Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090579
ISSN1525-2191
AutoresValerie Saw, Ifeoma Offiah, Robin Dart, Grazyna Galatowicz, John Dart, Julie T. Daniels, Virginia L. Calder,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoInterleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts. Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts. Autoimmune conjunctivitis caused by ocular mucous membrane pemphigoid (MMP), also known as ocular cicatricial pemphigoid, is a blinding disease characterized by recurrent episodes of inflammation and aggressive conjunctival fibrosis. It is part of a spectrum of immunobullous disease associated with linear deposition of IgG and IgA autoantibodies directed against epithelial basement membrane zone proteins, which most commonly involves oral and ocular mucosa. In ocular mucosa, unlike oral mucosa, scarring is a clinical hallmark of the disease.1Chan LS Ahmed AR Anhalt GJ Bernauer W Cooper KD Elder MJ Fine JD Foster CS Ghohestani R Hashimoto T Hoang-Xuan T Kirtschig G Korman NJ Lightman S Lozada-Nur F Marinkovich MP Mondino BJ Prost-Squarcioni C Rogers III, RS Setterfield JF West DP Wojnarowska F Woodley DT Yancey KB Zillikens D Zone JJ The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators.Arch Dermatol. 2002; 138: 370-379Crossref PubMed Google Scholar Although the mechanisms have not been clearly demonstrated, autoantibodies are thought to cause blistering by disrupting basement membrane adherence via activation of complement, neutrophils and pro-inflammatory cytokines.2Black AP Wojnarowska F Ogg GS Role of T cells in the pathogenesis of mucous membrane pemphigoid.Expert Rev Dermatol. 2006; 1: 25-30Crossref Scopus (2) Google Scholar In the eye, blisters are infrequently seen, and the disease manifests as chronic recurrent cicatricial conjunctivitis. The functional consequences of the inflammatory lesions that heal with scarring are most evident in the eye, where patients with severe conjunctival fibrosis become blind in 30% of cases.3Hardy KM Perry HO Pingree GC Kirby Jr, TJ Benign mucous membrane pemphigoid.Arch Dermatol. 1971; 104: 467-475Crossref PubMed Scopus (198) Google Scholar Developing effective anti-fibrotic agents would therefore have therapeutic potential, but is challenging because the cellular and molecular mechanisms of conjunctival scarring in MMP are incompletely understood. Given that conjunctival fibrosis can still progress, despite apparent clinical control of inflammation by conventional immunosuppressive therapy,4Miserocchi E Baltatzis S Roque MR Ahmed AR Foster CS The effect of treatment and its related side effects in patients with severe ocular cicatricial pemphigoid.Ophthalmology. 2002; 109: 111-118Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 5Saw VP Dart JK Rauz S Ramsay A Bunce C Xing W Maddison PG Phillips M Immunosuppressive therapy for ocular mucous membrane pemphigoid strategies and outcomes.Ophthalmology. 2008; 115: 253-261Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar better understanding of the changes in treated tissue compared with actively inflamed tissue might assist and guide in the development of adjunctive local therapies to target conjunctival fibrosis. Interleukin-13 is a key profibrotic mediator that has been identified as the dominant effector cytokine of fibrosis in experimental models of skin, hepatic, and pulmonary fibrosis and airway remodeling in asthma.6Aliprantis AO Wang J Fathman JW Lemaire R Dorfman DM Lafyatis R Glimcher LH Transcription factor T-bet regulates skin sclerosis through its function in innate immunity and via IL-13.Proc Natl Acad Sci USA. 2007; 104: 2827-2830Crossref PubMed Scopus (98) Google Scholar, 7Chiaramonte MG Donaldson DD Cheever AW Wynn TA An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response.J Clin Invest. 