(Received for publication, September 3, 1996, and in revised form, December 20, 1996)
From the Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Activated macrophages play a critical role in
controlling chronic tissue inflammation through the release of a
variety of mediators including cytokines, chemokines, growth factors,
active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly
understood. A hallmark of chronic inflammation is the turnover of
extracellular matrix components, and recent work has suggested that
interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the
effect of low molecular weight fragments of the extracellular matrix
glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS
mRNA, protein and activity alone, and markedly synergize with
interferon- to induce iNOS gene expression in murine macrophages. In
addition, we found that resident tissue alveolar macrophages respond
minimally, but inflammatory alveolar macrophages exhibit a marked
induction in iNOS expression in response to HA fragments. Finally, we
demonstrate that the mechanism of HA fragment-induced expression of
iNOS requires activation of the transcriptional regulator nuclear
factor
B. These data support the hypothesis that HA may be an
important regulator of macrophage activation at sites of chronic tissue
inflammation.
Nitric oxide (NO·)1 mediates a
number of the host defense functions of activated macrophages,
including antimicrobial and tumoricidal activity (1-7). NO· and
its metabolites have also been implicated in the pathogenesis of the
tissue damage associated with acute and chronic inflammation (8-12).
Macrophages generate NO· from the guanido moiety of
L-arginine through a reaction catalyzed by the inducible
form of nitric- oxide synthase (2). In contrast to the constitutive,
calcium-dependent form of the enzyme found in the central
nervous system and endothelial cells, iNOS can be induced by numerous
immune stimuli. Maximal, synergistic iNOS induction occurs in response
to the combination of a priming stimulus, such as IFN, and a
triggering stimulus, examples of which include LPS, tumor necrosis
,
and interleukin-2 (6, 13, 14).
Hallmarks of chronic inflammation include the accumulation of activated macrophages and of macrophage-derived mediators. However, the mechanisms of macrophage activation in this setting have not been clearly defined. The ECM undergoes increased degradation and turnover during inflammation, and fragments of ECM molecules have been found to possess biological activities distinct from their parent compounds (15-17). It has therefore been proposed that ECM fragments may be responsible for activating macrophages that infiltrate chronically inflamed tissues (18). We have recently demonstrated that fragments of the ECM component HA can bind to macrophages and induce the expression of a number of inflammatory genes (32), suggesting that HA fragments may be capable of activating macrophages at non-infectious sites of inflammation.
HA is a high molecular weight, nonsulfated linear glycosaminoglycan
composed of repeating units of (,1-
4)-D-glucuronic
acid-(
,1-
3)-N-acetyl-D-glucosamine (19).
Increased concentrations of HA have been found at sites of chronic
inflammation (20, 21), and when the size of this HA has been studied
the molecular weight range has been found to include lower molecular
weight species (22, 23). Chronically inflamed tissues also contain
elevated levels of the soluble, chemotactic cytokines known as
chemokines (24-31), many of which are major products of activated
macrophages. We have found that HA fragments of a size comparable with
that found in inflammation, but not HA in the physiologic, higher
molecular weight size range, induce the expression of chemokine genes
in macrophages through a mechanism involving the cellular HA receptor,
CD44 (32). We have also demonstrated that HA fragments induce
activation of the transcriptional regulator NF-
B in murine
macrophages (41). Since NF-
B mediates the expression and regulation
of numerous inflammatory genes, including LPS-induced iNOS (33-38),
the ability of HA fragments to induce this transcriptional regulator
provides further evidence that HA fragments are likely to play a role
in the inflammatory response.
The purpose of the present study was to investigate the hypothesis that
HA fragments induce the expression of iNOS in inflammatory macrophages
and to characterize the molecular mechanism of this induction. We
report here that murine macrophages produce iNOS in response to HA
fragments and that this induction is synergistically enhanced by
IFN. Normal alveolar macrophages respond to HA fragments with only
minimal iNOS gene expression, but inflammatory AM produce significant
levels of iNOS gene products when stimulated with HA fragments.
Additionally, our findings indicate that HA
fragment-dependent iNOS gene induction occurs through an
NF-
B-dependent mechanism.
