Division of Molecular and Cellular Medicine, Departments of Medicine and Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human orbital fibroblasts from patients with severe
thyroid-associated ophthalmopathy are particularly susceptible to the actions of a variety of proinflammatory molecules. In this study, we
demonstrate that the inductions of prostaglandin endoperoxide H
synthase-2 (PGHS-2), interleukin (IL)-1, and IL-1
by
leukoregulin, a product of activated T lymphocytes, are far more robust
in orbital fibroblasts than those observed in dermal fibroblasts. These
actions of leukoregulin are mediated through an intermediate induction of IL-1
. In contrast, leukoregulin also induces IL-1-receptor antagonist (IL-1ra) expression in orbital fibroblasts, but this induction is considerably greater in dermal fibroblasts (2.3- vs.
8.5-fold). Interrupting the effects of IL-1
, either with a
neutralizing antibody or with exogenous IL-1ra, can block the induction
of PGHS-2 by leukoregulin. Leukoregulin increases PGHS-2 gene
transcription in orbital fibroblasts but exerts the major effect on
cyclooxygenase expression by enhancing the stability of mature PGHS-2
mRNA. The cytokine triggers nuclear translocation of nuclear
factor-
B (NF-
B) p50/p50 homodimers and p50/p65 heterodimers, and
an inhibitor of this transcriptional factor,
pyrrolidinedithiocarbamate, can attenuate the PGHS-2 induction. Thus
differential inducibility of the members of the IL-1 family of genes in
orbital fibroblasts would appear to underlie, at least in part, the
differences in PGHS-2 induction observed in orbital and dermal
fibroblasts. NF-
B plays an important role in mediating the effects
of leukoregulin on PGHS-2 expression.
cyclooxygenase; inflammation; ophthalmopathy; Graves' disease
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HUMAN FIBROBLASTS represent a diverse population of
cells that vary with regard to the anatomic region from which they are derived (47). Orbital fibroblasts, especially those derived from
patients with severe thyroid-associated ophthalmopathy (TAO), exhibit
exaggerated responses to several proinflammatory cytokines (29). We
hypothesize that this particular susceptibility to cytokine action may
underlie the often intense inflammatory response and the dramatic
tissue remodeling observed in TAO (32). In that disease, there occurs a
disordered accumulation of the nonsulfated glycosaminoglycan hyaluronan
(32). We have reported that leukoregulin, a 50-kDa product of activated
T lymphocytes (9), and interferon- can dramatically upregulate the
synthesis of hyaluronan in orbital fibroblast cultures (34, 39). The
magnitude of increase with leukoregulin, up to 15-fold above the levels
observed in untreated cultures, is unprecedented and is severalfold
greater than that observed in dermal fibroblasts (39). Other cellular
responses also appear to be more robust in orbital fibroblasts than in
those from extra-orbital anatomic regions. Leukoregulin (16),
transforming growth factor-
(4), and interferon-
(31, 35) can
induce the expression of the serine protease inhibitor, plasminogen
activator inhibitor type 1 (PAI-1), in orbital fibroblasts, and these
effects are considerably greater than those in dermal fibroblasts.
Because PAI-1 modulates the pericellular proteolytic environment, the differential inducibility of the protein suggests that the
extracellular matrix economy of orbital fibroblasts differs from that
of its dermal counterparts.
Another response that is far more robust in orbital fibroblasts is the induction of the inflammatory cyclooxygenase prostaglandin endoperoxide H synthase-2 (PGHS-2; EC 1.14.99.1) by leukoregulin (21, 42). PGHS are a family of two bifunctional proteins that catalyze the conversion of arachidonate to PGH2 through a pair of discrete biochemical reactions involving different active sites on the enzymes (30, 41). PGHS-1 is a constitutive protein that appears to be responsible for the basal prostaglandin formation in most tissues. It is only modestly upregulated by proinflammatory molecules such as phorbol 12-myristate 13-acetate (PMA; see Ref. 45). PGHS-1 migrates as a 68-kDa protein and is encoded by a 5-kb mRNA in some tissues (10, 15) and a 2.8-kb transcript in others (12). In contrast, PGHS-2 is expressed at very low levels in most normal tissues but can be induced substantially by cytokines, growth factors, and serum (1, 18, 20, 22, 44). PGHS-2 mRNA, a 4.8-kb species, and PGHS-2 protein, usually a doublet at 72 kDa (20, 47), are undetectable in most untreated cells, but, when they are exposed to activational molecules, the expression of this cyclooxygenase is dramatically enhanced. The upregulated expression of PGHS-2 is often accompanied by increases in the production of PGE2.
With regard to cultured orbital fibroblasts, PGHS-2 is dramatically induced by leukoregulin, and the effects are mediated at the pretranslational level (42). Moreover, prostanoid generation after treatment with leukoregulin can be attenuated by the potent synthetic glucocorticoid dexamethasone and by SC-58125, a PGHS-2 selective inhibitor (42). The induction of PGHS-2 results in a twofold increase in gene transcription, as assessed by nuclear "run-on" assays, but the increase in steady-state mRNA levels is ~50-fold. Thus the cytokine is acting primarily on some other level(s) to upregulate the cyclooxygenase transcript. PGE2 production in the leukoregulin-activated orbital fibroblast, in turn, influences the morphology of the cells expressing high levels of PGHS-2 (21, 42). There is a change from typical fibroblast-like morphologies to a stellate appearance with multiple cellular processes. This same arborized appearance can be provoked by treating the orbital fibroblasts with exogenous PGE2 and with prostanoids that specifically bind to the EP2 subclass receptors (40, 43). Prostaglandins appear to exert diverse influences on immune responses and therefore condition the interplay between the immune system and other tissues. Thus, in the context of TAO, the orbital fibroblast could play an important immunomodulatory role by virtue of the prostanoids and other small molecules it elaborates and releases.