1999; 104: 777-785Crossref PubMed Scopus (498) Google Scholar, 8Keane MP Gomperts BN Weigt S Xue YY Burdick MD Nakamura H Zisman DA Ardehali A Saggar R Lynch III, JP Hogaboam C Kunkel SL Lukacs NW Ross DJ Grusby MJ Strieter RM Belperio JA IL-13 is pivotal in the fibro-obliterative process of bronchiolitis obliterans syndrome.J Immunol. 2007; 178: 511-519PubMed Google Scholar, 9Kumar RK Herbert C Yang M Koskinen AM McKenzie AN Foster PS Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma.Clin Exp Allergy. 2002; 32: 1104-1111Crossref PubMed Scopus (159) Google Scholar It is produced by type 2 helper T cell (Th2) cells, mast cells, and basophils, and is a potent stimulator of eosinophil-, lymphocyte- and macrophage-rich inflammation, mucosal metaplasia, tissue fibrosis, and parenchymal remodeling.10Hershey GK IL-13 receptors and signaling pathways: an evolving web.J Allergy Clin Immunol. 2003; 111: 677-690Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 11Zhu Z Homer RJ Wang Z Chen Q Geba GP Wang J Zhang Y Elias JA Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.J Clin Invest. 1999; 103: 779-788Crossref PubMed Scopus (1470) Google Scholar Fibrosis associated with repetitive injury in chronic inflammatory disease is strongly linked with CD4+ Th2 responses involving interleukin (IL)-4, IL-5, IL-13, and IL-21.12Wynn TA Fibrotic disease and the T(H)1/T(H)2 paradigm.Nat Rev Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1172) Google Scholar As CD4+ and CD8+ T cells are the predominant cells in the conjunctival subepithelial infiltrate in ocular MMP,13Bernauer W Wright P Dart JK Leonard JN Lightman S The conjunctiva in acute and chronic mucous membrane pemphigoid. An immunohistochemical analysis.Ophthalmology. 1993; 100: 339-346Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14Rice BA Foster CS Immunopathology of cicatricial pemphigoid affecting the conjunctiva.Ophthalmology. 1990; 97: 1476-1483Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 15Sacks EH Jakobiec FA Wieczorek R Donnenfeld E Perry H Knowles Jr, DM Immunophenotypic analysis of the inflammatory infiltrate in ocular cicatricial pemphigoid. Further evidence for a T cell-mediated disease.Ophthalmology. 1989; 96: 236-243Abstract Full Text PDF PubMed Scopus (64) Google Scholar it has been hypothesized that T cells play an important role in the pathogenesis of conjunctival fibrosis in MMP. As in most chronic immune-mediated fibroproliferative disorders,16Azouz A Razzaque MS El Hallak M Taguchi T Immunoinflammatory responses and fibrogenesis.Med Electron Microsc. 2004; 37: 141-148Crossref PubMed Scopus (40) Google Scholar both Th1 (interferon [IFN]γ) and Th2 cytokines (IL-4, IL-5) are present in MMP lesions,17Bernauer W Wright P Dart JK Leonard JN Lightman S Cytokines in the conjunctiva of acute and chronic mucous membrane pemphigoid: an immunohistochemical analysis.Graefes Arch Clin Exp Ophthalmol. 1993; 231: 563-570Crossref PubMed Scopus (33) Google Scholar, 18Caproni M Calzolari A Salvatore E Giomi B Volpi W D'Agata A Santucci M Fabbri P Cytokine profile and supposed contribution to scarring in cicatricial pemphigoid.J Oral Pathol Med. 2003; 32: 34-40Crossref PubMed Scopus (21) Google Scholar, 19Elder MJ Dart JK Lightman S Conjunctival fibrosis in ocular cicatricial pemphigoid–the role of cytokines.Exp Eye Res. 1997; 65: 165-176Crossref PubMed Scopus (50) Google Scholar, 20Razzaque MS Ahmed BS Foster CS Ahmed AR Effects of IL-4 on conjunctival fibroblasts: possible role in ocular cicatricial pemphigoid.Invest Ophthalmol Vis Sci. 2003; 44: 3417-3423Crossref PubMed Scopus (31) Google Scholar but it is not known whether the Th2 cytokine IL-13 plays a role in the autoimmune conjunctivitis and fibrosis observed in ocular MMP. IL-13 is unique in that it is not thought to exert any control over T cell function,10Hershey GK IL-13 receptors and signaling pathways: an evolving web.J Allergy Clin Immunol. 