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• W2022199933 abstract "Persistent inflammation is a well-known determinant of progressive tissue fibrosis; however, the mechanisms underlying this process remain unclear. There is growing evidence indicating a role of the cytokine IL-1β in profibrotic responses. We previously demonstrated that fibroblasts stimulated with IL-1β increased their generation of the polysaccharide hyaluronan (HA) and increased their expression of the HA synthase enzyme (HAS-2). The aim of this study was to determine the significance of IL-1β–induced changes in HA and HAS-2 generation. In this study, we found that stimulation of fibroblasts with IL-1β results in the relocalization of HA associated with the cell to the outer cell membrane, where it forms HAS2- and CD44-dependent cell membrane protrusions. CD44 is concentrated within the membrane protrusions, where it co-localizes with the intracellular adhesion molecule 1. Furthermore, we have identified that these cell protrusions enhance IL-1β–dependent fibroblast-monocyte binding through MAPK/ERK signaling. Although previous data have indicated the importance of the HA-binding protein TSG-6 in maintaining the transforming growth factor β1–dependent HA coat, TSG-6 was not essential for the formation of the IL-1β–dependent HA protrusions, thus identifying it as a key difference between IL-1β– and transforming growth factor β1–dependent HA matrices. In summary, these data suggest that IL-1β–dependent HA generation plays a role in fibroblast immune activation, leading to sequestration of monocytes within inflamed tissue and providing a possible mechanism for perpetual inflammation. Persistent inflammation is a well-known determinant of progressive tissue fibrosis; however, the mechanisms underlying this process remain unclear. There is growing evidence indicating a role of the cytokine IL-1β in profibrotic responses. We previously demonstrated that fibroblasts stimulated with IL-1β increased their generation of the polysaccharide hyaluronan (HA) and increased their expression of the HA synthase enzyme (HAS-2). The aim of this study was to determine the significance of IL-1β–induced changes in HA and HAS-2 generation. In this study, we found that stimulation of fibroblasts with IL-1β results in the relocalization of HA associated with the cell to the outer cell membrane, where it forms HAS2- and CD44-dependent cell membrane protrusions. CD44 is concentrated within the membrane protrusions, where it co-localizes with the intracellular adhesion molecule 1. Furthermore, we have identified that these cell protrusions enhance IL-1β–dependent fibroblast-monocyte binding through MAPK/ERK signaling. Although previous data have indicated the importance of the HA-binding protein TSG-6 in maintaining the transforming growth factor β1–dependent HA coat, TSG-6 was not essential for the formation of the IL-1β–dependent HA protrusions, thus identifying it as a key difference between IL-1β– and transforming growth factor β1–dependent HA matrices. In summary, these data suggest that IL-1β–dependent HA generation plays a role in fibroblast immune activation, leading to sequestration of monocytes within inflamed tissue and providing a possible mechanism for perpetual inflammation. Fibrosis is a pathological process that underlies a multitude of organ-specific diseases in the lung, liver, and kidneys, leading to a wide variety of clinical disorders.1Green F.H. Overview of pulmonary fibrosis.Chest. 2002; 122: 334S-339SCrossref PubMed Scopus (82) Google Scholar, 2Chapman H.A. Disorders of lung matrix remodeling.J Clin Invest. 2004; 113: 148-157Crossref PubMed Scopus (168) Google Scholar, 3Eddy A.A. Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 4Bedossa P. Paradis V. Liver extracellular matrix in health and disease.J Pathol. 2003; 200: 504-515Crossref PubMed Scopus (211) Google Scholar It is characterized by the accumulation of extracellular matrix and by the persistence of myofibroblasts in affected tissues and is causally linked to increased circulating levels of the cytokine transforming growth factor (TGF)-β1.5Border W.A. Noble N.A. Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (3007) Google Scholar, 6Border W.A. Noble N.A. Fibrosis linked to TGF-beta in yet another disease.J Clin Invest. 1995; 96: 655-656Crossref PubMed Scopus (48) Google Scholar, 7Blobe G.C. Schiemann W.P. Lodish H.F. Role of transforming growth factor beta in human disease.N Engl J Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2182) Google Scholar, 8Gabbiani G. The myofibroblast in wound healing and fibroconnective tissue disease.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1249) Google Scholar Persistent inflammation after mechanical, microbial, or physiologic tissue injury is a well-known determinant of progressive fibrosis; however, the mechanistic processes underlying this are poorly understood. Persistent inflammation leads to a progressive shift in the type of cells present at the site of injury. As inflammation progresses, the presence of macrophages become dominant in the injured tissue.9Heymann F. Trautwein C. Tacke F. Monocytes and macrophages as cellular targets in liver fibrosis.Inflamm Allergy Drug Targets. 2009; 8: 307-318Crossref PubMed Scopus (145) Google Scholar, 10Wynn T.A. Barron L. Macrophages: master regulators of inflammation and fibrosis.Semin Liver Dis. 2010; 30: 245-257Crossref PubMed Scopus (929) Google Scholar, 11Vernon M.A. Mylonas K.J. Hughes J. Macrophages and renal fibrosis.Semin Nephrol. 2010; 30: 302-317Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar In addition, an abnormal expansion and phenotypic activation of the fibroblastic cell population becomes more apparent as fibrous tissue is progressively laid down.8Gabbiani G. The myofibroblast in wound healing and fibroconnective tissue disease.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1249) Google Scholar, 12Gabbiani G. Ryan G.B. Majne G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction.Experientia. 1971; 27: 549-550Crossref PubMed Scopus (1170) Google Scholar Previous evidence suggests that complex interactions between fibroblasts and monocytes/macrophages play an important role in the progression of fibrous disease.13Adamson I.Y. Letourneau H.L. Bowden D.H. Enhanced macrophage-fibroblast interactions in the pulmonary interstitium increases fibrosis after silica injection to monocyte-depleted mice.Am J Pathol. 1989; 134: 411-418PubMed Google Scholar, 14Clark J.G. Kostal K.M. Marino B.A. Bleomycin-induced pulmonary fibrosis in hamsters: an alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production.J Clin Invest. 1983; 72: 2082-2091Crossref PubMed Scopus (41) Google Scholar, 15Lemaire I. Beaudoin H. Masse S. Grondin C. Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis.Am J Pathol. 1986; 122: 205-211PubMed Google Scholar Hence, monocytes and fibroblasts are considered to be key players in fibrogenesis. In addition, a variety of cytokines and growth factors have been demonstrated to be present at sites of chronic inflammation and active fibrosis. Of these, TGF-β1 is largely recognized as the most important profibrotic cytokine in terms of direct stimulation of fibroblast activation and matrix generation, which typifies fibrosis.16Vaughan M.B. Howard E.W. Tomasek J.J. Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast.Exp Cell Res. 2000; 257: 180-189Crossref PubMed Scopus (398) Google Scholar, 17Yoshioka K. Takemura T. Murakami K. Okada M. Hino S. Miyamoto H. Maki S. Transforming growth factor-beta protein and mRNA in glomeruli in normal and diseased human kidneys.Lab Invest. 1993; 68: 154-163PubMed Google Scholar, 18Nagy P. Schaff Z. Lapis K. Immunohistochemical detection of transforming growth factor-beta 1 in fibrotic liver diseases.Hepatology. 