In the current paper, we report the results of studies designed at
characterizing the induction by leukoregulin of PGHS-2 expression in
orbital fibroblasts. We find that leukoregulin substantially upregulates interleukin (IL)-1 and IL-1
synthesis and elicits a
very modest increase in IL-1-receptor antagonist (IL-1ra) levels in
these cells. The enhanced synthesis of IL-1
represents a critical intermediate step in the induction of PGHS-2. Compared with orbital fibroblasts, those from the dermis exhibit a more modest increase in
IL-1
and -
but a markedly more robust IL-1ra induction. We hypothesize that it is the discrepancy in the inducibility of the IL-1
family of proteins that underlies the exaggerated PGHS-2 induction in
orbital fibroblasts and leads to intense orbital inflammation in TAO.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents. SC-58125 was a kind gift of
Dr. Peter Isakson (Searle, Skokie, IL). Dexamethasone (1,4 pregnadien-9-fluoro-16-methyl-11
,17
,21-triol-3,20-dione), pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear factor-
B (NF-
B; see Ref. 13), and cycloheximide were from Sigma (St. Louis,
MO). Leukoregulin was prepared from peripheral blood leukocytes as
described (9) and was kindly supplied by Dr. Charles H. Evans (NCI,
Bethesda, MD). Briefly, normal lymphocytes were stimulated with
phytohemagglutinin for 48 h. The cytokine was purified by subjecting
cellular material through several chromatographic steps, as described
in Ref. 9. Leukoregulin has been shown to be free from
contamination with other known cytokines such as IL-1
, IL-1
, tumor necrosis factor (TNF)-
, and TNF-
(9). PGHS-1 and PGHS-2 cDNAs were kindly provided by Dr. Donald A. Young (University of
Rochester, Rochester, NY), and IL-1
cDNA was from Dr. J. A. Melendez. Monoclonal anti-PGHS-1 and -2 antibodies were purchased from
Cayman (Ann Arbor, MI), IL-1
and IL-1
enzyme-linked immunosorbent assays (ELISA) were from Immunotech (Westbrook, ME), and the IL-1ra assay and IL-1
and IL-1
neutralizing antibodies were from R & D
Systems (Minneapolis, MN). Recombinant IL-1ra was a gift from Amgen
(Boulder, CO).
Cell culture. Human orbital and dermal fibroblasts were cultivated as described previously (27, 33). Briefly, orbital tissue explants were obtained from surgical waste from individuals with severe TAO who were undergoing decompressive surgery. Dermal fibroblasts were obtained from punch biopsies of normal-appearing skin. These activities were approved by the Institutional Review Board of the Albany Medical College. The explants were allowed to adhere to plastic culture plates, and fibroblasts began to proliferate from these tissue fragments. The fibroblasts were allowed to expand, and monolayers were disrupted with gentle treatment with trypsin/EDTA, replated, and covered with Eagle's medium supplemented with 10% FBS, antibiotics, and glutamine. Cultures were maintained in a 37°C, humidified incubator in a 5% CO2 environment. Medium was changed every 3-4 days, and cultures were used for experiments between the 3rd and 12th passages.
Western blot analysis of proteins. The
relative levels of proteins were assessed by immunoblot analysis using
monoclonal antibodies directed against IL-1, PGHS-1, and PGHS-2, as
described previously (42). Confluent cultures of fibroblasts, usually
in 60-mm-diameter plastic culture plates, were shifted from growth
medium containing 10% FBS to medium supplemented with 1% serum for
1-2 days. They were then treated with the test compounds, added at
the times indicated in the text and in the figures. Monolayers were
washed and harvested in ice-cold buffer containing 15 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM EDTA, 20 mM Tris · HCl (pH 7.5), 10 µg/ml soybean trypsin inhibitor, and 10 µM phenylmethylsulfonyl
fluoride (PMSF). Lysates were taken up in Laemmli buffer and subjected
to SDS-PAGE, and the separated proteins were transferred to
polyvinylidene difluoride membranes. The primary antibodies (10 µg/ml) were incubated with the membranes for 2 h at room temperature
(RT). Membranes were then washed extensively and
reincubated with secondary, peroxidase-labeled antibodies for 2 h.
After another round of washes, the membranes were subjected to the
enhanced chemiluminescence (Amersham) detection system, and the signals
generated on X-Omat film were analyzed with a densitometric BioImage
scanner (Milligen).