The mouse alveolar macrophage cell line MH-S (39) and the mouse macrophage-like cell line RAW 264.7 were purchased from the American Type Culture Collection, Rockville, MD. Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated low-LPS fetal bovine serum and 1% penicillin/streptomycin/1% glutamine (Biofluids, Rockville, MD) at 37 °C under 5% CO2. Mouse bone marrow-derived macrophages were isolated as described previously (40) from female C3H/HeJ, LPS hyporesponsive mice purchased from the Jackson Laboratory (Bar Harbor, ME). Bleomycin experiments were performed using male Harlan Sprague Dawley rats obtained from Harlan (Indianapolis, IN). All experiments were carried out in the presence of the LPS inhibitor polymyxin B (10 µg/ml) unless stated otherwise in order to exclude the effects of contaminating LPS on inflammatory gene expression.
Chemicals and ReagentsPurified HA fragments from human
umbilical cords were purchased from ICN Biomedicals, Inc., Costa Mesa,
CA. The HA-ICN preparation is free of protein (<2%) and free of
chondroitin sulfate (<3%), and we have previously determined its peak
molecular size to be approximately 200,000 Da (41). Escherichia
coli 011:B4 LPS prepared by the Westphal method, the antioxidant
pyrrolidinedithiocarbamate (PDTC), and the serine protease inhibitor
N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK) were obtained from Sigma. Polymyxin B was purchased from
Calbiochem. Recombinant mouse IFN (specific activity, 3.0 × 105 units/ml; endotoxin level less than 0.2 ng/µg) was
from Genzyme Corp., Cambridge, MA. The proteasome inhibitor MG132 was a
generous gift of MyoGenics, Inc., Cambridge, MA. Bleomycin sulfate and chondroitinase ABC from Proteus vulgaris were purchased from
Sigma.
Bleomycin was administered to male Harlan Sprague Dawley rats according to published methods (42). Rats were anesthetized using inhaled Isofluorane. Following tracheostomy, 500 µl of sterile normal saline with 1 unit of bleomycin sulfate was instilled into the lungs through a 25-gauge needle inserted between the cartilagenous rings of the trachea. Control animals received saline lavage alone. The tracheostomy site was sutured, and the animals were allowed to recover until the time of BAL. Rats were killed with a lethal injection of sodium pentathol (Ampro Pharmaceutical, Arcadia, CA) at specified time points following intratracheal instrumentation.
BAL was performed by cannulating the trachea and instilling and retrieving approximately 50 ml of sterile normal saline in 5-ml aliquots. The entire lavage volume was centrifuged, and the cell pellet was resuspended in RPMI supplemented with fetal bovine serum and antibiotics. A differential cell count performed on the lavage fluid by Wright-Giemsa stain prior to centrifugation and repooling of cells consistently showed at least 95% macrophages. Cell viability was determined using trypan blue exclusion. Alveolar macrophages were purified by adherence to plastic tissue culture dishes for 1 h at 37 °C in RPMI without serum or antibiotics under 5% CO2; nonadherent cells were removed by aspiration, and adherent macrophages were washed once in 1 × sterile phosphate-buffered saline. All subsequent experiments were performed in RPMI without serum or antibiotics.
Northern Analysis of mRNA ExpressionRNA was extracted
from confluent cell monolayers using 4 M guanidine
isothiocyanate and purified by centrifugation through 5.7 M
cesium chloride for 12-18 h at 35,000 rpm as described (40). Ten-fifteen µg of total RNA was electrophoresed under denaturing conditions through a 1% formaldehyde-containing agarose gel, and RNA
was transferred to Nytran (Schleicher & Schuell) or Zetaprobe (Bio-Rad)
hybridization filters. Blots were briefly rinsed in 5 × SSC, and
RNA was cross-linked to the filter by UV cross-linking (Stratagene, La
Jolla, CA). Northern blots were hybridized overnight with
106 cpm/ml of the iNOS cDNA (43) labeled with
[32P]dCTP by the random prime method (Amersham Corp.).
Following hybridization, blots were washed once in 2 × SSC, 0.1%
SDS at room temperature for 30 min with shaking and then washed twice in 0.1 × SSC, 0.1% SDS at 50 °C with shaking (30 min each
wash). Blots were exposed at 70 °C against Kodak XAR diagnostic
film. Differences in RNA loading were documented by hybridizing
selected blots with 32P-labeled cDNA for
glyceraldehyde-3-phosphate dehydrogenase or aldolase (kindly provided
by Dr. M. Shin, University of Maryland School of Medicine, Baltimore).
Densitometric scanning was performed using a Molecular Dynamics
Personal Densitometer SI (Sunnyvale, CA).
