Interleukin-1 regulation of procollagenase mRNA and protein in periodontal fibroblasts in vitro

An in vitro model is used to investigate the hypothesis that activated fibroblasts produce collagenolytic activity in inflammatory sites. Interleukin-1, a cytokine present in the gingiva and crevicular fluid of periodontitis patients, has multiple biologic activities including the ability to stimulate collagenase in dermal and synovial fibroblasts. In this report IL-1 is tested on gingival (GF) and periodontal ligament fibroblasts (PLF) for its ability to increase collagenolytic activity and procollagenase mRNA and protein. GF produce a 3- to 7-fold increase in collagenase activity, while PLF collagenase activity is rarely increased above control amounts by IL-1 treatment. In contrast, both cell types demonstrate an increase in procollagenase protein production with IL-1 treatment. RNA from both GF and PLF contain procollagenase mRNA as demonstrated when northern blots of fibroblast total RNA are hybridized with the cDNA for human procollagenase. Treatment with IL-1 increases the steady-state levels of this message in GF by up to 10-fold in 48 hours when measured with dot blot analysis standardized for poly-A RNA. PLF also produce up to 7 times more message at the same dose and time. Since fibroblasts present in the lesion are exposed to inflammatory cell products it is possible that the production of collagenase by these cells could result in the destruction of the periodontal fibrous attachment.     

Stimulation of human periodontal ligament fibroblast collagenase production by a gingival epithelial cell-derived factor

To examine whether cell-to-cell interactions between human gingival epithelial cells (HGE) and periodontal ligament fibroblasts (PLF) or gingival fibroblasts (GF) take place in the periodontium, the effects on collagenase production by PLF and GF were analyzed after adding several concentrations of HGE-con-ditioned medium (HGE-CM) to PLF or GF culture. Collagenase production by both cell populations was stimulated by adding HGE-CM, which stimulated collagenase production by PLF to a greater extent than that by GF. The HGE-derived stimulatory factor had a molecular mass of approximately 20 kDa, and its stimulant effect was inhibited markedly in the presence of an anti-human interleukin-lα (IL-lα) neutralizing antibody, indicating that the factor was identical to, or antigenically cross-reactive with, IL-lα. These results suggest that epithelial apical migration in the periodontium may occur after interstitial resident cells have released tissue-degrading enzymes, such as collagenase, and damaged the extracellular matrix, once a sufficient amount of IL-lα-like factor for stimulating the production of proteolytic enzyme has been released by HGE in periodontal lesions.     

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-lα, IL-1β and tumor necrosis factor (TNF)-α stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-lα, IL-lβ, or TNF-α-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-l or TNF-α-induced IL-6 production, and that the enhancement of IL-6 production by IL-l or TNF-α may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kB) activation, markedly inhibited IL-l (α or β) or TNF-α-induced IL-6 production; so this production may be partially mediated through NF-kB. IL-l (α or β) and TNF-α enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-lβ was augmented by the addition of interferon (IFN)-(gama), but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-l (α or β)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-l or TNF-α, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-(gama) or IL-4, and glucocorticoids.     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1β

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1β (IL-β). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1β, IL-6 and IL-8. IL-1β activation of GF cells showed that IL-1β not only induces the expression of IL-6, IL-8 and TNF-α, but also acts in an autocrine manner on GF cells and induces IL-1βexpression. Furthermore, the continuous presence of IL-1β in GF cell cultures did not down regulate the response of GF cells to IL-1β. Pretreatment of GF cells with IL-lβ resulted in the enhanced synthesis of TNF-α in response to additional IL-lβ. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

interleukin-1β and tumor necrosis factor-α stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament

The present study demonstrates that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1β and TNF-α induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-γ. The peak expression of the MCP-1 gene by IL-1β or TNF-α was observed at 3 h after initiation of their treatment. Furthermore, IL-1β in combination with TNF-α synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1β also stimulated synergistically MCP-1 protein expression in combination with TNF-α. These results suggest that IL-1β and TNF-α may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.     