1991; 14: 269-273Crossref PubMed Scopus (105) Google Scholar, 19Anscher M.S. Peters W.P. Reisenbichler H. Petros W.P. Jirtle R.L. Transforming growth factor beta as a predictor of liver and lung fibrosis after autologous bone marrow transplantation for advanced breast cancer.N Engl J Med. 1993; 328: 1592-1598Crossref PubMed Scopus (305) Google Scholar However, there is accumulating evidence to indicate that a variety of other proinflammatory cytokines can also promote fibrogenesis, either through enhancing the profibrotic actions of TGF-β1 or through direct profibrotic effects on cells. A growing body of evidence indicates a pivotal role for the inflammatory cytokine IL-1β in profibrotic responses.20Kanangat S. Postlethwaite A. Hasty K. Kang A. Smeltzer M. Appling W. Schaberg D. Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections.Arthritis Res Ther. 2006; 8: R176Crossref PubMed Scopus (50) Google Scholar, 21Vesey D.A. Cheung C. Cuttle L. Endre Z. Gobe G. Johnson D.W. Interleukin-1beta stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-beta-dependent mechanism.J Lab Clin Med. 2002; 140: 342-350Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Vesey D.A. Cheung C.W. Cuttle L. Endre Z.A. Gobe G. Johnson D.W. Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production.Kidney Int. 2002; 62: 31-40Crossref PubMed Scopus (97) Google Scholar In vitro studies have demonstrated that it can exert a variety of profibrotic effects on a range of cell types, including stimulation of proliferation and extracellular matrix production; induction of stimuli, such as TGF-β1, nitric oxide, and reactive oxygen species; and induction of adhesion molecule expression.23Tesch G. Nikolic-Paterson D.J. Main I.W. Lan H.Y. Atkins R.C. The role of interleukin-1 in mesangial proliferation.Contrib Nephrol. 1995; 111: 144-148PubMed Google Scholar, 24Nikolic-Paterson D.J. Main I.W. Tesch G.H. Lan H.Y. Atkins R.C. Interleukin-1 in renal fibrosis.Kidney Int Suppl. 1996; 54: S88-S90PubMed Google Scholar, 25Raghow R. Postlethwaite A.E. Keski-Oja J. Moses H.L. Kang A.H. Transforming growth factor-beta increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts.J Clin Invest. 1987; 79: 1285-1288Crossref PubMed Scopus (368) Google Scholar, 26Postlethwaite A.E. Raghow R. Stricklin G.P. Poppleton H. Seyer J.M. Kang A.H. Modulation of fibroblast functions by interleukin 1: increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta.J Cell Biol. 1988; 106: 311-318Crossref PubMed Scopus (198) Google Scholar, 27Keski-Oja J. Raghow R. Sawdey M. Loskutoff D.J. Postlethwaite A.E. Kang A.H. Moses H.L. Regulation of mRNAs for type-1 plasminogen activator inhibitor, fibronectin, and type I procollagen by transforming growth factor-beta: divergent responses in lung fibroblasts and carcinoma cells.J Biol Chem. 1988; 263: 3111-3115Abstract Full Text PDF PubMed Google Scholar, 28Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide.J Biol Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar, 29Tiggelman A.M. Boers W. Linthorst C. Sala M. Chamuleau R.A. Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1 beta. IL-4, TGF beta and IFN gamma.J Hepatol. 1995; 23: 307-317Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 30Radeke H.H. Meier B. Topley N. Floge J. Habermehl G.G. Resch K. Interleukin 1-alpha and tumor necrosis factor-alpha induce oxygen radical production in mesangial cells.Kidney Int. 1990; 37: 767-775Crossref PubMed Scopus (277) Google Scholar, 31Jiang B. Haverty M. Brecher P. N-acetyl-L-cysteine enhances interleukin-1beta-induced nitric oxide synthase expression.Hypertension. 1999; 34: 574-579Crossref PubMed Scopus (38) Google Scholar, 32Phillips A.O. Topley N. Steadman R. Morrisey K. Williams J.D. Induction of TGF-beta 1 synthesis in D-glucose primed human proximal tubular cells by IL-1 beta and TNF alpha.Kidney Int. 1996; 50: 1546-1554Crossref PubMed Scopus (83) Google Scholar We have previously reported that IL-1β can enhance TGF-β1 signaling.33Luo D.D. Fielding C. Phillips A. Fraser D. Interleukin-1 beta regulates proximal tubular cell transforming growth factor beta-1 signalling.Nephrol Dial Transplant. 2009; 24: 2655-2665Crossref PubMed Scopus (44) Google Scholar Moreover, several studies in humans and animals have reported that IL-1β staining is strongly associated with fibrotic lesions in liver disease, in parenchymal lung disorders, and with the severity of tubulointerstitial lesions and proteinuria in the kidney.24Nikolic-Paterson D.J. Main I.W. Tesch G.H. Lan H.Y. Atkins R.C. Interleukin-1 in renal fibrosis.Kidney Int Suppl. 