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J Biol Chem, Vol. 273, Issue 52, 35088-35094, December 25, 1998


Hyaluronan Fragments Synergize with Interferon-gamma to Induce the C-X-C Chemokines Mig and Interferon-inducible Protein-10 in Mouse Macrophages*

Maureen R. HortonDagger , Charlotte M. McKeeDagger , Clare Bao§, Fang Liao§, Joshua M. Farber§, Jennifer Hodge-DuFour, Ellen Puré, Bonnie L. Oliver**Dagger Dagger , Timothy M. Wrightparallel , and Paul W. Noble**Dagger Dagger §§

From the Dagger  Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the § Laboratory of Clinical Investigation, NIAID, National Institutes of Health, Bethesda, Maryland 20892, the  Wistar Institute, Philadelphia, Pennsylvania 15261, the parallel  Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 19104, the ** Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, and the Dagger Dagger  Department of Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Hallmarks of chronic inflammation and tissue fibrosis are increased influx of activated inflammatory cells, mediator release, and increased turnover and production of the extracellular matrix (ECM). Recent evidence has suggested that fragments of the ECM component hyaluronan play a role in chronic inflammation by inducing macrophage expression of chemokines. Interferon-gamma (IFN-gamma ), an important regulator of macrophage functions, has been shown to induce the C-X-C chemokines Mig and IP-10. These chemokines affect T-cell recruitment and inhibit angiogenesis. The purpose of this investigation was to determine the effect of hyaluronan (HA) on IFN-gamma -induced Mig and IP-10 expression in mouse macrophages. We found a marked synergy between HA and IFN-gamma on Mig and IP-10 mRNA and protein expression in mouse macrophages. This was most significant with Mig, which was not induced by HA alone. The synergy was specific for HA, was not dependent on new protein synthesis, was not mediated by tumor necrosis factor-alpha , was selective for Mig and IP-10, and occurred at the level of gene transcription. These data suggest that the ECM component HA may influence chronic inflammatory states by working in concert with IFN-gamma to alter macrophage chemokine expression.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Activated macrophages play an essential role in inflammation through the release of a variety of mediators, including reactive oxygen species, reactive nitrogen species, proteases, chemokines, cytokines, and growth factors (1-5). Although the mechanisms controlling macrophage activation in inflammatory states are incompletely understood, recent studies suggest a role for extracellular matrix (ECM)1 components in the activation of inflammatory macrophages (6). In normal, healthy tissues, the ECM is composed of a complex array of proteins, proteoglycans, and glycosaminoglycans that play important roles in homeostasis (7) and maintenance of matrix structure. During an inflammatory response, there is increased production and degradation of the ECM, resulting in the accumulation of breakdown products of ECM components such as fibronectin, collagen, and glycosaminoglycans (8). Interestingly, these lower molecular mass ECM components have been shown to have different biological activities compared with their larger, native precursors (9-11). In fact, fragments of the ECM components collagen and fibronectin have been recently shown to have proinflammatory properties (12). Likewise, recent work from our laboratory and others has shown that low molecular weight fragments of the ECM glycosaminoglycan hyaluronan (HA) may play a role in macrophage activation (13-16)

HA is a ubiquitously distributed component of the ECM. In its native form, it exists as a high molecular weight nonsulfated glycosaminoglycan polymer (17, 18) made up of repeating disaccharide units of (beta ,1-4)-D-glucuronic acid-(beta ,1-3)-N-acetyl-D-glucosamine. High molecular mass HA is believed to have many functions in healthy tissue, such as water homeostasis, plasma protein distribution, and matrix structuring (17). However, at sites of inflammation and tissue injury, there is an accumulation of lower molecular mass HA species (19-21), which have different biological functions than their high molecular mass precursors (13-16). Recent studies have suggested that these lower molecular weight forms of HA may stimulate macrophages recruited to sites of inflammation to produce important mediators of tissue injury and repair (13-16). This effect is mediated, in part, by interaction with the HA receptor CD44 (13-16).

In the inflammatory milieu, there are numerous cytokines and chemokines that also influence macrophage expression of inflammatory genes. The cytokine IFN-gamma is an important modulator of macrophage effector functions and regulator of the inflammatory response (22). In addition to its antiviral activity, IFN-gamma also enhances certain macrophage functions, such as microbicidal and tumoricidal activity, through the production of reactive oxygen intermediates and reactive nitrogen intermediates (23, 24). Yet despite its many proinflammatory roles, IFN-gamma also inhibits macrophage expression of certain LPS-induced chemokines, such as monokine chemoattractive protein-1 and KC (24).