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites

Interleukin-l (IL-1) molecules, IL-lα and IL-lβ are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-lα, IL-1β, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-lα, IL-1bT and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-lα, IL-1β and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.     

Interleukin-1α produced in human gingival fibroblasts induces several activities related to the progression of periodontitis by direct contact

Previous observations suggest that interleukin-1 (IL-1) may play an important role in the progression of periodontitis. In the present study, we investigated whether a cell-associated IL-1α (CAIL-lα) produced in human gingival fibroblasts (HGF) induces biological activities related to the progression of periodontitis. HGF were treated with recombinant human IL-1β (rhIL-1β) for 12 h. After that, the cell layers of HGF were washed 3 times with fresh medium and were then fixed with 1% paraformaldehyde. The fixed cell layers of HGF were used for assays for bone resorbing activity, prostaglandin E₂ (PGE₂) production and collagenase activity. Fixed cell layers of HGF treated with rhIL-1β enhanced not only calcium release from BALB/c mouse calvaria but also PGE₂ production and collagenase activity in HGF and human periodontal ligament fibroblasts (HPLF) cultured on the fixed cell layers. These activities were neutralized by treatment with monoclonal mouse anti-human IL-1α antibody, but monoclonal mouse antihuman IL-1β antibody showed no effects on these activities. The induction of these activities by fixed cell layers of HGF required direct contact between the fixed cell layers and the calvaria, HGF, or HPLF. These results suggest that CAIL-1α produced in HGF treated with rhIL-1β induces bone resorbing activity, PGE₂ production and collagenase activity in the target cells by direct contact; CAIL-lα may play an important role in the progression of periodontitis.     

Effects of serum on hepatocyte growth factor secretion and activation by periodontal ligament and gingival fibroblasts

BACKGROUND: Hepatocyte growth factor (HGF)/scatter factor is a paracrine growth factor secreted by mesenchymal cells, which exerts an effect on a variety of epithelial cell types. Our recent study revealed that periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) cultured in the presence of serum which contains various stimulants produced HGF or HGF-like factor, a predominant chemoattractant for gingival epithelial cells, and suggested that it could be involved in epithelial down-growth in periodontitis. METHODS: To clarify whether serum in medium stimulates PLF and GF to synthesize or activate HGF, the effect of fetal bovine serum (FBS) on HGF production was determined by enzyme-linked immunoabsorbent assay (ELISA), and its chemotactic activity for gingival epithelial cells was examined by modified Boyden chamber assay. RESULTS: One to 10% FBS in the culture medium stimulated HGF secretion in a dose-dependent manner and the chemotactic activity was decreased by treatment with anti-hHGF neutralizing antibody. Furthermore, fibroblast-conditioned medium incubated with FBS and aprotinin reduced its chemotactic activity. Interestingly, serum-free culture of PLF and GF produced potent chemoattractants for gingival epithelial cells other than HGF. CONCLUSIONS: These results show that FBS stimulates both HGF secretion and activation by PLF and GF.     

Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells

To ascertain whether periodontal fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of periodontal ligament fibroblast-conditioned medium (PLF-CM) and gingival fibroblast-conditioned medium (GF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. Both PLF-CM and GF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) neutralizing antibody. Furthermore, the chemotactic activity of PLF-CM and GF-CM was well correlated with HGF content. These results show that PLF and GF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.     

The effects of interleukin 1 on collagenolytic activity and prostaglandin-E secretion by human periodontal-ligament and gingival fibroblast

Fibroblasts of the periodontium may be involved in extracellular matrix degradation in response to inflammatory cytokines produced by mononuclear phagocytes. Interleukin 1 (IL1), one of these biologically-active agents, is produced by such cells when stimulated by lipopolysaccharide (LPS). Periodontal-ligament (PLF) and gingival fibroblasts responded to recombinant human IL1 beta and to media conditioned by LPS-stimulated mononuclear phagocytes by secreting prostaglandin E (PGE). This response was dose- and time-dependent. Stimulated gingival fibroblasts also produced about five- to ten-fold as much collagenolytic activity when compared to controls but PLF produced no more activity. On mixing the conditioned media from both fibroblast types, inhibitory activity was found in the PLF-culture medium. Thus gingival fibroblasts in particular may be involved in the pathogenesis of periodontal disease by responding to factors produced by inflammatory phagocytes.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.     