1996; 54: S88-S90PubMed Google Scholar, 34Kolb M. Margetts P.J. Anthony D.C. Pitossi F. Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.J Clin Invest. 2001; 107: 1529-1536Crossref PubMed Scopus (605) Google Scholar, 35Koda K. Niitsu Y. Bunya M. Ito N. Sasagawa Y. Owada M. Morita K. Kohgo Y. Urushizaki I. [Increased production of interleukin-1 by macrophage from the patients with chronic liver disease and its implication for liver fibrosis].Nippon Shokakibyo Gakkai Zasshi. 1985; 82: 1527-1532PubMed Google Scholar, 36Gieling R.G. Wallace K. Han Y.P. Interleukin-1 participates in the progression from liver injury to fibrosis.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1324-G1331Crossref PubMed Scopus (194) Google Scholar Hyaluronan (HA) is a ubiquitous connective tissue polysaccharide known for its role in maintaining matrix stability. It is synthesized by HA synthase (HAS) enzymes of which three vertebrate genes have been isolated and characterized: HAS1, HAS2, and HAS3.37Spicer A.P. McDonald J.A. Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family.J Biol Chem. 1998; 273: 1923-1932Crossref PubMed Scopus (290) Google Scholar, 38Meyer M.F. Kreil G. Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.Proc Natl Acad Sci U S A. 1996; 93: 4543-4547Crossref PubMed Scopus (88) Google Scholar In addition to its homeostatic functions, it also has a role in regulating cell functions through interaction with cell-surface receptors (principally CD44 and RHAMM); hence, it plays a recognized role in mediating cell-cell adhesion, migration, proliferation, and differentiation.39Kosaki R. Watanabe K. Yamaguchi Y. Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity.Cancer Res. 1999; 59: 1141-1145PubMed Google Scholar, 40Legg J.W. Lewis C.A. Parsons M. Ng T. Isacke C.M. A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility.Nat Cell Biol. 2002; 4: 399-407Crossref PubMed Scopus (205) Google Scholar, 41Itano N. Atsumi F. Sawai T. Yamada Y. Miyaishi O. Senga T. Hamaguchi M. Kimata K. Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration.Proc Natl Acad Sci U S A. 2002; 99: 3609-3614Crossref PubMed Scopus (267) Google Scholar, 42Ito T. Williams J.D. Al-Assaf S. Phillips G.O. Phillips A.O. Hyaluronan and proximal tubular cell migration.Kidney Int. 2004; 65: 823-833Crossref PubMed Scopus (33) Google Scholar, 43Camenisch T.D. Schroeder J.A. Bradley J. Klewer S.E. McDonald J.A. Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors.Nat Med. 2002; 8: 850-855Crossref PubMed Scopus (276) Google Scholar, 44Zoltan-Jones A. Huang L. Ghatak S. Toole B.P. Elevated hyaluronan production induces mesenchymal and transformed properties in epithelial cells.J Biol Chem. 2003; 278: 45801-45810Crossref PubMed Scopus (185) Google Scholar As a result, it is an important regulator of tissue remodeling and has been implicated in a number of biological and pathological processes, including wound healing, embryonic development, tumor growth, and now fibrosis.45Chen W.Y. Abatangelo G. Functions of hyaluronan in wound repair.Wound Repair Regen. 1999; 7: 79-89Crossref PubMed Scopus (902) Google Scholar, 46Toole B.P. Hyaluronan in morphogenesis.J Intern Med. 1997; 242: 35-40Crossref PubMed Scopus (280) Google Scholar, 47Stern R. Hyaluronan metabolism: a major paradox in cancer biology.Pathol Biol (Paris). 2005; 53: 372-382Crossref PubMed Scopus (141) Google Scholar, 48Toole B.P. Wight T.N. Tammi M.I. Hyaluronan-cell interactions in cancer and vascular disease.J Biol Chem. 2002; 277: 4593-4596Crossref PubMed Scopus (429) Google Scholar, 49Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. Involvement of hyaluronan in regulation of fibroblast phenotype.J Biol Chem. 2007; 282: 25687-25697Crossref PubMed Scopus (119) Google Scholar, 50Meran S. Thomas D.W. Stephens P. Enoch S. Martin J. Steadman R. Phillips A.O. Hyaluronan facilitates transforming growth factor-beta1-mediated fibroblast proliferation.J Biol Chem. 2008; 283: 6530-6545Crossref PubMed Scopus (105) Google Scholar, 51Simpson R.M. Meran S. Thomas D. Stephens P. Bowen T. Steadman R. Phillips A. Age-related changes in pericellular hyaluronan organization leads to impaired dermal fibroblast to myofibroblast differentiation.Am J Pathol. 