2003; 111: 677-690Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar so unlike IL-4, it does not appear to be important in the initial differentiation of CD4+T cells into Th2-type cells, but rather appears to be important in the effector phase of inflammation and repair, via its effects on monocytes/macrophages, dendritic cells, fibroblasts, and other inflammatory and stromal cells. IL-13 has been shown to exert direct pro-fibrotic effects on fibroblasts isolated from human lung and skin.21Ingram JL Rice A Geisenhoffer K Madtes DK Bonner JC Interleukin-13 stimulates the proliferation of lung myofibroblasts via a signal transducer and activator of transcription-6-dependent mechanism: a possible mechanism for the development of airway fibrosis in asthma.Chest. 2003; 123: 422S-424SCrossref PubMed Google Scholar, 22Kohyama T Liu X Wen FQ Kobayashi T Abe S Rennard SI IL-4 and IL-13 induce chemotaxis of human foreskin fibroblasts, but not human fetal lung fibroblasts.Inflammation. 2004; 28: 33-37Crossref PubMed Scopus (6) Google Scholar, 23Liu X Kohyama T Wang H Zhu YK Wen FQ Kim HJ Romberger DJ Rennard SI Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells.Am J Physiol Lung Cell Mol Physiol. 2002; 282: L1049-L1056PubMed Google Scholar, 24Saito A Okazaki H Sugawara I Yamamoto K Takizawa H Potential action of IL-4 and IL-13 as fibrogenic factors on lung fibroblasts in vitro.Int Arch Allergy Immunol. 2003; 132: 168-176Crossref PubMed Scopus (121) Google Scholar Fibroblasts isolated from different bodily sites exhibit different functional properties and site-specific gene expression,25Flavell SJ Hou TZ Lax S Filer AD Salmon M Buckley CD Fibroblasts as novel therapeutic targets in chronic inflammation.Br J Pharmacol. 2008; 153: S241-S246PubMed Google Scholar so these effects cannot necessarily be extrapolated to fibroblasts from other tissues such as the conjunctiva. Although the effects of IL-13 on normal human conjunctival fibroblast proliferation, apoptosis, and matrix metalloproteinase expression have been reported,26Fujitsu Y Fukuda K Kimura K Seki K Kumagai N Nishida T Protection of human conjunctival fibroblasts from NO-induced apoptosis by interleukin-4 or interleukin-13.Invest Ophthalmol Vis Sci. 2005; 46: 797-802Crossref PubMed Scopus (18) Google Scholar, 27Fukuda K Fujitsu Y Kumagai N Nishida T Inhibition of matrix metalloproteinase-3 synthesis in human conjunctival fibroblasts by interleukin-4 or interleukin-13.Invest Ophthalmol Vis Sci. 2006; 47: 2857-2864Crossref PubMed Scopus (31) Google Scholar its effects on other functional activities carried out by normal conjunctival fibroblasts have not been fully investigated. Fibrosis is an excessive repair response, where the replacement of normal parenchymal tissue by connective tissues is uncontrolled, and there is an imbalance between accumulation of extracellular matrix, and remodeling and collagen turnover. Fibrosis typically results from chronic inflammation due to a persistent irritant that damages tissues.12Wynn TA Fibrotic disease and the T(H)1/T(H)2 paradigm.Nat Rev Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1172) Google Scholar, 28Stramer BM Mori R Martin P The inflammation-fibrosis link? A Jekyll and Hyde role for blood cells during wound repair.J Invest Dermatol. 2007; 127: 1009-1017Crossref PubMed Scopus (187) Google Scholar Inflammatory cells, including macrophages and Th2 lymphocytes, play important roles in the chronic inflammation resulting in fibrosis, by releasing cytokines, chemokines, and growth factors, and also through direct interaction with fibroblasts.12Wynn TA Fibrotic disease and the T(H)1/T(H)2 paradigm.Nat Rev Immunol. 