Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-31, Japan.
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ABSTRACT |
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The physiological inflammatory response can provide an effective
mechanism for delivering the baby at the time of parturition. We
characterized the mechanisms by which hyaluronic acid (HA) regulates
interleukin-1 (IL-1
), tumor necrosis factor-
(TNF-
), and
interleukin-8 (IL-8) production in human uterine fibroblasts. A
dose-dependent increase in cytokine release was observed over an HA
concentration range of 10 µg/ml to 1 mg/ml. The action of HA on the
cytokine production is mediated by CD44. Under serum-free conditions,
HA-induced cytokine generation was significantly less compared with
production in the presence of serum, suggesting involvement of serum
proteins. Addition of inter-
-trypsin inhibitor (ITI) under
serum-free conditions enhanced the HA-induced synthesis of TNF-
,
which stimulated the temporary release of IL-8. In addition, HA and
IL-1
stimulated the release of hyaluronidase by the fibroblasts. These results indicate that cytokine production in human uterine fibroblasts is regulated in a CD44-HA-ITI-specific fashion. HA may be
involved in the regulation of delivery in part through the selective
release of cytokines that contribute to uterine cervical ripening.
hyaluronan; interleukin-1; tumor necrosis factor-; interleukin-8
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INTRODUCTION |
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CYTOKINES SUCH AS interleukin-1 (IL-1
) and tumor
necrosis factor-
(TNF-
) are known to exert numerous effects on
various target tissues. It is well recognized that various substrates, including lipopolysaccharides, can stimulate macrophages and/or fibroblasts to produce IL-1
(11, 12). IL-1
is
considered to be a potent stimulator of hyaluronic acid (HA) production
by fibroblasts in vitro (17). The local accumulation of HA
concentrations could function to facilitate the migration of
inflammatory cells into the reaction site (4, 21). HA is able to retain
large amounts of water within its molecular domain on surrounding cells and matrix constituents. HA is synthesized early during the course of
an inflammatory reaction and is later degraded by fibroblasts during
the healing phase of inflammation (15, 27).
The cell surface adhesion molecule CD44 is the principal receptor for HA (3, 23). The CD44 family has several different isoforms that are derived from a single gene by alternative splicing of the mRNA. The NH2-terminal domain of CD44, which shows homology to the link protein of cartilage, binds to a six-sugar sequence of HA. Antibodies that block the interaction between HA and CD44 prevent the binding of HA to the cell surface, its subsequent uptake into the cytoplasm, and its eventual degradation in lysosomes. CD44 is an extremely versatile protein that may carry out several functions through HA binding (14).
It has been reported that a serum factor is required to organize HA
within the extracellular matrix (5, 7). The intercellular space between
fibroblasts increases due to the production of a mucoelastic
extracellular matrix in HA. This factor, which is important for
organizing HA within the extracellular matrix, has been recently
identified as a protein belonging to the inter--trypsin inhibitor
(ITI) family (6, 8, 16, 25, 31). Purified ITI was able to replace serum
in supporting pericellular matrix coats, and it was proposed that ITI
provides an integral structural component of the matrix by interacting
directly with HA. Previous studies showed that hyaluronidase (HAase)
and protease disorganized and partially degraded the microstructure of
the matrix, indicating that interactions between proteins and HA are
important for stability of the extracellular network between the cells
(5-8, 16, 25, 31).
At the time of parturition, the physiological inflammatory response can
provide an effective mechanism for delivering the baby. With uterine
cervical dilatation and softening (ripening), increases of HA
concentrations and collagenase activity and a conspicuous decrease of
collagen in the uterine cervix have been reported (9). The ripening of
the cervix is a necessary prerequisite for a normal labor. It is an
interesting fact that IL-1 promotes collagenase production and HA
synthesis as well as a depression of collagen synthesis, since these
actions could be associated with the uterine cervical
ripening process at parturition. HA synthesis is accelerated soon after
onset of labor. Cellular migration has been correlated with elevations
of HA concentrations in uterine cervix. However, endogenous cytokine
inducers during the ripening of the uterine cervix are not fully
clarified.