2009; 175: 1915-1928Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 52Webber J. Meran S. Steadman R. Phillips A. Hyaluronan orchestrates transforming growth factor-beta1-dependent maintenance of myofibroblast phenotype.J Biol Chem. 2009; 284: 9083-9092Crossref PubMed Scopus (109) Google Scholar In our attempts to delineate the cell biology of fibrosis, our research has previously focused on the phenotypic activation of fibroblasts by TGF-β1. This earlier work demonstrated that phenotypic activation of fibroblasts to myofibroblasts, characterized by increased α-smooth muscle actin (α-SMA) expression, was dependent on TGF-β1 and was mediated by HA.49Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. Involvement of hyaluronan in regulation of fibroblast phenotype.J Biol Chem. 2007; 282: 25687-25697Crossref PubMed Scopus (119) Google Scholar Specifically, we demonstrated that stimulation of fibroblasts with TGF-β1–induced HAS enzyme expression and HA synthesis and resulted in the formation of HA-dependent pericellular matrices (HA coats). The assembled HA coats were subsequently found to function in mediating TGF-β1–driven phenotypic differentiation in fibroblasts. We also have previous data demonstrating that, similar to TGF-β1, stimulation of fibroblasts with IL-1β also induces HAS enzyme expression and HA synthesis and leads to the formation of HA pericellular matrices.49Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. Involvement of hyaluronan in regulation of fibroblast phenotype.J Biol Chem. 2007; 282: 25687-25697Crossref PubMed Scopus (119) Google Scholar, 51Simpson R.M. Meran S. Thomas D. Stephens P. Bowen T. Steadman R. Phillips A. Age-related changes in pericellular hyaluronan organization leads to impaired dermal fibroblast to myofibroblast differentiation.Am J Pathol. 2009; 175: 1915-1928Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar However, in contrast to the TGF-β1–induced coats, the IL-1β–induced HA coats were not clearly related to a profibrotic phenotype. The functional significance of IL-1β–induced HA changes are therefore unclear. In this article, we study the functional effects of IL-1β–induced HA in the fibroblast. We report that stimulation of fibroblasts with IL-1β results in a HA pericellular coat that is distinct from the HA coat assembled after TGF-β1. IL-1β induces the fibroblast cell membranes to form multiple spiculated protrusions from which cell-associated HA projects in a linear manner. CD44 is critical to the formation of these cell structures and is redistributed to be concentrated within the membrane spicules, where it co-localizes with intercellular adhesion molecule (ICAM)-1. Functionally, these HA-rich structures appear to enhance ICAM-1 and monocyte interactions and promote fibroblast-monocyte binding. All reagents were purchased from Sigma (St. Louis, MO) or Invitrogen unless otherwise stated. Reverse transcription reagents, siRNA transfection reagents, and quantitative PCR (qPCR) primers and reagents were purchased from Invitrogen (Paisley, UK) and Applied Biosystems (Cheshire, UK). Radioisotopes were purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK), and the ERK (MEK) inhibitor was purchased from Calbiochem (Nottingham, UK). Human lung fibroblasts (AG02262) were purchased from Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 2 mmol/L l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin supplemented with 10% fetal bovine serum (Biological Industries Ltd., Cumbernauld, UK). The cells were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2, and fresh growth medium was added to the cells every 3 to 4 days until the cells were ready for experimentation. The cells were incubated in serum-free medium for 48 hours before use in all