Western blot analysis was performed as described (44). Briefly, 200 µg of macrophage total cell lysate was fractionated by SDS-polyacrylamide gel electrophoresis (10%), transferred to a nylon membrane, blocked and washed, incubated with polyclonal anti-iNOS antibody (45) at a dilution of 1:2500, and developed with a chemiluminescent system according to the manufacturer's instructions (Amersham Corp.).
Assay for NO· ProductionProduction of NO·
was determined by measurement of NO2,
a stable product of the reaction of NO· with molecular oxygen,
in culture supernatants as described previously (6, 46). In brief, 100 µl of supernatant was mixed with 100 µl Greiss reagent (0.5%
sulfanilamide, 0.05% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H3PO4) and incubated
at 22 °C. Optical densities of samples were determined at 5 and 30 min at 540 nm. NO2
concentrations were
determined by comparison with a standard curve prepared with
NaNO2.
Nuclear extracts were
prepared from macrophage monolayers stimulated in the absence of serum
using the technique of Andrews and Faller (47). Confluent macrophage
monolayers were stimulated on 10-cm Falcon tissue culture dishes in the
absence of serum, rinsed once in cold 1 × phosphate-buffered
saline, scraped, resuspended in 400 µl of Buffer A (10 mM
HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 M phenylmethanesulfonyl fluoride), and incubated on ice for
10 min. Nuclei were sedimented by centrifugation, resuspended in a
suitable volume of Buffer C (20 mM HEPES-KOH, pH 7.9, 25%
glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.2 M phenylmethanesulfonyl fluoride), and
incubated on ice for 20 min. The protein concentration of the extracts
was determined using the BCA method (Pierce). Extracts were kept at
70 °C.
Gel shift assays were performed as described previously (41). Five µg
of nuclear extract in 2-3 µl was incubated for 5 min at room
temperature with 1 µl of 1 mg/ml bovine serum albumin in AP-1 buffer
(10 mM HEPES-KOH, pH 7.5, 16% glycerol, 20 mM
NaCl, 4 mM MgCl2, 0.1 mM EDTA, 2 mM dithiothreitol, 2 mM spermidine (48)) for 5 min at room temperature with 2 µg of poly(I·C) (Pharmacia Biotech
Inc.) and for 15 min at 37 °C with 1 µl of 10 × TBE (890 mM Tris base, 890 mM boric acid, 20 mM EDTA, pH 8.0) plus 1 µl of 32P-labeled
(50,000 cpm) or 2 µl of unlabeled double-stranded oligonucleotide containing the proximal NF-B site from the murine iNOS promoter. The
double-stranded oligonucleotide containing the
B site extending from
85 to
76 was made by annealing the following two oligonucleotides: 5
AACTGGGGACTCTCCCTTTG3
and 3
CAAAGGGAGAGTCCCCAGTT5
. The
double-stranded oligonucleotide containing the mutated
B site was
made by annealing the following two oligonucleotides,
AACTGGAAACTCTCCCTTTG and CAAAGGGAGAGTTTCCAGTT,
with the mutated bases in boldface. The mixture was electrophoresed at
4 °C on a 6% polyacrylamide gel in 0.4 × TBE at 200 V. Gels
were dried and exposed to XAR Kodak film at
70 °C. To identify the
protein components of the NF-
B complex, 1 µl of specific
antibodies to p50, p65, or c-REL (Santa Cruz Biotechnology, Santa Cruz,
CA) was added prior to addition of labeled oligonucleotide and
incubated on ice for 10 min.
The transcriptional regulation of
iNOS was assayed using a reporter plasmid containing 1700 base pairs of
the iNOS promoter region placed immediately upstream of the
luc cDNA in the pGL2-Basic vector (Promega, Madison, WI)
(46). Another iNOS promoter-luciferase construct was also used in which
the B site was mutated by site-directed mutagenesis (Transformer
Kit, Clontech) so that base pairs
83 to
84 were changed from GG to
AA. For liposomal mediated transfection, 0.5 µg of iNOS reporter
plasmid and 0.5 µg of pSV-
-galactosidase control vector (Promega)
were mixed with the 15 µl of Lipofectin (Life Technologies, Inc.),
brought to a volume of 200 µl with Opti-MEM (Life Technologies,
Inc.), incubated for 20 min at 22 °C, brought to 1 ml with Opti-MEM,
and then added to aspirated 6-well plates of subconfluent RAW 264.7 macrophages. After 16 h incubation, the transfected macrophages
were stimulated with HA, cultured for 24 h, and then harvested in
lysis buffer (Promega). Luciferase activity was assayed in 40 µg of
protein lysate in a luminometer following the manufacturer's
recommendation (Promega) and standardized by dividing by
-galactosidase activity also measured in 200 µg of protein lysate
(Promega).