RNA extraction and Northern blot analysis of
steady-state mRNA levels. Fibroblasts were cultivated
in 100-mm-diameter plastic plates to a state of confluence in medium
with 10% FBS, and then they were shifted to medium supplemented with
1% serum for 1-2 days. They were then treated with the test
compounds indicated, the monolayers were washed, and the RNA was
extracted by the method described by Chomczynski and Sacchi (6) using
Ultraspec reagent (Biotecx, Houston, TX). Northern analysis was
performed as described previously (42) by electrophoresing RNA on
denaturing 1% agarose-formaldehyde gels. The integrity of the RNA was
verified by routinely determining the 260 to 280 nm ratio and by
staining the electrophoresed samples with ethidium bromide and
inspecting them under ultraviolet light. Samples were
transferred to a Zeta-probe membrane (Bio-Rad), and the immobilized RNA
was allowed to hybridize with
[32P]dCTP-labeled cDNA
probes. With regard to cyclooxygenase probes, PGHS-1 was generated from
a 1.6-kb cDNA, whereas PGHS-2 was synthesized from a 1.4-kb fragment.
Hybridizations were allowed to proceed in a solution containing
5× saline sodium citrate, 50% formamide, 5× Denhardt's
solution, 50 mM phosphate buffer, pH 6.5, 1% SDS, and 0.25 mg/ml
salmon sperm at 48°C overnight. Membranes were washed under
high-stringency conditions, and then radioactive hybrids were
visualized by autoradiography on X-Omat film (Eastman Kodak, Rochester,
NY) exposed at 70°C, and the radioactive bands were scanned
densitometrically. Membranes were stripped of radioactivity according
to the manufacturer's instructions and were rehybridized with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe for
standardization. The density of the PGHS-2 signals was normalized to
that resulting from the hybridization with the GAPDH probe.
Electrophoretic mobility shift assay.
Nuclei from untreated orbital and dermal fibroblasts and those treated
with leukoregulin (1 U/ml) and the other test compounds indicated were
prepared as follows. Two 100-mm-diameter plates of confluent cells were washed and scraped in PBS, and cell layer material was transferred to
1.5-ml microcentrifuge tubes. Cell pellets were suspended in 0.5 ml
buffer A [10 mM HEPES, pH 7.8, 15 mM KCl, 2 mM MgCl2, 0.1 mM
EDTA, 1 mM dithiothreitol (DTT), and 1 mM PMSF]. Samples were centrifuged at 750 g for 5 min. The
supernatant was removed, the pellet was resuspended in 200 µl of
buffer A and incubated at 4°C for
10 min. Nonidet P-40 was added to a final concentration of 0.5%, and
the samples were subjected to centrifugation at 14,000 g for 15 min. The resultant pellet was
suspended in 15 µl of buffer B
[20 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 0.5 mM DTT, 0.42 M NaCl, 0.2 mM EDTA, 25% (vol/vol) glycerol, and 0.5 mM PMSF]. After a 15-min incubation at 4°C, the suspension was centrifuged at 14,000 g for 10 min, and 15 µl of the
supernatant were combined with 35 µl of buffer
C (20 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.5 mM
DTT, and 0.5 mM PMSF). The nuclear pellets were frozen until assay at
80°C.
A double-stranded oligonucleotide conforming to the consensus NF-B
binding site (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and one
with a single mutation (5'-AGT TGA GGC GAC TTT CCC AGG
C-3') were purchased from Santa Cruz. Complementary strands were
synthesized with 5' overhanging ends. They were annealed, and
double-stranded probes were end-labeled with
[32P]dATP using T4
polynucleotide kinase. Aliquots of nuclear extract (2 µg
protein/sample) were preincubated with 2 µl of 5× gel shift binding buffer (Promega, Madison, WI) containing 20% glycerol, 5 mM
MgCl2, 2.5 mM EDTA, 2.5 mM DTT,
250 mM NaCl, 50 mM Tris · HCl, pH 7.5, and 0.25 mg/ml
poly(dI-dC) · poly(dI-dC) in a total reaction volume of 10 µl for 10 min. The reaction was stopped by
adding 1 ml loading buffer (250 mM Tris · HCl, pH
7.5, 0.2% bromphenol blue, 0.2% xylene cyanol, and 20% glycerol),
and the samples were loaded on native 8% polyacrylamide gels and run
at 100 V for 3 h. Gels were dried and exposed to X-Omat film.
Supershift assays were performed by incubating the nuclear extract with
anti-p50 and anti-p65 antibodies (1 µg/ml, final concentration) for
20 min at RT.
Assays for the expression of IL-1,
IL-1
, and IL-1ra. Fibroblasts were
allowed to proliferate to confluence in 24-well culture plates covered
with medium containing 10% FBS. Monolayers were shifted to medium with
1% serum for 1-2 days, and then the test compounds indicated were
added to the medium. After the incubation period, media were removed,
and the cell layers were washed and harvested in a buffer containing 15 mM CHAPS, 1 mM EDTA, 20 mM Tris · HCl, pH 7.5, 10 µg/ml soybean trypsin inhibitor, 10 µM PMSF, 3 µg/ml aprotinin,
and 0.5% Nonidet P-40. Cellular proteins (5-10 µg/sample) were
subjected to specific ELISAs for IL-1
, IL-1
, and IL-1ra according
to the manufacturer's instructions.