IFN-gamma has recently been shown to induce macrophage expression of the novel chemokines monokine induced by interferon-gamma (Mig) and interferon-inducible protein-10 (IP-10) (25, 26). Both Mig and IP-10 are members of the C-X-C chemokine family and may play roles in chronic inflammation (27, 28), as well as in viral and protozoan infections (25, 29, 30). In chronic infectious states, expression of Mig and IP-10 has been shown to correlate with IFN-gamma expression (29). Mig and IP-10 have each been implicated in T-cell trafficking, chemotaxis, and activation (31-33). Mig and IP-10 have also been shown to have angiostatic properties (34-36) and thus may play an important role in regulating tissue granulation and remodeling by inhibiting angiogenesis. IP-10 is induced in macrophages by alpha , beta , and IFN-gamma , as well as by LPS (25, 37, 38). To date, Mig has only been shown to be induced in macrophages by IFN-gamma (26, 39). Recent investigations have described synergistic enhancement of IFN-gamma -induced Mig and IP-10 expression by TNF-alpha in fibroblasts (40, 41).

In this report, we examined the effect of low molecular mass HA fragments on IFN-gamma -induced expression of Mig and IP-10. We observed a striking synergy between HA and IFN-gamma on the expression of Mig and IP-10 mRNA and protein levels in murine macrophages that occurs at the level of gene transcription. These results identify a previously uncharacterized mechanism by which the ECM, acting in conjunction with IFN-gamma , may regulate the immune response by influencing the expression of specific chemokines.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells, Mice, and Cell Lines-- The mouse alveolar macrophage cell line MH-S (42) was purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium 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 (16), from female C3H/HeJ LPS hyporesponsive mice purchased from The Jackson Laboratory (Bar Harbor, ME). After harvest, cells (11 × 106 cells/dish) were cultured for 5 days in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated low LPS fetal bovine serum, 15% L-cell media, and 1% penicillin-streptomycin/1% glutamine (Biofluids) at 37 °C under 8% CO2. Thioglycollate-elicited peritoneal macrophages were lavaged from female C3H/HeJ mice or TNF-alpha (-/-) mice (43) 4 days after injection of 2 ml of sterile thioglycollate (Sigma). The cells were allowed to adhere overnight in RMPI 1640 medium supplemented with 10% heat-inactivated low LPS fetal bovine serum and 1% penicillin-streptomycin/1% glutamine before use. To exclude effects of contaminating LPS on experimental conditions, cell stimulation was carried out in the presence of B 10 µg/ml polymixin (Calbiochem, La Jolla, CA).

Chemicals and Reagents-- Purified HA fragments from human umbilical cords were purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). The HA-ICN preparation was free of protein (<2%) and other glycosaminoglycans with a peak molecular mass of 200,000 Da (44). Recombinant mouse IFN-gamma (specific activity, 3.0 × 105 units/ml; endotoxin level less than 0.2 ng/mg) was from Genzyme Corporation (Cambridge, MA), the recombinant mouse TNF-alpha was purchased from R & D Systems, the anti-TNF-alpha was from Cappel (Aurora, OH), and the cycloheximide (10 µg/ml) was from Sigma. Polymixin B was purchased from Calbiochem. Stock solutions of reagents were tested for LPS contamination using the Limulus amebocyte assay (Sigma).

Northern Analysis of mRNA Production-- RNA 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 (16). Ten µg of total RNA was electrophoresed under denaturing conditions through a 1% formaldehyde-containing agarose gel and RNA was transferred to Nytran (Schleicher and Schuell) hybridization filters. Blots were briefly rinsed in 5× SSC, RNA was cross-linked to the filter by UV cross-linking (Stratagene, La Jolla, CA), and blots were hybridized overnight with 106 cpm/ml of 32P-labeled DNA labeled by the random prime method (Amersham Pharmacia Biotech). 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 for 20 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 aldolase (45). Densitometric scanning was performed using a Molecular Dynamics Personal Densitometer SI (Sunnyvale, CA).