Laminin- and fibronectin-like molecules produced by periodontal ligament fibroblasts under serum-free culture are potent chemoattractants for gingival epithelial cells

Previously, we revealed that hepatocyte growth factor (HGF) or an HGF-like factor secreted by periodontal ligament fibroblasts (PLF) and gingival fibroblasts cultured in the presence of serum was a major chemoattractant for gingival epithelial cells, and suggested that it might play a role in epithelial invasion. However, our recent study showed that serum-free culture of PLF and gingival fibroblasts produced potent chemoattractants other than HGF for gingival epithelial cells. To identify these chemoattractants, PLF-conditioned medium (PLF-CM) from serum-free cultures was obtained, concentrated, and separated by gel filtration column chromatography, and the chemotactic activity for gingival epithelial cells of each eluted fraction was monitored by a modified Boyden chamber assay. The chemoattractant activity was eluted at a molecular mass of around 600 kDa, which would include laminin and fibronectin, but not HGF, determined by ELISA. The chemotactic activity was reduced by treatment with antilaminin and/or antifibronectin polyclonal antibodies. Western blots using both antibodies revealed that the PLF-CM contained laminin- and fibronectin-like molecules. Along with HGF, these large glycoprotein molecules produced by PLF may be involved in the pathogenesis and progression of periodontitis by inducing the apical migration of epithelial cells.     

Inhibitors of collagenolytic enzymes synthesized by fibroblasts and epithelial cells from porcine and macaque periodontal tissues

Fibroblasts and epithelial cells from periodontal tissues were grown in monolayer culture and the culture media analysed for collagenolytic enzyme activity and inhibitors of these activities. Collagenase was detected only in gingival fibroblast cultures whereas nonspecific and specific gelatinase activities were present in both ligament and gingival fibroblast cultures. The collagenase and non-specific gelatinase enzymes were present in the culture media in latent forms that could be activated by organo-mercurials or trypsinization. Proteins capable of inhibiting these enzymes were synthesized by gingival fibroblasts and ligament epithelial cells. The inhibitory activity was blocked by organo-mercurials or the trypsin treatment used to activate the latent enzymes, suggesting that the latent enzymes are enzyme-inhibitor complexes. High mol. wt inhibitory activity against the same enzymes was also produced by the ligament fibroblasts. This activity was not affected by organo-mercurials or trypsinization. This type of inhibition may explain the inability to detect latent collagenase activity in some fibroblast cultures.     

Differential chemotactic effect of cementum attachment protein on periodontal cells

Selective re-population of the root surface by periodontal ligament cells is considered a key factor in Periodontal regeneration. A recently isolated cementum attachment protein (CAP) has been shown to enhance fibroblast attachment. In the present study the potential of CAP to selectively attract periodontal ligament cells (PLC) was studied in vitro in a micro-chemotaxis system. Human periodontal ligament cells and gingival fibroblasts (GF) were compared for their chemotactic response to either cementum attachment protein or to fibronectin. Murine dermal fibroblasts (MDF) served as control, irrelevant to the periodontium. The chemotactic response of PLC to fibronectin at 10^?8 M was of a similar magnitude as that of GF (16 ± 5 and 11 ± 3 cells/field, respectively), but both were significantly lower than the response of MDF (28 ± 3 cells/field). The chemotactic response of periodontal ligament cells to the cementum attachment protein at 10^?7 M was higher (36 ± 5 cells/field) than that of gingival fibroblasts or murine dermal fibroblasts (14 ± 2 and 16 ± 2 cells/field, respectively). These results suggest that cementum attachment protein can influence the selective re-population of root surfaces by periodontal ligament cells.     