Here, we report that HA induces cytokine production in human uterine
fibroblasts and may contribute to pathological and physiological changes in connective tissues. HA stimulates the release of IL-1 and
TNF-
from uterine fibroblasts. IL-1
could stimulate neighboring connective tissue fibroblasts to produce increased quantities of HA.
IL-1
and TNF-
synthesized by fibroblasts after stimulation by
endogenously produced or exogenously applied HA could act directly as a
positive feedback to release additional HA from fibroblasts.
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MATERIALS AND METHODS |
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Materials.
Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, Hanks'
balanced salt solution, fetal calf serum (FCS), trypsin-EDTA, and
gentamicin were obtained from GIBCO (Grand Island, NY). HA, with
molecular masses of 800 and 2,000 kDa, were kindly
supplied by Chugai Pharmaceutical and Kaken Chemical, respectively;
both had a purity of at least 99%. Intra-articular
injection therapy with HA is widely used in Japan for degenerative
gonarthrosis, and it has shown favorable clinical results in terms of
improving articular symptoms. The HA used in the present experiments
was derived from chick's comb. HA is a pure polysaccharide. Combining 12,500 disaccharide units produces an HA molecule of ~5 × 109 molecular weight, similar to
the HA present in cartilaginous tissues. The normal size range of HA
secreted by uterine fibroblasts is also 3-5 × 109 molecular weight. When HA is
dissolved in physiological saline, every molecule touches another one
and a continuous molecular network is formed. HA is not subjected to
conformational changes by physiological saline. HA binding protein
(HABP, 40 kDa) was also kindly supplied by Chugai Pharmaceutical. A
monoclonal antibody (MAb) against IL-1 and an MAb against TNF-
were obtained from Genentech. Heparan sulfate, dermatan sulfate,
chondroitin sulfate, and HAase (which was from
Streptomyces hyaluronicus) were
purchased from Seikagaku Kogyo (Tokyo, Japan). Anti-CD44 antibody was
obtained from Cosmo Bio (Tokyo, Japan).
N-acetyl-D-glucosamine,
glucuronic acid,
N-acetyl-D-mannosamine,
N-acetyl-D-galactosamine,
and D-glucosamine were obtained
from Sigma Chemical (St. Louis, MO).
Cell culture. Human uterine fibroblasts were obtained from surgery specimens using an explant culture. Cells were maintained in vitro in 75-cm2 flasks (Nunc, Tokyo, Japan) in DMEM supplemented with 10% FCS and antibiotics (50 µg/ml gentamicin and 2.5 µg/ml amphotericin). The cells were maintained at saturated humidity in an atmosphere of 95% air-5% CO2. Fibroblasts were studied during passages 2-5.
Confluent fibroblasts were washed in phosphate-buffered saline (PBS), trypsinized (0.05% trypsin, 0.1% EDTA) for 2 min at 37°C, diluted in medium containing 10% FCS to inactivate the trypsin, and centrifuged for 10 min at 250 g. Cell pellets were resuspended in culture medium (DMEM containing 10% FCS), and quadruplicate samples of 1-ml aliquots of cell suspension were added to 24-well plates (Costar). The cell number added to each well was ~2 × 105 cells. After 24 h, the cells were washed three times with Hanks' balanced salt solution and serum-free DMEM supplemented with 0.1 mg/ml bovine serum albumin (BSA). The cells were maintained in fresh complete medium containing either no additions (control) or HA in the presence or absence of anti-CD44 antibody. All conditions were examined in triplicate. After the indicated periods of time, the culture supernatants were harvested and stored atEnzyme-linked immunosorbent assays for IL-1,
TNF-
, and IL-8.
Cytokines were measured in fibroblast culture supernatants by specific
enzyme-linked immunosorbent assays (ELISA; TFB, Tokyo, Japan). The
ELISA had a lower detection limit of 6.3 pg/ml (IL-1
), 1.9 pg/ml
(TNF-
), or 3.1 pg/ml (IL-8). The intra-assay variations of these
assays were <10%.