PGE2 assay. Fibroblasts were cultivated to confluence in 24-well plastic culture arrays in medium with 10% FBS and then were shifted to medium containing 1% serum for 16- 24 h. The test compounds indicated were added to the medium for the times specified. The final 30 min of the treatment periods were conducted by removing the medium and adding PBS with the respective additives. After this incubation, the PBS samples were collected, centrifuged, and subjected to a specific RIA for PGE2 (Amersham) following the manufacturer's instructions.
Statistics. Data are generally presented as means ± SE of at least three independent replicates. Where appropriate, data were subjected to Student's t-test. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Leukoregulin treatment results in a
dramatic upregulation of PGHS-2 expression in orbital
fibroblasts. Orbital fibroblasts appear to play a
central role in the pathogenesis of TAO by virtue of the array of
biosynthetic activities they exhibit. With regard to cyclooxygenase
expression, we have reported that PGHS-2 expression in these cells
under control culture conditions is at a very low level (42). In
contrast, PGHS-1 protein and mRNA are expressed at easily detectable
levels, and a substantial fraction of basal PGE2 production can be attributed
to the activity of PGHS-1 (42). Treatment of orbital fibroblasts with
leukoregulin (1 U/ml) for 16 h results in a substantial enhancement of
PGE2 production (Fig. 1A).
The magnitude of the increase is 88-fold above control cultures (P < 0.0001). This increase is
considerably greater than that observed in dermal fibroblasts (6-fold,
P < 0.0002 vs. control), and the
levels of PGE2 were considerably
greater than those attained in the dermal fibroblasts
(P < 0.001). The two types of
fibroblasts were treated and cultured under the same conditions. When
these fibroblasts are treated with leukoregulin and the cellular
proteins were subjected to Western blot analysis for cyclooxygenase
protein levels, the cytokine elicits a dramatic upregulation in PGHS-2 protein as the blot in Fig. 1B
demonstrates. The magnitude of the induction is severalfold greater
than that observed in dermal fibroblasts cultured under identical
conditions. PGHS-2 induction by leukoregulin results from a massive
increase in the levels of steady-state PGHS-2 mRNA levels (42). Figure
1 also demonstrates the remarkably constant expression of PGHS-1
protein in both orbital and dermal fibroblasts.
|
Leukoregulin upregulates IL-1 and
IL-1
in orbital fibroblasts, but the increase in IL-1ra
is modest. Because we and others have demonstrated that
the IL-1 family of polypeptides is expressed in many cell types,
including orbital fibroblasts (5), we determined whether leukoregulin
could influence the synthesis of these cytokines. As the data contained
in Fig. 2 suggest, leukoregulin increases, in a time-dependent manner, the production of IL-1
(A), IL-1
(B), and IL-1ra
(C). The increases of IL-1
and
IL-1
appear to peak after 12 h of exposure to leukoregulin and begin
to fall thereafter. In contrast, IL-1ra levels continue to rise so that by 48 h, the duration of the study, the cytokine-receptor antagonist is
at its highest (11-fold above control values). We next compared the
effects of leukoregulin on the expression of IL-1-related polypeptides
in orbital and dermal fibroblasts. Figure 3
contains results of side-by-side experiments where the two cell
populations were compared. As those data demonstrate, the effects of
leukoregulin on IL-1
and IL-1
production were considerably
greater in orbital than in dermal fibroblasts (Fig. 3,
A and
B). The levels of IL-1
achieved
after leukoregulin treatment for 16 h were 369 ± 36 pg/10 µg
protein in orbital and 154 ± 24 pg/10 µg protein in dermal fibroblasts (P < 0.002), whereas the
levels of IL-1
achieved were 279 ± 46 and 70 ± 12 pg/10 µg
protein, respectively (P < 0.002).
Of great interest was the substantially higher level of IL-1ra achieved
in the dermal fibroblasts treated with leukoregulin for 12 h where the
levels were 181 ± 50 pg/10 µg protein in orbital cultures vs.
1,006 ± 84 pg/10 µg protein in dermal cells
(P < 0.0002). The induction of
IL-1
protein was concentration dependent in the range of 0-3
U/ml (Fig.
4A) and
occurred at a pretranslational level. Steady-state IL-1
mRNA levels,
migrating as a 1.7-kb transcript on Northern blot analysis, were
upregulated within 4 h of the initiation of leukoregulin treatment
(Fig. 4B) and were maximal at 8 h.
At 12 h, the levels of IL-1
mRNA had fallen. Leukoregulin also
appears to upregulate IL-1
at a pretranslational level, based on
increases in transcript determined by RT-PCR (data not shown).
|
|
|
IL-1 family members can induce each other and themselves (8). We
therefore determined whether neutralizing either IL-1 or IL-1
could influence the effects of leukoregulin on IL-1 protein expression.
Addition of neutralizing anti-IL-1
antibodies (1 µg/ml) could
block the upregulation of both IL-1
and IL-1
protein (Fig.
5, A and
B). In contrast, IL-1
neutralizing antibodies failed to influence the synthesis of either
IL-1 family member. Addition of exogenous IL-1ra (500 ng/ml) to
leukoregulin-activated fibroblasts also blocked both IL-1
and
IL-1
production. These results suggest that IL-1
but not IL-1
serves as a critical intermediate protein in the induction of
leukoregulin-dependent IL-1 expression in orbital fibroblasts. As the
Northern blot in Fig. 5C suggests, the
involvement of IL-1
in leukoregulin's effects on IL-1
expression
is mediated at the pretranslational level. Addition of either
anti-IL-1
or IL-1ra to leukoregulin-treated fibroblasts attenuates
the induction of IL-1
mRNA.
|
Interruption of IL-1 but not IL-1
appears to attenuate the induction by leukoregulin of PGHS-2 and
cytokine-dependent PGE2 production in
orbital fibroblasts.