We first
investigated the ability of HA fragments to induce iNOS in the mouse
alveolar macrophage-like cell line MH-S. MH-S cells are resident
alveolar macrophages that have been transformed with SV40 (39), and
when stimulated with HA fragments of peak molecular size of 200,000 Da,
these cells expressed iNOS gene products in a
time-dependent fashion (Fig. 1a).
Maximal mRNA expression was observed at 6 h. Related
glycosaminoglycans, including chondroitin 4-sulfate, dermatan
sulfate, chondroitin 6-sulfate, and heparan sulfate all failed to
induce iNOS gene expression in MH-S cells, as did the individual
saccharide components of HA (data not shown). We have previously
demonstrated that HA disaccharides are unable to induce inflammatory
gene expression in murine macrophages (32), and Fig. 1b
shows that HA fragment-dependent iNOS gene induction was
significantly reduced following digestion of HA fragments to
disaccharides with chondroitinase ABC (49). High molecular weight HA
(with a peak molecular size of approximately 6 × 106
Da) also failed to induce iNOS mRNA expression (data not shown). HA
fragment-induced iNOS mRNA expression in MH-S cells was accompanied by the production of iNOS protein, as shown in Fig. 1c. The
time course of HA fragment-dependent nitrite production in
MH-S cells is shown in Fig. 1d, demonstrating that HA
fragments induced functional NOS enzyme activity in these cells.
Synergy Between HA Fragments and IFN
Next we determined the effect of
the addition of IFN on HA fragment-dependent iNOS
induction in MH-S cells. IFN
alone induced little or no iNOS
mRNA in these cells, as seen in Fig. 2a.
However, HA fragment-induced iNOS mRNA expression was dramatically
increased by the addition of IFN
. Fig. 2b demonstrates
that addition of IFN
allowed detection of iNOS gene expression at
doses of HA fragment as low as 1 µg/ml. IFN
similarly enhanced HA
fragment-dependent iNOS protein production, shown in Fig.
2c. These results suggest that HA fragments function as a
triggering stimulus for iNOS production in macrophages, acting together
with the priming stimulus IFN
to induce iNOS expression.
HA Fragments Synergize with IFN
The in vivo
macrophage response to an immune stimulus involves the interaction of
multiple macrophage populations. Inflammation triggers the recruitment
and activation of peripheral monocytes, which differentiate into tissue
macrophages and become activated in situ. We investigated
the ability of HA fragments and IFN to induce iNOS in primary BMDMs
which, when cultured for 5 to 7 days in the presence of colony
stimulating factor-1, represent a mature macrophage population capable
of responding to inflammatory stimuli. The time course of HA
fragment-dependent iNOS gene expression in these cells is
shown in Fig. 3a, with maximal mRNA
levels seen at 3 h. IFN
alone induced very low levels of iNOS
gene expression in BMDMs (Fig. 3b), but the combination of
HA fragments and IFN
induced gene expression in a synergistic
fashion. These data demonstrate that HA fragments synergize with IFN
to induce iNOS in primary BMDMs.
HA Fragments Induce iNOS Gene Expression in Inflammatory Alveolar Macrophages
We next asked whether tissue inflammation affects the
capacity of macrophages to produce iNOS in response to HA fragments. To
address this question we collected AM by BAL from normal rats and from
rats that had received intratracheal bleomycin, which induces a series
of well-characterized inflammatory changes in the lungs of experimental
animals (50, 51). Normal AM expressed minimal iNOS mRNA in response
to HA fragments (Fig. 4) and were much less responsive
than either the alveolar cell line MH-S or primary BMDMØ. However, AM
isolated 5 days after treatment with bleomycin responded dramatically
to HA fragments. In fact, the response to HA fragments alone was equal
to that of HA fragments plus IFN, suggesting that the inflammatory
AM were already maximally primed. This response persisted at day 9 following treatment. These results suggest that the presence of an
inflammatory stimulus such as HA fragments is necessary but not
sufficient to induce production of iNOS by macrophages and that
macrophages acquire the capacity to respond to HA fragments in the
setting of inflammation.