2004; 4: 583-594Crossref PubMed Scopus (1172) Google Scholar, 16Azouz A Razzaque MS El Hallak M Taguchi T Immunoinflammatory responses and fibrogenesis.Med Electron Microsc. 2004; 37: 141-148Crossref PubMed Scopus (40) Google Scholar, 28Stramer BM Mori R Martin P The inflammation-fibrosis link? A Jekyll and Hyde role for blood cells during wound repair.J Invest Dermatol. 2007; 127: 1009-1017Crossref PubMed Scopus (187) Google Scholar Rather than simply being structural cells, it is also recognized that fibroblasts play an active role in the persistence of inflammation by secreting cytokines and chemokines, recruiting and maintaining the survival of T cells and other inflammatory cells via both soluble factors, and by direct cross talk with lymphocytes via the CD40/CD154 pathway.25Flavell SJ Hou TZ Lax S Filer AD Salmon M Buckley CD Fibroblasts as novel therapeutic targets in chronic inflammation.Br J Pharmacol. 2008; 153: S241-S246PubMed Google Scholar, 29Filer A Parsonage G Smith E Osborne C Thomas AM Curnow SJ Rainger GE Raza K Nash GB Lord J Salmon M Buckley CD Differential survival of leukocyte subsets mediated by synovial, bone marrow, and skin fibroblasts: site-specific versus activation-dependent survival of T cells and neutrophils.Arthritis Rheum. 2006; 54: 2096-2108Crossref PubMed Scopus (72) Google Scholar It has previously been shown that IFNγ up-regulates CD40 expression on human lung fibroblasts, and that IL-13 increases CD154 levels in human lung fibroblasts, but the effects of IL-13 on co-stimulatory molecule expression by human conjunctival fibroblasts has yet to be elucidated. In the present study, we sought to explore the potential role of IL-13 in ocular MMP, and its effects on conjunctival fibroblast-related profibrotic and pro-inflammatory activity. Bulbar conjunctival biopsies obtained from patients with ocular MMP were used to study the expression of IL-13. The patients were classified according to ocular disease activity as having active disease with acute inflammation (n = 11), or chronic disease (n = 10) without clinically apparent inflammation following treatment with immunosuppressive therapy. The diagnosis of MMP was based on clinical presentation and direct immunofluorescence of the conjunctiva showing linear IgG, IgA, and/or complement deposition along the basement membrane zone. Conjunctivae from nine normal individuals undergoing routine cataract surgery were used as controls. Details regarding the patients and normal controls are shown in Table 1.30Tauber J Jabbur N Foster CS Improved detection of disease progression in ocular cicatricial pemphigoid.Cornea. 1992; 11: 446-451Crossref PubMed Scopus (80) Google Scholar Institutional research and ethics committee approval was granted and informed consent was obtained from all patients and normal controls participating in the study.Table 1Details of Patients and Controls,30Tauber J Jabbur N Foster CS Improved detection of disease progression in ocular cicatricial pemphigoid.Cornea. 1992; 11: 446-451Crossref PubMed Scopus (80) Google Scholar and IL-13 Staining ResultsDiagnosisAgeSexDisease duration (yrs)Bulbar inflammation grade (0 to 4)Tauber stage30Tauber J Jabbur N Foster CS Improved detection of disease progression in ocular cicatricial pemphigoid.Cornea. 