Because of the important role of IL-1 as an inducer of PGHS-2 and
prostanoid production in many cell types, we next assessed whether
either IL-1
or IL-1
synthesis could be implicated in the
upregulation by leukoregulin of the cyclooxygenase in orbital fibroblasts. Addition of neutralizing antibodies directed at IL-1
or
IL-1
(1 µg/ml each) or a high concentration of exogenous IL-1ra (500 ng/ml) to the culture medium of orbital fibroblasts treated with leukoregulin (1 U/ml) resulted in an interesting
pattern of prostanoid production and PGHS-2 responses. Both
anti-IL-1
and IL-1ra could attenuate the upregulation by
leukoregulin of PGE2 synthesis
(Fig.
6A). In
cultures receiving leukoregulin alone, PGE2 levels were 2,030 ± 310 pg/ml compared with 617 ± 138 and 172 ± 100 pg/ml in
cultures receiving leukoregulin in addition to IL-1ra or anti-IL-1
neutralizing antibodies, respectively. The effects of leukoregulin on
PGHS-2 mRNA (Fig. 6B) and protein (Fig. 6C) were also markedly
attenuated by either IL-1ra or anti-IL-1
antibody. In contrast,
anti-IL-1
antibodies failed to block the induction of PGHS-2 protein
(Fig. 6C) or to influence
cytokine-dependent PGE2 generation
(data not shown). Neither anti-IL-1
antibodies nor IL-1ra could
block the induction of PGHS-2 by PMA or TNF-
(data not shown). Thus
it would appear that IL-1
is a critical intermediate protein
involved in the upregulation of PGHS-2 expression by leukoregulin. This
finding is also similar to our previous observation that IL-1
but
not IL-1
serves as an intermediate in the induction of PGHS-2
through the CD40/CD40 ligand activational bridge in orbital fibroblasts
(5).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Orbital fibroblasts are particularly susceptible to the actions of
proinflammatory cytokines such as IL-1, interferon-, and leukoregulin (31, 47). Of particular potential importance is the
substantial induction by cytokines of PGHS-2, the inflammatory cyclooxygenase. We hypothesize that it is the inducibility of PGHS-2 in
orbital fibroblasts that underlies, at least in part, the dramatic
inflammation observed in orbital connective tissue in the setting of
TAO. It appears that this response is a point of divergence of the
orbital fibroblast phenotype from fibroblasts derived from elsewhere in
the human body. It would seem, on the basis of the results we report
here, that the differential inducibility of IL-1 and IL-1ra may
underlie at least some of the attributes associated with orbital
fibroblasts. Our findings suggest that the local generation of IL-1
polypeptides in orbital fibroblasts could also impact cellular
neighbors, since IL-1
is released from the cell synthesizing it (8).
This cytokine can function as a paracrine factor. Because IL-1
induction appears to be critical to the induction of PGHS-2 by both
leukoregulin (Fig. 6) and the CD40/CD40 ligand activational bridge,
interrupting production of this cytokine or its actions might prove to
be a useful therapeutic target for disease modification.
An earlier report had attempted to demonstrate differential
upregulation of both intracellular and soluble forms of IL-1ra in
normal and TAO-derived orbital fibroblasts (19). Unfortunately, the
studies described in that paper utilized RT-PCR to assess the levels of
these IL-1ra mRNAs, and thus the data were not quantitative. The
findings did suggest a considerable difference in the induction by
IL-1 and lipopolysaccharide of soluble IL-1ra protein in normal and
TAO fibroblasts as measured by an assay similar to the one used here.
Thus it would appear that the substantial differences in IL-1ra
inducibility in TAO orbital fibroblasts compared with dermal cultures
we report are, at least in part, a consequence of the disease state in
addition to the anatomic derivation of the cells.
The finding that leukoregulin represents a strong inducer of PGHS-2 in
orbital fibroblasts has potential mechanistic relevance with regard to
the cross talk between these cells and orbital T lymphocytes found in
the tissues affected by TAO (7, 11). In addition to its effects on
prostanoid biosynthesis in fibroblasts, leukoregulin can dramatically
increase hyaluronan synthesis (39). This increase is very specific in
that the other abundant glycosaminoglycans, all sulfated, are
uninfluenced (39). Moreover, the effects on hyaluronan are
anatomic-site selective in that macromolecular synthesis in dermal
fibroblasts is enhanced to a far lesser degree. We have reported very
recently that lymphocytes can signal orbital fibroblasts through
another pathway that apparently does not involve leukoregulin, but this
cross talk also results in the upregulation of multiple orbital
fibroblast genes. CD40 ligand, when engaging CD40 displayed by these
fibroblasts, elicits a striking induction of both PGHS-2 expression and
hyaluronan synthesis (5). The expression of IL-6 and IL-8 in orbital
fibroblasts can also be enhanced through the CD40/CD40 ligand bridge
(24). The induction of at least some of these genes appears to be
related to NF-B (24). Taken together, it would appear that the
orbital fibroblast can be activated in an anatomic site-selective
manner by multiple molecular signals.