Western Analysis of Protein Secretion-- Western blot analysis was performed as described (46). Briefly, 200 µg of macrophage-conditioned media was fractionated by SDS-polyacrylamide gel electrophoresis (10%), transferred to a nylon membrane, blocked and washed, incubated with the polyclonal anti-Mig antibody at a dilution of 1:2500 or polyclonal anti-crg-2 (murine IP-10 antibody from R & D Systems) at a dilution of 1:3000, and developed with a chemiluminescent system according to the manufacturer's instructions (Amersham Pharmacia Biotech). The recombinant Mig protein was prepared as described previously (26), and the recombinant crg-2 (murine IP-10) was purchased from R & D Systems.

Nuclear Run-on-- Nuclei from confluent monolayers of MH-S cells were harvested by scraping in ice-cold phosphate-buffered saline and subsequently isolated by centrifugation through a sucrose cushion (47). Nuclei were then incubated for 30 min with 1 M dithiothreitol, 20 mM NTPs, and 100 µCi of [32P]UTP in transcription buffer. The nuclei received a cold UTP chase for 10 min before the reaction was stopped by the addition of termination buffer, DNase (Promega, Madison, WI) and RNase-Inhibitor (Boehringer Mannheim). The nuclei were then incubated with tRNA (Sigma) for 15 min before the addition of 10% SDS, 0.2 M EDTA and proteinase K (Sigma). After 15 min of incubation, the RNA was extracted with phenol:chloroform:isoamyl alcohol, precipitated with 20% trichloroacetic acid, washed with 5% trichloroacetic acid/5% PPi, dissolved in 0.1% SET, and precipitated for a second time with 4 M NaAc and 100% ETOH at -80 °C for 30 min. Purified radiolabeled RNA was washed once in 70% ETOH, dried with a speed vacuum concentrator (Savant), and resuspended in 100 µl of water. 5 µl of the radioactive RNA was counted, and all samples were normalized for counts using hybridization fluid. Normalized samples were hybridized with prehybridized Optitran-S membranes (Schleicher and Schuell) containing the cDNAs of interest. Blots were hybridized for 3-4 days and then washed once in 2× SSC/0.1% SDS at room temperature for 5 min with shaking, and then washed twice in 0.1× SSC/0.1% SDS at 50 °C with shaking (20 min each wash). The blots were then exposed and quantitated with a PhosphorImager (Molecular Dynamics).

Transient Transfections-- Transient transfections of the MH-S cells were performed using Lipofectin as described elsewhere (48). Briefly, 2.5 × 106 cells were plated in 60-mm tissue culture dishes 1 day prior to transfection. The cells were washed twice with Opti-MEM (Life Technologies, Inc.) and incubated with the DNA/Lipofectin solution (5 µg of DNA and 15 µg of Lipofectin) for 7 h. The DNA/Lipofectin solution was then aspirated, 3 ml of Opti-MEM was added, and the cells were incubated overnight at 37 °C. The contents of the dishes were scraped in 8 ml of fresh medium and divided between four 60-mm culture dishes. After incubation for 4 h to allow the cells to adhere, one dish from each transfection group received medium alone, one received HA (100 µg/ml), the third received IFN-gamma alone (300 units/ml), and the last received HA + IFN-gamma . The cells were harvested for analysis of chloramphenicol acetyltransferase (CAT) expression after 18 h of stimulation. The -1117/+43 Mig/firefly luciferase, gamma RE-1 × 4 Mig/firefly luciferase, and -299/+7 IP-10/CAT construct were prepared as described previously (49, 50).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