1992; 11: 446-451Crossref PubMed Scopus (80) Google Scholar (Upper stage/Lower stage)Topical therapyActive MMP54F0.53IIcIIId/IIcIIIdHypromellose, acetylcysteine, yellow soft paraffin59M32.5I/IIaHypromellose, lacrilube76M53IIaIIIa(1)/IIbIIIb(2)Hypromellose, chloramphenicol80M103IIbIIIc(2)/IIdIIId(2)Carmellose55M133IIcIIIc(2)/IIdIIId(1)Dexamethasone, carmellose60F23I/IIcIIIc(3)Carmellose76F22.5IIc/IIbIIIa(2)Prednisolone, hypromellose, carmellose51F0.13I/IIIa(1)Nil57M0.53IIb/IIcIIIb(2)Dexamethasone, lacrilube64F12IIa/IIbIIIc(2)Prednisolone83F73IIa/IIbIIIb(2)Brimonidine, latanoprost, timolol, dorzolamide, carmelloseTreated uninflamed MMP59F101IIbIIIb(2)/IIdIIId(2)Carbomer 98086F151IIb/IIcIIIb(2)Nil78F101IIbIIIb(2)/IIdIIId(2)Nil84F20IIb/IIcIIIc(2)Nil66F20IIb/IIcIIIb(2)Nil59M41I/IIaHypromellose, carmellose, liquid paraffin, chloramphenicol76M61.5IIdIIId(2)/IIbIIIb(2)Chloramphenicol, hypromellose, retinoic acid, acetylcysteine60F31I/IIaIIIa(1)Carmellose62F0.750.5IIa/IIcIIIa(1)Hyaluronate51F11I/IIIa(1)NilNormal control50F—0—Nil76M—0—Nil65M—0—Nil70F—0—Nil82F—0—Nil65M—0—Nil75F—0—Nil73F—0—Nil62M—0—NilSystemic therapyOther eye pathologyStromal IL-13 staining (grade N to ++++)Epithelial IL-13 staining (grade N to ++++)Nil+++++++Nil++++++MycophenolateAmblyopia+++/NMycophenolate + dapsone++++++Mycophenolate + dapsone + deflazocort++++Mycophenolate + dapsone+++NMycophenolate + dapsoneSicca, blepharitis+NNil++NNil+++NNil+++++++Mycophenolate + doxycyclineGlaucoma+++/NDapsone+++++Nil++Nil+NMycophenolate++Dapsone++++Cyclophosphamide++++CyclophosphamideBlepharitis+++Cyclophosphamide + dapsone+++Prednisolone+++++Cyclophosphamide + prednisolone+++++NilCataract++++NilCataractN+NilCataractN++NilCataract+/NNNilCataract+/NNNilCataract+/NNNilCataractN+NilCataractNNNilCataract+/N+MMP mucous membrane pemphigoid, F = female, M = male, N = Nil.+, minimal extent and intensity of staining; ++, mild extent and intensity of staining; +++, moderate extent and intensity of staining; ++++, marked extent and intensity of staining. Open table in a new tab MMP mucous membrane pemphigoid, F = female, M = male, N = Nil. +, minimal extent and intensity of staining; ++, mild extent and intensity of staining; +++, moderate extent and intensity of staining; ++++, marked extent and intensity of staining. Immunohistochemistry was performed on glycol methacrylate resin-embedded sections of conjunctiva, prepared as described previously.13Bernauer W Wright P Dart JK Leonard JN Lightman S The conjunctiva in acute and chronic mucous membrane pemphigoid. An immunohistochemical analysis.Ophthalmology. 1993; 100: 339-346Abstract Full Text PDF PubMed Scopus (70) Google Scholar In brief, fresh biopsy tissue was fixed overnight in acetone and protease inhibitors at −20°C, post-fixed in acetone then dehydrated with methylbenzoate. The specimens were embedded in glycol methacrylate resin using the JB4 kit (TAAB Laboratories, Aldermaston, UK) and stored at −70°C. Serial sections 2 μm thick were cut, mounted on poly-l-lysine-coated slides and air dried. Endogenous peroxidase was inhibited using 0.3% hydrogen peroxide in 0.1% sodium azide, followed by incubation with 10% fetal calf serum to block nonspecific binding. The sections were incubated overnight at room temperature with a rabbit antibody against human IL-13 (Biogenesis Ltd, Poole UK [5378–8530]), 1:100 dilution. After washing with PBS, biotinylated goat anti-rabbit immunoglobulin (Dako, Cambridgeshire UK) was applied to the sections, followed by incubation with streptavidin-peroxidase (Dako), and finally amino-ethyl carbazole (AEC, Dako), forming a red AEC reaction product. For IL13-CD3 double staining, the slides were washed with PBS and the immunohistochemistry process repeated using a mouse antibody against human CD3 (Dako) 1:10 dilution, and 3,3′diaminobenzidine (DAB), forming a brown DAB reaction product. The specimens were counterstained with Meyer’s hematoxylin (Dako) and mounted with glycergel mounting medium (Dako). Double staining was identified by a brown or combined red-black color, due to the combination of a positive reaction to AEC and DAB. Human tonsil sections were used as positive controls, and the two negative controls used were fetal calf serum and an isotype