Another aspect that emerges from the current report is the substantial
impact that leukoregulin has on the stability of mature PGHS-2 mRNA.
Unlike many of the other cell types studied thus far, where the
inducibility of PGHS-2 has been characterized, it is the turnover of
the transcript in orbital fibroblasts rather than its rate of synthesis
that represents the predominant target for cytokine action. In some
experimental models, transcriptional upregulation accounts for the
greatest influence on steady-state mRNA levels, whereas, in ECV304
endothelial cells, increases in both gene transcription and mRNA
stability appear to contribute substantially (22). We have reported
previously that the rate of PGHS-2 gene transcription in orbital
fibroblasts changes only slightly after treatment with leukoregulin,
approximately twofold (42). The current studies demonstrate the
dramatic enhancement of the PGHS-2 mRNA half-life after leukoregulin
treatment. Thus it would appear that the mechanisms involved in the
upregulation of PGHS-2 and therefore of prostanoid production are
cell-type specific. Leukoregulin also enhances NF-B activity in
orbital fibroblasts, and attenuating this pathway blocks the induction of PGHS-2. Thus the relatively small increase in PGHS-2 gene
transcription might prove critical to the substantial increases in
steady-state mRNA levels. Alternatively, the importance of the NF-
B
pathway might relate to leukoregulin's upregulation of IL-1
or some
other intermediate protein, the promoter of which contains NF-
B
binding sites. It is of interest to note that cytokine stimulation of orbital fibroblasts results in a twofold increase in PGHS-2 promoter activity in cells transfected with a reporter gene (unpublished observations).
Orbital fibroblasts display a profile of receptors (36), gangliosides (2, 38), morphology (33), and responses to hormones (26, 33) and cytokines (14, 47) that distinguishe them from other fibroblast populations. We have suggested that it is the diverse array of molecules that fibroblasts synthesize and release that suggests broad roles for fibroblasts as sentinel cells that help coordinate complex events in wound repair and inflammation (25). The strikingly characteristic repertoire of biosynthetic activities ascribed to orbital fibroblasts underlies the unique remodeling of the orbital connective tissue observed in TAO and in other orbital inflammatory processes.
The highly inducible PGHS-2 in orbital fibroblasts implies a substantial capacity for these cells to produce large amounts of prostanoids in the context of inflammation. PGE2 has been shown to bias the differentiation of naive T lymphocytes away from the TH-1 phenotype and toward that of TH-2 lymphocytes (3). PGE2 also influences mast cell behavior and B cell differentiation (17, 23). Thus the high levels of PGE2 production found by orbital fibroblasts suggest that these cells might condition the immune responses occurring in the orbit, both under normal conditions and in disease. We have reported very recently that mast cells can directly activate gene expression in orbital fibroblasts, and at least some of this interplay between the two cell types is mediated through IL-4 (37). Thus the PGE2 generated by orbital fibroblasts might feed back on the mast cell, in turn modulating its activities. While not defining the role for prostaglandin production in TAO or in other situations of disease or health, our current findings do begin to identify the molecular events surrounding the exaggerated induction of PGHS-2 in orbital fibroblasts. Clearly, the role of PGE2 and other cyclooxygenase products in this disease will require further studies, perhaps some involving in vivo models. Should the cyclooxygenase prove to be overexpressed in TAO, therapeutic trials with the newly developed, specific PGHS-2 inhibitors would become warranted.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Julia Rozenblit and Heather Meekins for technical assistance. We are grateful to Dr. C. H. Evans for providing the leukoregulin used in these studies.
![]() |
FOOTNOTES |
---|
These studies were supported in part by National Eye Institute Grants EY-08976 and EY-11708 and by a Merit Review award from the Department of Veterans Affairs.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. J. Smith, Div. of Molecular Medicine, Dept. of Medicine, Harbor-UCLA Medical Center, 1000 West Carson St., Torrance, CA 90509.
Received 15 June 1999; accepted in final form 4 August 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Appleby, S. B.,
A. Ristimäki,
K. Neilson,
K. Narko,
and
T. Hla.
Structure of the human cyclo-oxygenase-2 gene.
Biochem. J.
302:
723-727,
1994[Medline].
2.
Berenson, C. S.,
and
T. J. Smith.
Human orbital fibroblasts in culture express ganglioside profiles distinct from those in dermal fibroblasts.
J. Clin. Endocrinol. Metab.
80:
2668-2674,
1995[Abstract].
3.
Betz, M.,
and
B. S. Fox.
Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines.
J. Immunol.
146:
108-113,
1991
4.
Cao, H. J.,
M. G. Hogg,
L. J. Martino,
and
T. J. Smith.
Transforming growth factor- induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts.
Invest. Ophthalmol. Vis. Sci.
36:
1411-1419,
1995[Abstract].
5.
Cao, H. J.,
H.-S. Wang,
Y. Zhang,
H.-Y. Lin,
R. P. Phipps,
and
T. J. Smith.
Activation of human orbital fibroblasts through CD40 engagement results in a dramatic induction of hyaluronan synthesis and prostaglandin endoperoxide H synthase-2 expression. Insights into potential pathogenic mechanisms of thyroid-associated ophthalmopathy.