HA Fragments Synergize with IFNgamma to Induce Mig and IP-10 mRNA Expression in Mouse Macrophages-- We investigated the effect of HA and IFN-gamma on Mig and IP-10 mRNA expression by mouse macrophages. Mig and IP-10 were originally identified as being induced by IFN-gamma stimulation of the mouse macrophage cell line RAW 264.7 (25, 26). We have recently shown that HA fragments (200,000 Da) induce the expression of a number of inflammatory mediators, including several members of the chemokine family (MIP-1alpha , MIP-1beta , IP-10, RANTES, monokine chemoattractive protein-1, IL-8, and KC), as well as the cytokines IL-1, TNF-alpha , IL-12, and inducible nitric oxide synthase (13-16). Furthermore, recent work from our laboratory has shown that IFN-gamma selectively inhibits HA-induced MIP-1alpha and MIP-1beta expression in primary mouse macrophages (51). We were therefore interested in the possible role for low molecular mass HA in regulating Mig and IP-10 expression in the presence of IFN-gamma . In order to assess the combined effect of HA and IFN-gamma on Mig and IP-10 gene expression, macrophages were simultaneously stimulated with HA and IFN-gamma for 6 h, mRNA was isolated, and Northern analysis was performed. As shown in Fig. 1A, HA alone had virtually no effect on Mig or IP-10 mRNA expression in inflammatory thioglycollate-elicited peritoneal macrophages from C3H/HeJ-LPS hyporesponsive mice, whereas IFN-gamma induced moderate expression of Mig and IP-10 mRNA. However, we unexpectedly found that HA dramatically influences the effect of IFN-gamma on Mig and IP-10 mRNA expression (Fig. 1A). Similar results were observed with MH-S cells, a murine alveolar macrophage cell line (Fig. 1B). In these alveolar macrophages, the synergy is most apparent for Mig where, in addition to no effect on Mig gene expression by HA alone, IFN-gamma alone induced only a faint signal (Fig. 1B). However, the combination of HA and IFN-gamma markedly induced Mig mRNA expression (Fig. 1B). In this alveolar macrophage cell line, HA minimally induced IP-10 mRNA, and IFN-gamma had a moderate effect on IP-10 mRNA, but there was still marked enhancement of IP-10 mRNA when cells were stimulated both with HA and IFN-gamma (Fig. 1B).


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  		Fig. 1.   HA and IFN-gamma synergistically induce Mig and IP-10 mRNA expression in murine macrophages. Northern analysis of mRNA derived from elicited peritoneal macrophages (EPM) (A) and MH-S (B) cells stimulated with HA (100 µg/ml) and IFN-gamma (300 units/ml) for 6 h. These data are representative of six experiments.

HA and IFNgamma Exhibit a Time- and Dose-dependent Synergistic Induction of Mig and IP-10 mRNA Levels-- To further delineate the effects of HA and IFN-gamma on Mig and IP-10 mRNA expression, we stimulated MH-S cells simultaneously with HA and IFN-gamma for varying time intervals and found that HA failed to independently induce Mig expression at any time point and only minimally induced IP-10 mRNA (data not shown). However, the synergistic effect of HA and IFN-gamma on Mig and IP-10 mRNA expression was seen as early as 3 h after simultaneous stimulation of MH-S cells, peaked after 6-9 h, and decreased toward baseline after 24 h of stimulation (data not shown).

We then determined the dose-response relationships for HA and IFN-gamma to induce Mig and IP-10 gene expression in MH-S cells. The effect of varying concentrations of HA with a constant concentration of IFN-gamma on Mig and IP-10 mRNA expression by the alveolar macrophage cell line MH-S is shown in Fig. 2A. The synergy between HA and IFN-gamma on Mig and IP-10 gene expression was observed with as little as 1 µg/ml HA and was maximal at 10 µg/ml HA in the presence of 300 units/ml IFN-gamma . The converse is shown in Fig. 2B using a concentration of 100 µg/ml HA. The synergy between HA and IFN-gamma occurs with as little as 1 unit/ml IFN-gamma and is maximal at 10 units/ml IFN-gamma .


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  		Fig. 2.   Dose response of HA and IFN-gamma for Mig and IP-10 gene expression in MH-S cells. A, Northern analysis of mRNA derived from MH-S cells stimulated with varing doses of HA ± IFN-gamma (300 units/ml) for 6 h. B, Northern analysis of mRNA derived from MH-S cells stimulated with varing doses of IFN-gamma  ± HA (100 µg/ml) for 6 h. These data are representative of four experiments.

IFNgamma Synergizes Specifically with HA Fragments to Induce Mig and IP-10 mRNA Expression in Mouse Macrophages-- In order to determine whether the synergy between HA and IFN-gamma was specific to low molecular mass HA fragments, we stimulated MH-S cells in the presence of IFN-gamma and numerous other glycosaminoglycans. Fig. 3 shows that IFN-gamma only synergizes with HA fragments and not high molecular mass HA, chondroitin sulfate A or B, or HA disaccharides to induce Mig or IP-10 mRNA expression. Thus, the synergy between HA and IFN-gamma on Mig and IP-10 gene expression in MH-S cells appears to be specific to the low molecular mass HA fragments.