Inhibitors of NF-
Recent evidence indicates that
the induction of iNOS by LPS, and by LPS plus IFN, is dependent upon
NF-
B (34, 36-38). Since HA fragments induce NF-
B DNA binding
activity (41), we investigated whether this transcriptional regulator
might also be involved in HA fragment-dependent iNOS gene
induction. Using an oligonucleotide probe containing the proximal
NF-
B site from the iNOS promoter, we performed electromobility shift
assays with nuclear extracts obtained from MH-S cells stimulated with
HA fragments. Fig. 5a demonstrates that HA
fragments induced NF-
B DNA binding activity in 1 h. In order to
characterize the composition of the NF-
B complex, we performed
super-shift assays using antibody to the p50 and p65 subunits of
NF-
B (Fig. 5b). p50 and p65 constituted the majority of
the HA fragment-induced DNA binding activity, whereas the related
family member c-REL appeared to contribute to this binding activity
only minimally.
HA fragment-induced NF-B DNA binding
activity and iNOS gene expression are blocked by inhibitors of NF-
B.
a, electromobility shift assays were performed using nuclear
extracts prepared from MH-S cells after 1 h stimulation with
medium alone (lane 2) or purified HA fragments at
concentrations of 1 µg/ml (lane 3), 10 µg/ml (lane
4), 25 µg/ml (lane 5), 50 µg/ml (lane
6), and 100 µg/ml (lane 7). Nuclear extracts were
incubated with 32P-labeled oligonucleotide containing the
proximal NF-
B binding site of the murine iNOS promoter as described
under "Experimental Procedures." Lane 1 represents
radiolabeled oligonucleotide probe alone, and lane 8 represents HA fragments (100 µg/ml) plus radiolabeled oligonucleotide
in the presence of 10 × excess unlabeled oligonucleotide. b,
electromobility shift assays were performed using nuclear extracts prepared from MH-S cells after 1 h stimulation with purified HA fragments alone (lane 2, 50 µg/ml) or HA fragments in the
presence of antibody to p50 (lane 3), p65 (lane
4), p50 and p65 (lane 5), and c-REL (lane
6). Lane 1 represents radiolabeled oligonucleotide probe alone. c, nuclear extracts were prepared from MH-S
cells after 1 h stimulation with purified HA fragments (50 µg/ml) or HA fragments plus IFN
(1000 units/ml), with or without
PDTC (100 µM), MG132 (15 µM), or TPCK (20 µM). Electromobility shift assays were performed as
described. d, MH-S cells were stimulated for 4 h with
purified HA fragments (100 µg/ml) or HA fragments plus IFN
(1000 units/ml) with or without PDTC (100 µM), MG132 (15 µM), or TPCK (20 µM). Total RNA was
collected, and Northern analysis was performed.
We then utilized three distinct classes of NF-B inhibitors in
order to determine the effect of NF-
B inhibition on HA
fragment-dependent iNOS induction. The serine
protease inhibitor TPCK and the ubiquitin-proteasome inhibitor MG132
both block activation of NF-
B by stabilizing the inhibitor I-
B
(34, 52-54). The antioxidant PDTC inhibits NF-
B activity by a
mechanism that is not fully understood but that is believed to be
related to the scavenging of oxygen radicals (55, 56). Both HA
fragment-dependent NF-
B DNA binding activity and iNOS
mRNA levels were reduced or eliminated by all three of these
inhibitors (Figs. 5, c and d). HA
fragment-induced NF-
B DNA binding activity and iNOS gene expression
were unaffected by the control serine protease inhibitor leupeptin
(data not shown). Interestingly, HA fragment-induced NF-
B DNA
binding activity was not enhanced by the addition of IFN
(Fig.
5c), indicating that the synergistic induction of iNOS
mRNA by HA fragments and IFN
is not due to increased NF-
B
binding at the proximal
B site. These data are in accordance
with published work demonstrating that IFN
regulates iNOS
transcription through interferon regulatory factor-1 (57, 58).
In order to investigate
more directly the effect of HA fragments on iNOS promoter activity, we
transiently transfected a construct consisting of 1700 base pairs of
the iNOS promoter, which contains two NF-B binding sites, upstream
of a luciferase reporter gene into the mouse macrophage-like cell line
RAW 264.7. As shown in Fig. 6, HA fragments induced a
greater than 3-fold increase over base line in wild-type promoter
activity. However, HA fragment-dependent increases in
promoter activity were no longer observed when the proximal NF-
B
binding site was mutated. This indicates that the proximal NF-
B
binding site is critical for HA fragment-dependent iNOS
gene expression.