J. Biol. Chem.
273:
29615-29625,
1998
6.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
7.
De Carli, M.,
M. M. D'Elios,
S. Mariotti,
C. Marcocci,
A. Pinchera,
M. Ricci,
S. Romagnani,
and
G. del Prete.
Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocytic infiltrates of Graves' ophthalmopathy.
J. Clin. Endocrinol. Metab.
77:
1120-1124,
1993[Abstract].
8.
Dinarello, C. A.
Biologic basis for interleukin-1 in disease.
Blood
87:
2095-2147,
1996
9.
Evans, C. H.,
A. C. Wilson,
and
B. A. Gelléri.
Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin.
Anal. Biochem.
177:
358-363,
1989[Medline].
10.
Funk, C. D.,
L. B. Funk,
M. E. Kennedy,
A. S. Pong,
and
G. A. Fitzgerald.
Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment.
FASEB J.
5:
2304-2312,
1991
11.
Grubeck-Loebenstein, B.,
K. Trieb,
A. Sztankay,
W. Holter,
H. Anderl,
and
G. Wick.
Retrobulbar T cells from patients with Graves' ophthalmopathy are CD8+ and specifically recognize autologous fibroblasts.
J. Clin. Invest.
93:
2738-2743,
1994[Medline].
12.
Hempel, S. L.,
M. M. Monick,
and
G. W. Hunninghake.
Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes.
J. Clin. Invest.
93:
391-396,
1994[Medline].
13.
Henkel, T.,
T. Machleidt,
I. Alkalay,
M. Krönke,
Y. Ben-Neriah,
and
P. A. Baeuerle.
Rapid proteolysis of I B-
is necessary for activation of transcription factor NF-
B.
Nature
365:
182-185,
1993[Medline].
14.
Higgins, P. J.,
and
T. J. Smith.
Pleiotropic action of interferon gamma in human orbital fibroblasts.
Biochim. Biophys. Acta
1181:
23-30,
1993[Medline].
15.
Hla, T.
Molecular characterization of the 5.2 KB isoform of the human cyclooxygenase-1 transcript.
Prostaglandins
51:
81-85,
1996[Medline].
16.
Hogg, M. G.,
C. H. Evans,
and
T. J. Smith.
Leukoregulin induces plasminogen activator inhibitor type 1 in human orbital fibroblasts.
Am. J. Physiol.
269 (Cell Physiol. 38):
C359-C366,
1995
17.
Hu, Z.-Q.,
K. Asano,
H. Seki,
and
T. Shimamura.
An essential role of prostaglandin E on mouse mast cell induction.
J. Immunol.
155:
2134-2142,
1995[Abstract].
18.
Kujubu, D. A.,
and
H. R. Herschman.
Dexamethasone inhibits mitogen induction of the TIS10 prostaglandin synthase/cyclooxygenase gene.
J. Biol. Chem.
267:
7991-7994,
1992
19.
Mühlberg, T.,
H.-J. Heberling,
W. Joba,
H.-D. Schworm,
and
A. E. Heufelder.
Detection and modulation of interleukin-1 receptor antagonist messenger ribonucleic acid and immunoreactivity in Graves' orbital fibroblasts.
Invest. Ophthalmol. Vis. Sci.
38:
1018-1028,
1997[Abstract].
20.
O'Banion, M. K.,
V. D. Winn,
and
D. A. Young.
cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.
Proc. Natl. Acad. Sci. USA
89:
4888-4892,
1992[Abstract].
21.
Reddy, L,
H.-S. Wang,
C. R. Keese,
I. Giaever,
and
T. J. Smith.
Assessment of rapid morphological changes associated with elevated cAMP levels in human orbital fibroblasts.
Exp. Cell Res.
245:
360-367,
1998[Medline].
22.
Ristimäki, A.,
S. Garfinkel,
J. Wessendorf,
T. Maciag,
and
T. Hla.
Induction of cyclooxygenase-2 by interleukin-1. Evidence for post-transcriptional regulation.
J. Biol. Chem.
269:
11769-11775,
1994
23.
Roper, R. L.,
D. H. Conrad,
D. M. Brown,
G. L. Warner,
and
R. P. Phipps.
Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis.
J. Immunol.
145:
2644-2651,
1990
24.
Sempowski, G. D.,
J. Rozenblit,
T. J. Smith,
and
R. P. Phipps.
Human orbital fibroblasts are activated through CD40 to induce proinflammatory cytokine production.
Am. J. Physiol.
274 (Cell Physiol. 43):
C707-C714,
1998
25.
Smith, R. S.,
T. J. Smith,
T. M. Blieden,
and
R. P. Phipps.
Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation.
Am. J. Pathol.
151:
317-322,
1997[Abstract].
26.
Smith, T. J.
Dexamethasone regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts. Similar effects of glucocorticoid and thyroid hormones.
J. Clin. Invest.
74:
2157-2163,
1984[Medline].
27.
Smith, T. J.
n-Butyrate inhibition of hyaluronate synthesis in cultured human fibroblasts.
J. Clin. Invest.
79:
1493-1497,
1987[Medline].
28.
Smith, T. J.
Glucocorticoid regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts: evidence for a receptor-mediated mechanism involving effects on specific de novo protein synthesis.