Flow cytometric analysis. Fibroblast cells growing as monolayers were detached with 0.02% EDTA in PBS. Complete detachment was confirmed by direct visualization. Single cell suspensions were made by repeated pipetting through a 0.4-mm-diameter cannula. Cells were washed with ice-cold PBS containing 0.1% BSA and resuspended. Aliquots of cells were incubated with anti-CD44 antibody for 30 min at 4°C. After two washes, cells were incubated with 100 µg/ml of HA (30 min, 23°C) and then stained with fluorescein isothiocyanate (FITC)-labeled HABP (5 µg/ml; 30 min, 23°C). HABP was used as a specific probe for HA. The cells were analyzed by flow cytometry (Coulter, Miami, FL). Cells were gated using forward vs. side scatter to select for HA expression on fibroblasts, excluding dead cells and debris. Net mean fluorescent intensity was expressed as channel number of HA expression after subtraction of the background fluorescence.
Preparation of ITI and its derivatives. Human ITI was isolated from serum according to the method of Salier et al. (28). ITI is comprised of three genetically different peptides: two heavy chains (HC1 and HC2) and a light chain (urinary trypsin inhibitor; UTI). The trypsin inhibitor activity of ITI is localized with UTI (10, 13). A highly purified preparation of human UTI with an activity of 2,330 U/mg protein and a molecular mass of 40 kDa [by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis] was kindly supplied by Mochida Pharmaceutical (Tokyo, Japan). The covalent structure of the polypeptide chain of the physiological inhibitor UTI has already been determined by Wachter and Hochstrasser (29).
HAase activity. HA-unstimulated and HA-stimulated fibroblast culture supernatants (100 µl of each) were dialyzed and then lyophilized. Samples to be examined for the presence of HAase were electrophoresed on 8.0% SDS acrylamide gel. HAase activity was determined by zymography using HA-impregnated gels as described previously (22).
Fragments of HA. Oligosaccharides of HA were prepared by degradation of HA with HAase and characterized by gel chromatography according to the manufacturer's instructions.
Statistical analysis. All data were analyzed by comparing the samples with a control group by the Student's t-test.
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RESULTS |
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HA-induced release of TNF- and IL-1
by fibroblasts.
Many cytokines are released either spontaneously or in response to
cellular activation. To determine whether HA (molecular mass of 800 kDa) affects cytokine production, IL-1
and TNF-
proteins released
into the conditioned medium of fibroblasts were quantitated by ELISA.
These data enabled an estimate to be made of the relative quantities of
newly synthesized cytokines released into the incubation medium.
|
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Involvement of CD44 in HA-induced cytokine release. We tested whether the anti-CD44 antibodies block the binding of HA to the cell surface CD44 (Fig. 3). A direct binding assay of HA and a competitive binding study between HA and the anti-CD44 antibodies to uterine fibroblasts were demonstrated. In the first experiment, direct binding assays using flow cytometry were performed. Binding of HA to the fibroblasts was determined by FITC-conjugated HABP as a specific probe. Binding of HA to the fibroblasts was dose dependent and saturable. In the next experiment, competitive binding assays were performed in which increasing amounts of anti-CD44 antibodies were preincubated with the cells. The binding of HA (10 µg/ml) was competed by the anti-CD44 antibodies but not by nonimmune immunoglobulin G (IgG); 50% inhibition of HA binding to the cells was obtained with 4 µg/ml anti-CD44 antibodies. This firmly confirms the data from Fig. 4.
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Effect of serum or ITI and its derivatives on HA-induced cytokine release. We investigated whether FCS affected HA-induced cytokine release (Fig. 5). In the absence of serum, the release of cytokine during a 24-h culture period was strongly diminished compared with the release in the presence of 10% FCS.
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Enhanced release of IL-8 by fibroblasts after stimulation with HA. Fibroblast monolayers also showed a significant stimulation of IL-8 release when the cells were exposed to HA (1 mg/ml) (Fig. 6). IL-8 release peaked at 24 h and remained elevated at 48 h. A dose-dependent increase in IL-8 release was observed over an HA concentration range of 100 µg/ml to 1 mg/ml (data not shown).