We present data supporting the hypothesis that fragments of the
ECM component HA induce iNOS expression in murine macrophages through
an NF-B-dependent mechanism and that IFN
synergistically enhances the HA fragment-dependent
induction of iNOS. These findings expand our understanding of the role
of HA fragments in macrophage activation and suggest that low molecular
weight HA may function as a macrophage triggering stimulus in the
setting of non-infectious inflammation. Together with our previous
observation that HA fragments induce the production of chemokines in
macrophages, the current findings support the hypothesis that HA
fragments participate in the development of a complex inflammatory
milieu characterized by the production of multiple macrophage-derived
inflammatory mediators that can in turn recruit additional macrophages
and directly influence effector cell functions.
Since the in vivo response to an inflammatory stimulus
involves complex interactions among distinct populations of
macrophages, we examined the ability of HA fragments to induce iNOS
expression in several different types of macrophages. We found that
cells from the transformed cell line MH-S, which are derived from
resident alveolar macrophages, and primary BMDMs both readily expressed iNOS mRNA when stimulated with HA fragments. However, we observed significant differences in the response to HA fragments between normal
AM and from bleomycin-exposed inflammatory AM. Normal AM responded
minimally, whereas inflammatory AM stimulated with HA fragments
exhibited a marked induction of iNOS mRNA. The dramatic induction
of iNOS mRNA in inflammatory AM was not further enhanced by IFN,
suggesting that these cells were already maximally primed. These
findings suggest that the response to HA fragments is dependent on the
state of macrophage activation. Additionally, these results suggest a
role for HA fragments in bleomycin-induced lung injury. HA levels are
increased in the lungs of experimental animals following treatment with
bleomycin (51), and although bleomycin-exposed AM have been shown to
express higher levels of iNOS at base line than normal (59), our data
indicate that HA fragments induce further elevations in the levels of
iNOS that may contribute to the pulmonary toxicity observed with this
antineoplastic agent.
Our results indicate that HA fragment-dependent iNOS
induction in murine macrophages occurs through an
NF-B-dependent mechanism. The role of NF-
B in
LPS-induced macrophage iNOS production has been well established
(36-38). However, what role if any NF-
B plays in the
IFN
-dependent synergistic induction of iNOS is not clear. There is evidence that NF-
B participates in the
IFN
-mediated enhancement of iNOS induction by LPS in epithelial
cells (60), but with rare exceptions (37) the studies reported to date
in macrophages do not specifically address mechanistic distinctions between induction of iNOS by LPS and by LPS plus IFN
. We found that
the addition of IFN
did not increase HA
fragment-dependent NF-
B DNA binding activity in MH-S
cells, and inhibitors of NF-
B were more effective in blocking HA
fragment-induced DNA binding activity and iNOS gene expression than in
blocking binding activity and gene expression induced by HA fragments
plus IFN
. These findings suggest that the synergistic enhancement of
HA fragment-dependent iNOS induction observed with IFN
is likely to be mediated by alternative transcriptional regulator(s).
These data are consistent with the results demonstrating a critical
role for interferon regulatory factor-1 in mediating the IFN
-induced
synergy with LPS on the expression of iNOS (57, 58). Induction of iNOS, through the generation of HA fragments at sites of inflammation, may
prove to be an important mechanism for the local production of nitric
oxide in inflamed tissues. Given the ability of HA fragments to
activate the NF-
B/I
B system and induce the production of chemokines by macrophages, HA fragments may play an important part in
propagating the inflammatory response. HA fragments may also contribute
to the pathological development of chronic inflammation through the
induction of NO· and other reactive nitrogen intermediates.
Peroxynitrite, for example, is a product of the reaction of NO·
and superoxide. This highly reactive species has been implicated in
protein nitrosylation and tissue damage (8, 61) and can degrade high
molecular weight HA to smaller fragments. It is intriguing to speculate
that HA fragments generated during the course of inflammation may
indirectly induce peroxynitrite production, which could in turn
generate more HA fragments and lead to the development of an ongoing
inflammatory state. Through their ability to activate macrophages and
induce inflammatory mediators and reactive intermediates, HA fragments
may be instrumental in establishing the pathological cycle of
inflammation that ultimately results in chronic inflammatory disorders.