Metabolism
37:
179-184,
1988[Medline].
29.
Smith, T. J.
The putative role of cytokine-orbital fibroblast interactions in the pathogenesis of thyroid-associated ophthalmopathy.
Orbit
15:
137-146,
1996.
30.
Smith, T. J.
Cyclooxygenases as the principal targets for the actions of NSAIDs.
Rheum. Dis. Clin. North Am.
24:
501-523,
1998[Medline].
31.
Smith, T. J.,
A. Ahmed,
M. G. Hogg,
and
P. J. Higgins.
Interferon- is an inducer of plasminogen activator inhibitor type 1 in human orbital fibroblasts.
Am. J. Physiol.
263 (Cell Physiol. 32):
C24-C29,
1992
32.
Smith, T. J.,
R. S. Bahn,
and
C. A. Gorman.
Connective tissue, glycosaminoglycans, and diseases of the thyroid.
Endocr. Rev.
10:
366-391,
1989[Medline].
33.
Smith, T. J.,
R. S. Bahn,
and
C. A. Gorman.
Hormonal regulation of hyaluronate synthesis in cultured human fibroblasts: evidence for differences between retroocular and dermal fibroblasts.
J. Clin. Endocrinol. Metab.
69:
1019-1023,
1989[Abstract].
34.
Smith, T. J.,
R. S. Bahn,
C. A. Gorman,
and
M. Cheavens.
Stimulation of glycosaminoglycan accumulation by interferon gamma in cultured human retroocular fibroblasts.
J. Clin. Endocrinol. Metab.
72:
1169-1171,
1991[Abstract].
35.
Smith, T. J.,
and
P. J. Higgins.
Interferon gamma regulation of de novo protein synthesis in human dermal fibroblasts in culture is anatomic site dependent.
J. Invest. Dermatol.
100:
288-292,
1993[Abstract].
36.
Smith, T. J.,
R. J. Kottke,
H. Lum,
and
T. T. Andersen.
Human orbital fibroblasts in culture bind and respond to endothelin.
Am. J. Physiol.
265 (Cell Physiol. 34):
C138-C142,
1993
37.
Smith, T. J.,
and
S. J. Parikh.
HMC-1 mast cells activate human orbital fibroblasts in co-culture: evidence for up-regulation of prostaglandin E2 and hyaluronan synthesis.
Endocrinology
140:
3518-3525,
1999
38.
Smith, T. J.,
G. D. Sempowski,
C. S. Berenson,
H. J. Cao,
H.-S. Wang,
and
R. P. Phipps.
Human thyroid fibroblasts exhibit a distinctive phenotype in culture: characteristic ganglioside profile and functional CD40 expression.
Endocrinology
138:
5576-5588,
1997
39.
Smith, T. J.,
H.-S. Wang,
and
C. H. Evans.
Leukoregulin is a potent inducer of hyaluronan synthesis in cultured human orbital fibroblasts.
Am. J. Physiol.
268 (Cell Physiol. 37):
C382-C388,
1995
40.
Smith, T. J.,
H.-S. Wang,
M. G. Hogg,
R. C. Henrikson,
C. R. Keese,
and
I. Giaever.
Prostaglandin E2 elicits a morphological change in cultured orbital fibroblasts from patients with Graves ophthalmopathy.
Proc. Natl. Acad. Sci. USA
91:
5094-5098,
1994[Abstract].
41.
Smith, W. L.,
R. M. Garavito,
and
D. L. DeWitt.
Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2.
J. Biol. Chem.
271:
33157-33160,
1996
42.
Wang, H.-S.,
H. J. Cao,
V. D. Winn,
L. J. Rezanka,
Y. Frobert,
C. H. Evans,
D. Sciaky,
D. A. Young,
and
T. J. Smith.
Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.
J. Biol. Chem.
271:
22718-22728,
1996
43.
Wang, H.-S.,
C. R. Keese,
I. Giaever,
and
T. J. Smith.
Prostaglandin E2 alters human orbital fibroblast shape through a mechanism involving the generation of cyclic adenosine monophosphate.
J. Clin. Endocrinol. Metab.
80:
3553-3560,
1995[Abstract].
44.
Xie, W.,
J. G. Chipman,
D. L. Robertson,
R. L. Erikson,
and
D. L. Simmons.
Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.
Proc. Natl. Acad. Sci. USA
88:
2692-2696,
1991[Abstract].
45.
Xu, X.-M.,
J.-L. Tang,
A. Hajibeigi,
D. S. Loose-Mitchell,
and
K. K. Wu.
Transcriptional regulation of endothelial constitutive PGHS-1 expression by phorbol ester.
Am. J. Physiol.
270 (Cell Physiol. 39):
C259-C264,
1996
46.
Yang, X.,
F. Hou,
L. Taylor,
and
P. Polgar.
Characterization of human cyclooxygenase 2 gene promoter localization of a TGF- response element.
Biochim. Biophys. Acta
1350:
287-292,
1997[Medline].
47.
Young, D. A.,
C. H. Evans,
and
T. J. Smith.
Leukoregulin induction of protein expression in human orbital fibroblasts: evidence for anatomical site-restricted cytokine-target cell interactions.
Proc. Natl. Acad. Sci. USA
95:
8904-8909,
1998