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  		Fig. 3.   Specificity of HA in synergy with IFN-gamma to induce Mig and IP-10 gene expression in MH-S cells. Northern analysis of mRNA derived from MH-S stimulated with HA (100 µg/ml) or alternative ECM components in the presence of IFN-gamma (300 units/ml) for 6 h. These data are representative of four identical experiments.

HA and IFNgamma Synergize to Induce Mig Protein Production in Mouse Macrophages-- Having identified the synergistic effect between HA and IFN-gamma on Mig and IP-10 mRNA levels, we investigated Mig and IP-10 production at the protein level. MH-S cells were simultaneously stimulated with HA in the presence of IFN-gamma for 20 h. Mig protein secretion was determined in the supernatants by Western blot analysis. Fig. 4A shows that there was little Mig protein present in the conditioned media from unstimulated cells or cells stimulated with HA or IFN-gamma alone. However, when the macrophages were stimulated with HA and IFN-gamma together, there was marked induction of Mig protein production in the conditioned media. Fig. 4B shows similar synergistic induction of IP-10 protein production by MH-S cells stimulated with both HA and IFN-gamma . However, unlike Mig, there was some IP-10 protein induced by HA and IFN-gamma alone. Thus, the synergy between HA and IFN-gamma is also demonstrated at the protein level.


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  		Fig. 4.   HA and IFN-gamma synergize to induce Mig and IP-10 protein secretion in MH-S cells. Western analysis was performed on supernatants from MH-S cells stimulated with HA (100 µg/ml) and IFN-gamma (300 units/ml) for 24 h. Recombinant Mig and IP-10 protein were included as controls. A, Mig; B, IP-10. These data are representative of three identical experiments.

Cycloheximide Has a Minimal Effect on the Synergy between HA and IFN-gamma on Mig or IP-10 Gene Expression in Mouse Macrophages-- In order to further dissect the mechanism for the observed synergy, we examined the role of new protein synthesis on the synergy between HA and IFN-gamma on Mig and IP-10 mRNA expression in MH-S cells. We pretreated MH-S cells with cycloheximide (CHX) for 30 min before the addition of HA and IFN-gamma for 6 h. As shown in Fig. 5, CHX minimally inhibited the synergistic expression of Mig and IP-10 mRNA by HA and IFN-gamma . Similarly, CHX had no effect on Mig or IP-10 gene expression from unstimulated cells or cells stimulated with HA alone, and it had little effect on cells stimulated with IFN-gamma alone (Fig. 5). Thus, the synergistic induction of Mig and IP-10 gene expression by HA and IFN-gamma does not appear to require new protein synthesis.


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  		Fig. 5.   Effect of CHX on the synergistic induction of Mig and IP-10 by HA and IFN-gamma . MH-S cells were pretreated with CHX (10 µg/ml) for 30 min before stimulation with CHX (10 µg/ml) ± HA (100 µg/ml), ± IFN-gamma (300 units/ml) for 6 h. mRNA was isolated, and Northern analysis was performed. These data are representative of four identical experiments.

The Synergistic Induction of Mig and IP-10 Gene Expression by HA and IFNgamma Is Not Dependent on TNF-alpha -- Recently, Ohmori et al. (40, 41) have provided data that TNF-alpha and IFN-gamma synergize to induce Mig and IP-10 mRNA in fibroblasts. We have previously shown that HA induces TNF-alpha expression by mouse macrophages (16). Therefore, we investigated the possible role of TNF-alpha in the synergistic induction of Mig and IP-10 gene expression by HA and IFN-gamma . First, using anti-TNF-alpha neutralizing antibodies, we found that TNF-alpha was not necessary for the induction of Mig or IP-10 mRNA by HA and IFN-gamma in MH-S cells (data not shown). In addition, we isolated thioglycollate-elicited peritoneal macrophages from mice that have had the TNF-alpha gene deleted (43). As shown in Fig. 6, HA and IFN-gamma synergize to induce Mig and IP-10 mRNA expression despite the complete absence of TNF-alpha . Similar results were also found in thioglycollate-elicited peritoneal macrophages from wild type littermate controls (Fig. 6). Thus, the synergy between HA and IFN-gamma on Mig and IP-10 gene expression occurs independently of TNF-alpha expression.