experiments, and all experiments were performed under serum-free conditions unless otherwise stated. All experiments were undertaken using cells at passage 6 to 10. Cells were grown to full confluence in T75 flasks. They were then growth arrested in serum-free medium for 48 hours. After this they were incubated with either serum-free medium containing 20 μCi/mL of 3H-glucosamine for 24 hours or serum-free medium containing 20 μCi/mL of 3H-glucosamine together with 1 ng/mL of recombinant human IL-1β (R&D Systems, Abingdon, UK) for 24 hours. The culture medium was then removed and the cell washed with PBS. The wash and medium were combined to form the conditioned medium extract. This extract was then treated with an equal volume of 200 μg/mL of Pronase in 100 mmol/L Tris-HCL, pH 8, 0.05% sodium azide for 24 hours. The samples were then passed over DEAE-Sephacel ion exchange columns equilibrated with 8 mol/L urea in 20 mmol/L of BisTris buffer, pH 6, containing 0.2% Triton X100 to remove any low-molecular-weight peptides and unincorporated radiolabel. HA was eluted in 8 mol/L urea buffer containing 0.3 mol/L NaCl. Each sample was split into two, and the HA was precipitated with three volumes of 1% potassium acetate in 95% ethanol in the presence of 50 μg/mL of each HA, heparin, and chondroitin sulfate as co-precipitants. The first half of each sample was resuspended in 500 μL of 4 mol/L guanidine buffer and analyzed on a Sephacryl S-500 column equilibrated with 4 mol/L guanidine buffer. To confirm that the chromatography profile generated was the result of radiolabeled HA, the second half of each sample was digested at 37°C overnight with 1 U of Streptomyces hyalurolyticus hyaluronidase (ICN Pharmaceuticals Ltd., Basingstoke, UK) in 200 μL of 20 mmol/L sodium acetate, pH 6, containing 0.05% sodium azide and 0.15 mol/L sodium chloride. The sample was then mixed with an equal volume of 4 mol/L guanidine buffer and analyzed on the same Sephacryl S-500 column equilibrated with 4 mol/L guanidine buffer. To produce the chromatography profile, the 3H activity for each half of the sample was normalized and corrected for dilution, and then the S. hyalurolyticus hyaluronidase–resistant counts were subtracted. The chromatography profiles only depict S. hyalurolyticus hyaluronidase–sensitive activity in each fraction plotted against fraction number. The column was calibrated with 3H-glucosamine hydrochloride, Mr 215; 35S-chondroitin sulfate glycosaminoglycans, Mr 25 × 103; decorin, Mr 10 × 104; and 35S-versican, Mr 1.3 × 106. RT-PCR and qPCR was used to assess HAS2, ICAM-1, CD45, TSG-6, and CD44 standard isoform (product code: Hs01075861_m1) mRNA expression. Primers and probes for these genes were commercially designed and purchased from Applied Biosystems. The cells were grown in 35-mm dishes and washed with PBS before lysis with Tri-reagent and RNA purification according to the manufacturer protocol. Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit according to the manufacturer protocol (Applied Biosystems). This kit uses the random primer method for initiating cDNA synthesis. As a negative control reverse transcription was performed with sterile H2O replacing the RNA sample. We performed qPCR using the 7900HT fast real-time PCR system from Applied Biosystems. PCR was performed in a final volume of 20 μL per sample as follows: 1 μL of reverse transcription product, 1 μL of target gene primers and probe, 10 μL of Taqman Universal PCR mastermix, and 8 μL of sterile water. Amplification was performed using a cycle of 95°C for 1 second and 60°C for 20 seconds for 40 cycles. As a negative control, PCR was performed with sterile H2O replacing the cDNA sample. PCR was simultaneously performed for ribosomal RNA (primers and probe commercially designed and purchased from Applied Biosystems) as a standard reference gene. The comparative CT method was used for relative quantification of gene expression. The CT (threshold cycle where amplification is in the linear range of the amplification curve) for the standard reference gene (ribosomal RNA) was