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Cytokine generation from fibroblasts after incubation with glycosaminoglycans. The effects of glycosaminoglycans and their components on cytokine generation were tested by incubation in cultured fibroblasts (Fig. 7). Fibroblast monolayers were incubated with soluble, structurally related glycosaminoglycans to provide some assessment of specificity for HA. The stimulatory effect was specific for HA and chondroitin sulfate to some extent. Other glycosaminoglycans, such as heparan sulfate and dermatan sulfate, had no significant effects.
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HA regulation of fibroblast HAase activity.
We measured the HAase activity of unstimulated and HA-stimulated
fibroblast culture supernatants (Fig.
8). Unstimulated
fibroblasts manifest modest levels of HAase activity. HA caused a
significant increase in HAase activity. The levels of HAase activity in
cells stimulated with HA were significantly greater than the levels seen in cells incubated with HA in the presence of anti-IL-1 antibody. Treatment of fibroblast monolayers with IL-1
caused a
significant increase in HAase activity. These studies suggested the
possibility that HA-stimulated IL-1
release may be involved in
regulating the pathway leading to HAase production.
|
Cytokine release from fibroblasts incubated with HA fragments of
different molecular masses.
We investigated the molecular basis for the effect of HA (Fig.
9). HA fragments ranging
from 800 to 2,000 kDa all stimulated TNF- generation. HA fragments
at lower molecular masses (10-100 kDa) stimulated the cytokine
generation significantly less. HA fragments of <5 kDa were without
effect, as were the glycosaminoglycans, N-acetyl-D-glucosamine,
glucuronic acid (these two are the components from which HA is built
up),
N-acetyl-D-mannosamine,
N-acetyl-D-galactosamine, and D-glucosamine. These results
demonstrated that the stimulatory effect of HA on cytokine generation
was restricted to HA of high molecular mass (
800 kDa).
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DISCUSSION |
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HA is a potent, cell-derived bioactive macromolecule thought to be involved in many cellular functions, such as cell migration, invasion, proliferation, transformation, and mitosis (19, 20, 30, 32). Dilatation of the uterine cervix at parturition is associated with an increase in cervical HA content. It has been reported that circulating HA levels increase significantly at parturition in the guinea pig (26). An accumulation of HA in the extracellular matrix may result in the observed softening and swelling of the uterine cervix because of its unique viscoelastic properties and its high avidity for water (26). More recently, it has been reported that HA acts on intracellular signal transduction via protein kinase C by the binding of HA to CD44 or its entry into the cytoplasm (internalization), suggesting that HA also exerts chemical effects, activating fibroblast metabolism at the cell level (2).
In the present study, we showed that the action of HA on the production
of cytokines, including IL-1, TNF-
, and IL-8, by uterine
fibroblasts is mediated by CD44 and that HA and IL-1
stimulate the
release of HAase by the fibroblasts. In addition, ITI enhanced the
HA-induced TNF-
synthesis under serum-free conditions. These
findings seem to be useful for understanding the roles of CD44, HA, and
ITI in uterine cervical ripening.
Cytokine production is regulated in an HA-specific fashion. However, HA fragments of molecular weight of <5,000 were without effect. Furthermore, the HA-enhanced generation of cytokines leads to postulated temporary release of HAase. These data are in accord with prior observations that HA is synthesized early during the course of an inflammatory reaction and is later degraded by fibroblasts during the healing phase of inflammation (15).
HA accumulation is regulated by the balance of biosynthesis and
degradation by HAase. This balance is altered in physiological (normal
delivery) and pathological (premature delivery) states. Increased
amounts of HA have been noted in uterine cervical tissues at
parturition (9, 26). Studies from several laboratories have shown that
a variety of inflammatory cytokines, including interferon-, TNF-
,
IL-1, and transforming growth factor-
, regulate fibroblast HA
production. These cytokines interact to alter the size as well as the
amounts of HA produced by fibroblasts. These alterations may be
mediated by alterations in fibroblast HA degradation. An interesting
aspect of this study is the demonstration that HA itself stimulates
fibroblast-derived HAase production. Cytokines may regulate fibroblast
HA degradation by altering fibroblast HA binding, and CD44 may also
play a role in this process. It is likely that exogenously applied HA
is depolymerized into lower-molecular-weight forms by
fibroblast-derived HAase. The effects of the molecular weight of HA on
binding to CD44 in fibroblasts have not been investigated, but
depolymerization of HA may affect binding and signal transduction.