matched, irrelevant monoclonal antibody (Dako). The number of cells stained in the stroma were counted in a masked fashion in five representative high power fields per patient, using a microscope with a graticule (Leica, Milton Keynes UK). Fibroblasts were grown from the conjunctival biopsies of normal human subjects patients as previously described.31Khaw PT Ward S Porter A Grierson I Hitchings RA Rice NS The long-term effects of 5-fluorouracil and sodium butyrate on human Tenon's fibroblasts.Invest Ophthalmol Vis Sci. 1992; 33: 2043-2052PubMed Google Scholar The explants were placed into tissue culture wells under a coverslip, and cultured with fibroblast culture medium (FCM) composed of Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat inactivated fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (all from Gibco Invitrogen Ltd, Paisley, UK) at 37°C with 5% (v/v) CO2 in air. The cells were passaged 1:3 with trypsin/EDTA. Cultures were used between passages 3 and 7 for experiments, and cultures were assessed for typical fibroblast morphology by phase contrast microscopy before every experiment. By passage 3 onwards, contaminating epithelial cells had been eliminated in cultures that had been fed only FCM, due to the fastidious nutritional requirements of epithelial cells. Serum-free medium (SFM) composed of Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine serum albumin (Sigma-Aldrich, Dorset, UK) was used where possible in all experiments where the effect of the addition of IL-13 was being evaluated. Testing under serum-free conditions eliminates the possibility that the observed responses could be due to the activity of unspecified cytokines and growth factors present in serum-containing medium. The optimal concentration of IL-13 used in experiments was initially determined from previous results.32Leonardi A Cortivo R Fregona I Plebani M Secchi AG Abatangelo G Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts.Invest Ophthalmol Vis Sci. 2003; 44: 183-189Crossref PubMed Scopus (68) Google Scholar, 33Leonardi A Curnow SJ Zhan H Calder VL Multiple cytokines in human tear specimens in seasonal and chronic allergic eye disease and in conjunctival fibroblast cultures.Clin Exp Allergy. 2006; 36: 777-784Crossref PubMed Scopus (159) Google Scholar If this level did not result in a significant response, a range of IL-13 concentrations (1, 10, 100 ng/ml) was then evaluated in a preliminary dose-response experiment. The concentration that showed the greatest response in this preliminary experiment was then selected as the optimal concentration for that assay. 3T3 cells (a fibroblast cell line) were used for initial calibration experiments that compared the negative control SFM versus the positive control FCM. To assess matrix contraction in the presence of IL-13, free-floating collagen lattice models were used. Three-dimensional fibroblast-populated type I rat tail collagen (5 mg/ml; Sigma-Aldrich, Dorset, UK) lattices were prepared with 16.7 × 105 cells/ml of lattice mixture as previously described.34Mazure A Grierson I In vitro studies of the contractility of cell types involved in proliferative vitreoretinopathy.Invest Ophthalmol Vis Sci. 1992; 33: 3407-3416PubMed Google Scholar The collagen lattices were incubated for 2 hours at 37°C to set in the culture wells, then the lattices were detached immediately after feeding with either 1) 10 ng/ml IL-13 (recombinant human IL-13, R&D systems, Abingdon, UK) in SFM, or 2) the positive control of 10% fetal calf serum-containing FCM, or 3) the negative control of SFM alone. Gelatin sepharose beads (Gelatin Sepharose 4B, Amersham Biosciences, Buckinghamshire, UK) were incubated in serum at a concentration of 10% with gentle rocking for 2 hours to produce gelatinase-free serum. Reduction