subtracted from the target gene CT to obtain the ΔCT. The mean ΔCT values for replicate samples were then calculated. The expression of the target gene in experimental samples relative to expression in control samples was then calculated using the equation:2−[ΔCT(1)−ΔCT(2)](1) where ΔCT(1) is the mean ΔCT calculated for the experimental samples, and ΔCT(2) is the mean ΔCT calculated for the control samples. Transient transfection of fibroblasts was performed with specific siRNA nucleotides (Applied Biosystems) targeting HAS2, CD44, or TSG-6. Transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) in accordance with the manufacturer protocol. Briefly, cells were grown to 70% confluence in antibiotic free medium in either 35-mm dishes or 8-well Permanox chamber slides. Five microliters of the transfection reagent was diluted in 250 μL of Opti-MEM reduced growth medium (GIBCO, Paisley, UK) and left to incubate for 5 minutes at room temperature. The specific siRNA oligonucleotides were diluted in Opti-MEM reduced growth medium to achieve a final concentration of 30 nm. The transfection agent and siRNA mixtures were then combined and incubated at room temperature for an additional 20 minutes. The newly formed transfection complexes were subsequently added to the cells and incubated at 37°C with 5% CO2 for 24 hours in serum-free medium before experimentation. As a control, cells were transfected with negative control siRNA (a scrambled sequence that bears no homology to the human genome) (Applied Biosystems). Cells were grown to 70% confluence in eight-well Permanox chamber slides. The culture medium was removed and the cells washed with sterile PBS before fixation in acetone-methanol (1:1 v/v) for 5 minutes at room temperature. After fixation, slides were blocked with 5% bovine serum albumin for 20 minutes before a further washing step with PBS. Subsequently, the slides were incubated with the primary antibody diluted in 0.1% bovine serum albumin and PBS for 2 hours at room temperature. After a further washing step, slides were incubated with fluorescein isothiocyanate (FITC)–conjugated and/or tetramethyl rhodamine isothiocyanate–conjugated secondary antibodies for 1 hour at room temperature (Dako, Cambridgeshire, UK). Cell nuclei were enhanced by staining with DAPI. Cells were then mounted and analyzed by confocal fluorescent microscopy. In some experiments, U937 cells in co-culture with fibroblasts were assessed. This is a monocyte cell line originally derived from a human histiocytic lymphoma purchased from the ATCC (Manassas, VA). Their use to study monocyte behavior has been well established previously.53Selbi W. de la Motte C. Hascall V. Phillips A. BMP-7 modulates hyaluronan-mediated proximal tubular cell-monocyte interaction.J Am Soc Nephrol. 2004; 15: 1199-1211Crossref PubMed Scopus (52) Google Scholar They were grown in suspension culture in RPMI medium supplemented with l-glutamine and penicillin/streptomycin and containing 5% fetal bovine serum until an appropriate cell density was achieved (1 × 106 cells/mL). They were then incubated for specified number of hours with fibroblasts grown to 70% confluence under specific experimental conditions (details are outlined in the figure legends). The cultures were then washed 10 times with 1000 μL of PBS to remove any unbound U937 cells. Subsequently, the cells were fixed with 1:1 acetone:methanol for 5 minutes at room temperature and blocked and treated with primary and secondary antibodies as described. Cell nuclei were enhanced by staining with DAPI, thereby facilitating localization of monocytes. The following primary antibodies were used: rat anti-human CD44 antibody (Calbiochem A020)" @default.
• W2022199933 created "2016-06-24" @default.
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• W2022199933 date "2013-06-01" @default.
• W2022199933 modified "2023-09-25" @default.
• W2022199933 title "Interleukin-1β Induces Hyaluronan and CD44-Dependent Cell Protrusions That Facilitate Fibroblast-Monocyte Binding" @default.
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