Specificity of Antimicrobial Peptide LL‐37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

Background: The antimicrobial peptide LL‐37 is known to have a potent lipopolysaccharide (LPS)‐neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL‐37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS‐neutralizing activity of LL‐37 to various periodontopathogenic LPS in interleukin‐8 (IL‐8) production after challenging them in human oral fibroblasts. Methods: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24‐well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL‐37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL‐8 production by enzyme‐linked immunosorbent assay. Results: All periodontopathogenic LPS statistically significantly induced IL‐8 production in both PDLF and GF (P <0.01). After neutralization with LL‐37, both PDLF and GF showed a statistically significant reduction in IL‐8 production compared with LPS‐treated groups without LL‐37 (P <0.01), and the percentage of reduction in IL‐8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL‐8 production varied considerably according to each periodontopathogenic LPS. Conclusions: The antimicrobial peptide LL‐37 had an ability to suppress periodontopathogenic LPS‐induced IL‐8 production in both PDLF and GF. Its LPS‐neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell‐surface components through the CD14/Toll‐like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin‐8 (IL‐8) responses of the cells to Salmonella lipopolysaccharide, a water‐soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll‐like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR‐related molecules, i.e. TLR2, TLR4, MD‐2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL‐8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL‐8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2‐ and TLR4‐independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti‐CD14 MAb.     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. K.GF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa. from gingival, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (PCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% PCS. Total RNA was isolated and used for Northern blotting with a P³²-labeled KGP cDNA. probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% PCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore. RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in non cultured periodontal ligament cells.     

人牙龈和牙周韧带成纤维细胞的体外培养及生物学特性研究——细胞形态、生长曲线及碱性磷酸酶活性测定

目的 :建立人牙龈和牙周韧带成纤维细胞体外培养模型并对其生物学特性作初步探讨。方法 :采用组织块法常规条件下分别进行牙龈和牙周韧带成纤维细胞的培养 ,通过光镜、透射电镜、生长曲线及碱性磷酸酶测定等手段对其部分生物学特性进行研究。结果 :两种培养的原代及传代细胞在光镜下细胞排列及结构无明显差别 ,传代培养时牙龈成纤维细胞有接触抑制现象 ,而牙周韧带成纤维细胞则可呈复层生长 ,牙周韧带成纤维细胞排列方向性较明显 ,生长曲线表明牙周韧带成纤维细胞增殖活性高于牙龈成纤维细胞 ;碱性磷酸酶活性测定及染色法显示两者有明显的差别。结论 :体外培养的两种细胞在形态及排列上相似 ,但在细胞亚型的组成上存在差异。     

The effect of interleukin-1 β on hyaluronic acid synthesized by adult human gingival fibroblasts in vitro

The effect of recombinant interleukin-1β (IL-lβ) on hyaluronic acid synthesis by human gingival fibroblasts was studied. IL-1bT caused a dose-dependent increase in the incorporation of (³lucosamine into hyaluronic acid. The ³⁵S/35H ratios of labeled macromolecules did not change regardless of the presence or absence of TL-lβ and indicates stimulation of hyaluronic acid synthesis. Inhibition of cell proliferation by hydroxyurea caused an increase in hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis in the presence of hydroxyurea was increased over untreated and IL-lβ-treated controls, but equivalent to the hydroxyurea-treated controls. Thus the effect of IL-1β on hyaluronic acid synthesis may be independent of cell proliferation. Furthermore, inhibition of prostaglandin E2 synthesis by indomethacin abolished the effect of IL-1β on hyaluronic acid synthesis. Inhibition of new protein synthesis by cycloheximide negated the effect of IL-β on hyaluronic acid synthesis. This may be related to inhibition of new hyaluronate synthetase synthesis, since IL-1β stimulated the level of hyaluronate synthetase activity. Sepharose CL-2B chromatography revealed that most of the newly synthesized hyaluronic acid was of large molecular size. The cells exposed to IL-1β retained more large molecular size hyaluronic acid in their cell layer environment than did the control cells. These responses by fibroblasts to IL-1β may be indicative of early tissue repair.