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  		Fig. 6.   TNF-alpha is not required for the synergistic induction of Mig or IP-10 by HA and IFN-gamma in elicited peritoneal macrophages. Thioglycollate-elicited peritoneal macrophages from TNF -/- and control C57BL6 mice were stimulated with HA (100 µg/ml) ± IFN-gamma for 6 h. mRNA was isolated, and Northern analysis was performed. This is a representative blot of two identical experiments.

HA and IFNgamma Synergize to Induce Mig and IP-10 Gene Expression in MH-S Cells at the Level of Gene Transcription-- We examined the direct effect of HA and IFN-gamma on the induction of Mig and IP-10 gene transcription by performing nuclear run-on assays. The mRNA transcribed in nuclei isolated from MH-S cells stimulated with HA, IFN-gamma , and HA + IFN-gamma for 3 h was radiolabeled. As shown in Fig. 7, there was little transcription of Mig mRNA in unstimulated cells or cells stimulated with HA or IFN-gamma alone. However, there was marked enhancement of Mig transcription in cells stimulated with the combination of HA and IFN-gamma . Similar results were found with IP-10, although the baseline levels of IP-10 mRNA transcription were higher (Fig. 7). At earlier time points, after 1 h stimulation, there was modest enhancement of Mig and IP-10 mRNA transcription in cells treated with IFN-gamma alone, but the synergistic enhancement with HA was not present (data not shown). Thus, HA and IFN-gamma synergize to induce Mig and IP-10 gene expression at the level of transcription


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  		Fig. 7.   The effect of HA and IFN-gamma on Mig and IP-10 gene transcription. MH-S cells were stimulated with HA (100 µg/ml) and/or IFN-gamma (300 units/ml) for 3 h. Nuclei were isolated, and nuclear run-on analysis was performed as described under "Experimental Procedures." This is a representative blot of four identical experiments.

To further elucidate the mechanism responsible for the synergy between HA and IFN-gamma on Mig promoter activity, we transiently transfected MH-S cells with a 5' promoter construct spanning base pairs -1117 to +43 upstream from a firefly luciferase reporter gene. As shown in Fig. 8A, IFN-gamma alone induced firefly luciferase activity by 12-fold, whereas stimulation of transfected cells with HA and IFN-gamma together showed over a 32-fold induction of luciferase activity over unstimulated cells. We also transiently transfected MH-S cells with a promoter construct containing four copies of the interferon-gamma -responsive site, -200 to -167, of the Mig promoter upstream of a firefly luciferase reporter gene (gamma RE-1 × 4). IFN-gamma alone induced firefly luciferase activity by 6.2-fold over unstimulated cells, whereas stimulation with HA + IFN-gamma only minimally induced (1.7-fold) luciferase activity. Thus, the gamma RE is not sufficient to account for the synergistic enhancement of Mig gene expression by HA fragments and IFN-gamma .


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  		Fig. 8.   HA and IFN-gamma synergize to induce Mig and IP-10 promoter activity. MH-S cells were transfected with constructs containing the 5' Mig promoter spanning base pairs -1117 to +43 (A), or four copies of the gamma RE-1 site from the Mig promoter upstream of a luciferase reporter 5' IP-10 promoter spanning the region of base pairs -299 to +7 upstream of a CAT reporter gene (B). Transfected cells were stimulated with HA (100 µg/ml) ± IFN-gamma (300 units/ml) for 18 h. Promoter activity was assessed by luciferase or CAT activity. Shown are the results of six identical experiments.