Under serum-free conditions, HA-induced cytokine generation was significantly less compared with production in the presence of serum, suggesting involvement of serum proteins. The serum factor has been recently identified as a protein belonging to the ITI family (5-8, 16, 25, 31). It has been reported that the presence of ITI in the culture medium is necessary for organizing HA in the extracellular matrix. The ITI family responsible for this effect has been identified by Chen et al. (8). We studied the effect of ITI and its derivative, UTI, on the HA-induced cytokine generation from cultured fibroblasts. Addition of ITI enhanced the HA-induced cytokine synthesis under serum-free conditions but had no effect in the presence of serum. On the other hand, UTI, known to possess protease-inhibiting activity within the ITI molecule, did not enhance HA-induced cytokine synthesis. Some structural components, such as ITI or heavy chains of ITI, that bind to HA with specificity for the HA oligomers are required to organize and stabilize HA within the matrix. The stabilization of the pericellular matrix with HA-ITI complexes may be required for cytokine generation from the cultured fibroblasts.
The cell-associated HA matrix dissociates in the absence of serum (7). Initially, the process is rapid. After the disaggregation process (after most of the HA has already been released into the culture medium), cytokine generation slowly declined and was completely abolished by 24 h of total culture (7). Once the fibroblast matrix is formed, then cooperation of several structural elements may be responsible for the stability of the pericellular matrix and the generation of cytokines. When ITI and HABP (the link protein family) cooperatively bind to HA on the surface of fibroblasts, the ternary aggregates (ITI-HA-link protein) may not be dissociated in the presence of exogenously applied HA. Cell-matrix interactions may be important to prime the cells so that they can respond to HA.
Uterine cervical ripening at parturition is a particularly interesting
state with regard to HA metabolism (9). The uterine cervix is
infiltrated with inflammatory cells that produce exaggerated amounts of
IL-1, TNF-
, and IL-8. These cytokines are, at least partly,
responsible for the abnormal HA accumulation noted during this
condition. Our finding that HA can stimulate the secretion of cytokines
has important implications for our understanding of the regulation of
cervical ripening. When pregnancy is close to term, the HA component
increases, leading to greater water retention and the soft, swollen
appearance of the term cervix. The dilatation and effacement (ripening)
at the cervix are necessary prerequisites for a normal labor (9). The
HA-enhanced generation of TNF-
that leads to postulated temporary
activation of IL-8 may share in a physiological mechanism for ripening
of the uterine cervix. If cervical ripening is induced by a neutrophil
infiltrate, one of the mediators is IL-8. An action of HA with a
relatively small constitutive production of IL-8 within the cervix is a
sufficient explanation of the ripening effect of endogenously produced
HA. A corollary of such a mechanism is that HA or IL-8 as vaginal suppositories could also be used to ripen the cervix, with the advantage that such an approach would be less likely to result in
hyperstimulation of the myometrium. IL-1
and TNF-
synthesized by
fibroblasts after stimulation by endogenously produced HA could act
directly as a positive feedback of further generation of HA from
neighboring connective tissue fibroblasts. The production of cytokines
in HA-stimulated fibroblasts implies that the autocrine stimulatory
loops may play a role in ripening the uterine cervix. Because HA is a
physiological potent stimulator of cytokines from uterine fibroblasts
and may contribute to cervical ripening, followed by dilatation of the
uterine cervix, HA could be used to control delivery.
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FOOTNOTES |
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Address for reprint requests: H. Kobayashi, Dept. of Obstetrics and Gynecology, Hamamatsu Univ. School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-31, Japan.
Received 18 December 1996; accepted in final form 3 June 1997.
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