally
potentiated exogenous IL-1ß- and TNF--induced IL-8, MCP-1, and
IL-6 production by HRPE cells, monocytes, and HRPE cell/monocyte
co-cultures. Synergistic effects of thrombin and IL-1ß on IL-8
production by HRPE cell/monocyte co-cultures were particularly
prominent. The mechanism of this synergism has not yet been defined.
However, Loppnow and colleagues31
has hypothesized that
thrombin might regulate IL-1 receptor expression or IL-1 might increase
thrombin receptor expression. Accordingly, some cytokine enhancement of
thrombin receptor expression and thrombin induction of cytokine
receptors have been published.41,42
In addition, in
vascular endothelial cells thrombin potentiates TNF--induced nuclear
factor-
B activity that is known to participate in the induction of
IL-8, MCP-1, and IL-6 gene transcription.43
Therefore,
thrombin and cytokines may affect each others receptor expression
and/or signaling pathways. The capacity of thrombin to potentiate
cytokine-induced chemokine production is likely to be biologically
important because it is likely that HRPE cells and monocytes in
diseased retinal tissue are exposed to both inflammatory cytokines and
thrombin, especially at sites of hemorrhage and breakdown of
blood-retina barrier in the eyes.
IFN- potentiated IL-8, MCP-1, and IL-6 secretion by HRPE
cell/monocyte co-cultures in the absence and presence of thrombin, but
it had different effects on the secretion of these cytokines by HRPE
cells and monocytes alone. IFN- has been shown to have
immunomodulatory effects on HRPE cells and monocytes, including
modulation of expression of cell surface molecules and
cytokines,44,45
which might enhance interaction between
HRPE cells and monocytes.
The basal surfaces of HRPE cells are adjacent to the choroid which contains circulating leukocytes. HRPE cells exhibit polarized secretion of chemokines toward the basal side, suggesting that HRPE-derived chemokines may recruit leukocytes efficiently.46,47 Our results show that once monocytes are recruited and contact with HRPE cells, IL-8 and MCP-1 secretion are enhanced, and further potentiated by thrombin. Therefore, the HRPE chemokine increases induced by thrombin are likely to have pathophysiologically significant effects.
Our findings suggest that chemokine induction by thrombin may exacerbate and perpetuate mechanisms of the blinding retinal diseases. This study may provide a novel molecular mechanism linking thrombosis and inflammation in the eye.
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Footnotes |
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Supported by the National Institutes of Health (grants EY09441, EY007003), and Research to Prevent Blindness–Olga Keith Weiss Award (to V. M. E.).
Accepted for publication June 11, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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