Similar results were found with transient transfections of MH-S cells with a 5' IP-10 promoter construct spanning base pairs -299 to +7 upstream from a CAT reporter gene. CAT activity was induced 10-fold by HA alone, 8-fold by IFN-gamma alone, and 28-fold after stimulation of transfected cells with HA and IFN-gamma (Fig. 8B). Together, these data suggest that the regulatory elements that convey synergy between HA and IFN-gamma on Mig gene transcription are contained within the approximately 1-kilobase proximal promoter, and for IP-10 gene transcription within the approximately 300-base pair proximal promoter.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The purpose of this study was to examine the effects of the ECM glycosaminoglycan HA on the expression of the inflammatory chemokines Mig and IP-10 in the presence of IFN-gamma . Previous work in our laboratory has shown that low molecular mass HA fragments can stimulate mouse macrophages to express numerous chemokines (13-15). Mig is unique in that no stimulus other than IFN-gamma has been shown to induce its expression in macrophages. IP-10, on the other hand, has been shown to be induced by IFN-alpha , -beta , and -gamma , as well as LPS. The results presented herein identify a new role for the ECM in enhancing IFN-gamma induced Mig and IP-10 gene expression. Interestingly, the effect of HA on IFN-gamma -inducible Mig and IP-10 expression appeared to be unique among the chemokines we examined. We have recently shown that IFN-gamma inhibits HA-induced expression of MIP-1alpha , MIP-1b, and KC, while having no significant effect on RANTES and monokine chemoattractive protein-1 (51).

HA alone did not induce Mig and only minimally induced IP-10 mRNA and protein production in a variety of murine macrophages. However, HA enhanced the IFN-gamma -induced steady state mRNA levels of Mig and IP-10 in both primary mouse macrophages (elicited peritoneal macrophages) and in MH-S cells, an alveolar macrophage cell line. The increased Mig and IP-10 mRNA levels were found to correlate with Mig and IP-10 protein production in cells stimulated with both HA and IFN-gamma .

To further characterize the mechanisms by which HA and IFN-gamma synergize to induce Mig and IP-10 gene expression in macrophages, we performed time course and dose-response experiments. The peak synergy between HA and IFN-gamma occurs after 6-9 h of stimulation and with as little as 1 µg/ml of HA and 1 unit/ml of IFN-gamma . Furthermore, the effect of HA on IFN-gamma induced Mig and IP-10 expression is specific to HA and not a general characteristic of glycosaminoglycans. Low molecular mass HA fragments alone, not high molecular mas native HA, chondroitin sulfate A or B, or HA disaccharides, influenced the effect of IFN-gamma on Mig or IP-10 expression. These results suggest that the synergistic effect of HA on IFN-gamma -induced Mig and IP-10 mRNA expression is immediate, specific to HA fragments, and quite sensitive as it occurs with low concentrations of these mediators.

In an attempt to delineate whether new protein synthesis was necessary for synergy, we performed a series of experiments in the presence of the protein synthesis inhibitor cycloheximide. Evaluation of these experiments revealed that HA- and IFN-gamma -induced Mig and IP-10 expression was minimally blocked by CHX, suggesting that the synergy does not require synthesis of a secondary mediator.

Recent investigations have determined that TNF-alpha and IFN-gamma synergize to induce Mig and IP-10 mRNA expression in fibroblasts (40, 41); therefore, we examined the role of TNF-alpha in mediating the effect of HA on IFN-gamma -inducible Mig and IP-10 expression. Although HA independently induces TNF-alpha expression in macrophages, sufficient concentrations of TNF-alpha blocking antibodies to inhibit all HA-induced TNF-alpha activity failed to inhibit the induction of Mig or IP-10 by HA and IFN-gamma in MH-S cells (data not shown). Furthermore, thioglycollate-elicited peritoneal macrophages from TNF-alpha -deficient mice exhibited the same synergy between HA and IFN-gamma on Mig and IP-10 expression as did littermate mice expressing TNF-alpha . Thus, unlike with fibroblasts, the synergistic effect of HA and IFN-gamma on Mig expression is independent of TNF-alpha .

The synergistic induction of Mig and IP-10 by HA and IFN-gamma is due to the up-regulation of Mig and IP-10 gene transcription. Nuclear run-on studies in the MH-S cells show that HA does not induce Mig mRNA transcription and only minimally induces IP-10. IFN-gamma alone has minimal effect on Mig and IP-10 transcription compared with unstimulated cells. However, cells stimulated with both HA and IFN-gamma have a marked enhancement of Mig and IP-10 gene transcription. Similarly, when MH-S cells were transfected with a Mig promoter construct containing the 5' regulatory region previously shown to convey the IFN-gamma responsiveness, there was over a 32-fold induction in Mig promoter activity compared with only a 12-fold induction in promoter activity by IFN-gamma alone. Similarly, when MH-S cells were transfected with a IP-10 promoter construct containing the 5' regulatory region previously shown to convey the IFN-gamma responsiveness, there was over a 28-fold induction in IP-10 promoter activity compared with only an 8-fold induction in promoter activity by IFN-gamma alone. Furthermore, in the case of Mig, the gamma RE, although enough to convey responsiveness to IFN-gamma , failed to account for the HA- and IFN-gamma -induced synergistic enhancement of Mig gene expression. Thus, the gamma RE is not sufficient to allow for the synergistic effects of HA and IFN-gamma . Together, these results suggest that HA profoundly enhances IFN-gamma induced Mig and IP-10 steady-state mRNA expression and does so at the level of Mig and IP-10 gene transcription.