in lattice area at days 1, 3, and 7 due to contraction was digitally photographed, and the gel areas calculated using image analysis software (Image J public domain Java image processing program http://rsb.info.nih.gov/ij/). Conditioned medium collected from contracting lattices was analyzed for matrix metalloproteinases (mmp)-1, mmp-2, mmp-3, mmp-8, mmp-10, and mmp-13; and tissue inhibitor of matrix metalloproteinases (timp)-1, timp-2, and timp-4 protein levels by using an antibody-coated membrane array (Raybiotech Inc, Norcross, GA) in accordance with the manufacturer’s instructions. The mmp and timp levels in baseline control medium were subtracted from the conditioned medium results. Conditioned medium collected from contracting lattices under both serum-free and serum-containing conditions, in the presence and absence of IL-13 [10 ng/ml], was analyzed for secretion of the C-terminal propeptide of type I collagen using an enzyme-linked immunosorbent assay (Quidel Corp, San Diego CA) performed according to the manufacturer’s instructions. Fibroblast cell division in the presence or absence of IL-13 was analyzed by flow cytometry using the fluorescent probe carboxyfluorescein diacetate, succinimidyl ester (CFSE; Vybrant CFDA SE Cell Tracer Kit, Invitrogen, Paisley UK). Serum-deprived fibroblasts were incubated with 1 μmol/L CFSE in serum-free medium at 37°C for 10 minutes, and ice-cold FCM was added to stop the reaction. The cell pellet was then resuspended in ice-cold FCM for 30 minutes to allow free CFSE to come out of the cells. The cell pellet was then resuspended at 2 × 105 cells/ml in FCM, and the cells were seeded into 6-well tissue culture plates at a density of 4 × 105 cells per well and stimulated with IL-13 10 ng/ml in FCM. After 72 hours, the cells were detached with trypsin and 10,000 events acquired for flow cytometry (Facscalibur, BD Pharmigen). Winlist software (Verity Software House, Topsham, ME) was used to analyze the number of divided cells per 5000 undivided cells. Cell culture inserts incorporating polyethylene terephthalate membranes with a pore size of 8 μm (BD Falcon, VWR International, Leicestershire UK), which fit into wells in a tissue culture plate, were used to assess fibroblast migration in the presence and absence of IL-13, with or without the addition of T cell culture supernatant (see below). Cells were seeded into the inserts at a density of 8000 per insert in 200 μl SFM and allowed to attach to the upper surface of the membrane for 4 hours. A volume of 700 μl of IL-13 [10 ng/ml] in SFM was added to the well in the tissue culture plate, so that the IL-13-containing medium was in contact with the undersurface of the membrane in the culture insert. SFM was used as a negative control, and 10% bovine serum FCM as a positive control. The cells were incubated for 16 hours to permit migration through the pores, to the undersurface of the membrane. The culture inserts were then washed with PBS to remove excess protein, fixed in 70% (v/v) methanol for 5 minutes, then stained with Mayer’s hematoxylin (Dako) for 30 minutes and rinsed in tap water. Settled cells on the upper surface of the membrane in the culture inserts were removed using a cotton swab. The number of migrated cells on the undersurface of the membrane was counted per ×10 objective field (average of five fields) was counted using an inverted microscope and cell counting software (Image J). Supernatant from culture of a conjunctival T cell line derived from an atopic keratoconjunctivitis patient stimulated with phytohaemagglutinin was obtained from previous experiments.35Calder VL Jolly G Hingorani M Adamson P Leonardi A Secchi AG Buckley RJ Lightman S Cytokine production and mRNA expression
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