Recent studies have suggested that IFN-gamma induction of Mig is mediated by the transcription factor gamma RF-1(50). gamma RE-1, the unique IFN-gamma -responsive cis element in the Mig promoter, is an imperfect palindrome consisting of the following sequence: 5'-TTxxxATAAACxxxxxGTTTATXXXAA-3'(50). gamma RF-1 has recently been shown to consist of a complex containing STAT-1alpha (49). The electrophoretic and binding properties of gamma RF-1 are distinct, however, from the previously described dimeric form of STAT-1 also known as gamma -interferon-activated factor (GAF) (reviewed in Ref. 52). In contrast to the ability of GAF to bind to a monomeric STAT-1 binding site, purified gamma RF-1 does not bind to the STAT-1 site (49) but rather is specific for the imperfect palindrome resembling a STAT binding site. These studies are further supported by the report by Ohmori et al. (41), which has shown that in STAT 1-deficient mice, IFN-gamma fails to induce Mig gene expression in fibroblasts (41). One possible mechanism for synergy between HA and IFN-gamma could be enhanced phosphorylation of STAT-1 in the presence of HA. We investigated this possibility and found no effect of HA on IFN-gamma -induced phosphorylation of STAT-1 (data not shown).

Similarly, investigators have defined the IFN-gamma -responsive element in the IP-10 promoter in macrophages (40). The IP-10 promoter contains an interferon stimulus response element, as well as two kappa B sites located at -228 to -102 in the 5' proximal promoter (40). Although the interferon stimulus response element is necessary and sufficient for IFN-gamma induced IP-10 gene expression, optimal response to IFN-gamma required both the interferon stimulus response element and one of two kappa B sites.

Low molecular mass HA fragments have been shown to activate the NF-kappa B/Ikappa -Balpha transcriptional regulatory system (53). Interestingly, analysis of the Mig promoter reveals that there are three NF-kappa B binding sites on the 5' promoter downstream from the gamma RE-1 and, as noted, two NF-kappa B sites downstream from the interferon stimulus response element on the 5' IP-10 promoter. Thus, HA may be synergizing with IFN-gamma to induce chemokine gene expression through interactions between a STAT-1alpha like protein and/or NF-kappa B for Mig and IP-10 gene expression. Studies to address this possibility are ongoing.

Interactions between ECM and cytokines in regulating inflammatory gene expression may have an important role in determining the resolution of an inflammatory response. Recent studies have shown that HA and IFN-gamma promote the expression of macrophage-derived IL-12 and reactive nitrogen intermediates (14, 15) while abrogating the ability of HA to induce MIP-1alpha , MIP-1beta , and KC (51). These new data add Mig and IP-10 to the subgroup of chemokines produced in the presence of low molecular mass HA and IFN-gamma . It is interesting to speculate that introducing IFN-gamma at the appropriate time in the inflammatory response will promote resolution through the direct effects on macrophage chemokine production. Thus, understanding the molecular mechanisms regulating the interaction between ECM- and IFN-gamma -`inducible genes may lead to new approaches to ameliorate chronic inflammation.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (K11HL02880, RO1HL60539, and 5F32HL09614-02) and the American Lung Association.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. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Yale University School of Medicine, Department of Veterans Affairs Connecticut Healthcare System, Pulmonary Section/111A, 950 Campbell Ave., West Haven, CT 06516. Tel.: 203-937-4977; Fax: 203-937-3455; E-mail: paul.noble{at}yale.edu.

The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; Mig, monokine induced by gamma -interferon; IP-10, interferon-inducible protein-10; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; EPM, elicited peritoneal macrophages; CHX, cycloheximide; TNF, tumor necrosis factor; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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