Profibrotic Phenotype of Conjunctival Fibroblasts from Mucous Membrane Pemphigoid
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.013
ISSN1525-2191
AutoresValerie Saw, Enno Schmidt, Ifeoma Offiah, Grazyna Galatowicz, Detlef Zillikens, John Dart, Virginia L. Calder, Julie T. Daniels,
Tópico(s)Urticaria and Related Conditions
ResumoOcular mucous membrane pemphigoid is an immunobullous disease in which excessive conjunctival fibrosis causes blindness, and the pathogenesis of scarring is incompletely understood. To establish whether profibrotic fibroblasts with an altered phenotype exist in ocular mucous membrane pemphigoid, we compared the functional characteristics of pemphigoid conjunctival fibroblasts to normal conjunctival fibroblasts with respect to cell division; migration; collagen contraction; matrix metalloproteinase, secretion of collagen and chemokines; and myofibroblast differentiation. We found that pemphigoid fibroblasts showed increased cell division (P = 0.01), increased migration in serum-free medium (72 ± 18 migrated cells versus 33 ± 11, P = 0.04), increased collagen contraction in the presence of 10 ng/ml tumor necrosis factor-α, increased collagen type I secretion (P = 0.03), increased secretion of matrix metalloproteinase-3 (P = 0.03), and increased secretion of eotaxin in response to interleukin-13 (P = 0.04). Differences between pemphigoid and normal conjunctival fibroblasts with respect to collagen contraction and MMP secretion in the presence of interleukin-13 were also observed. Together, these findings indicate that pemphigoid conjunctival fibroblasts have a profibrotic phenotype that is maintained in vitro. No differences between pemphigoid fibroblasts obtained from acutely inflamed versus clinically uninflamed conjunctiva were observed. Developing effective antifibrotic therapies will require understanding of the mechanisms that both induce and maintain the profibrotic phenotype. Ocular mucous membrane pemphigoid is an immunobullous disease in which excessive conjunctival fibrosis causes blindness, and the pathogenesis of scarring is incompletely understood. To establish whether profibrotic fibroblasts with an altered phenotype exist in ocular mucous membrane pemphigoid, we compared the functional characteristics of pemphigoid conjunctival fibroblasts to normal conjunctival fibroblasts with respect to cell division; migration; collagen contraction; matrix metalloproteinase, secretion of collagen and chemokines; and myofibroblast differentiation. We found that pemphigoid fibroblasts showed increased cell division (P = 0.01), increased migration in serum-free medium (72 ± 18 migrated cells versus 33 ± 11, P = 0.04), increased collagen contraction in the presence of 10 ng/ml tumor necrosis factor-α, increased collagen type I secretion (P = 0.03), increased secretion of matrix metalloproteinase-3 (P = 0.03), and increased secretion of eotaxin in response to interleukin-13 (P = 0.04). Differences between pemphigoid and normal conjunctival fibroblasts with respect to collagen contraction and MMP secretion in the presence of interleukin-13 were also observed. Together, these findings indicate that pemphigoid conjunctival fibroblasts have a profibrotic phenotype that is maintained in vitro. No differences between pemphigoid fibroblasts obtained from acutely inflamed versus clinically uninflamed conjunctiva were observed. Developing effective antifibrotic therapies will require understanding of the mechanisms that both induce and maintain the profibrotic phenotype. Ocular mucous membrane pemphigoid (ocular MMP), also known as ocular cicatricial pemphigoid, is a blinding disease characterized by recurrent episodes of inflammation and aggressive conjunctival fibrosis.1Ahmed M. Zein G. Khawaja F. Foster C.S. Ocular cicatricial pemphigoid: pathogenesis, diagnosis and treatment.Prog Retin Eye Res. 2004; 23: 579-592Crossref PubMed Scopus (85) Google Scholar, 2Chan L.S. Ahmed A.R. Anhalt G.J. Bernauer W. Cooper K.D. Elder M.J. Fine J.D. Foster C.S. Ghohestani R. Hashimoto T. Hoang-Xuan T. Kirtschig G. Korman N.J. Lightman S. Lozada-Nur F. Marinkovich M.P. Mondino B.J. Prost-Squarcioni C. Rogers III, R.S. Setterfield J.F. West D.P. Wojnarowska F. Woodley D.T. Yancey K.B. Zillikens D. Zone J.J. 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 It is part of a spectrum of immunobullous disease associated with linear deposition of IgG and IgA autoantibodies directed against epithelial basement membrane zone (BMZ) proteins, in which subepithelial bullae and scarring are the clinical hallmarks.2Chan L.S. Ahmed A.R. Anhalt G.J. Bernauer W. Cooper K.D. Elder M.J. Fine J.D. Foster C.S. Ghohestani R. Hashimoto T. Hoang-Xuan T. Kirtschig G. Korman N.J. Lightman S. Lozada-Nur F. Marinkovich M.P. Mondino B.J. Prost-Squarcioni C. Rogers III, R.S. Setterfield J.F. West D.P. Wojnarowska F. Woodley D.T. Yancey K.B. Zillikens D. Zone J.J. 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, the autoantibodies are thought to cause subepithelial blisters by disrupting basement membrane adherence via a process involving complement, neutrophils, and proinflammatory cytokines.3Black A.P. Wojnarowska F. Ogg G.S. Role of T cells in the pathogenesis of mucous membrane pemphigoid.Expert Rev Dermatol. 2006; 1: 25-30Crossref Scopus (2) Google Scholar The resulting loss of BMZ adhesion, and inflammation secondary to the BMZ separation process, leads to fibrosis. In the eye, blisters are rarely seen clinically, and the disease usually manifests as a chronic, recurrent cicatricial conjunctivitis. Fibrosis of subepithelial tissues appears as fine, white striae most easily seen surrounding the superficial vessels of substantia propria. Contracture of these striae ultimately leads to bands of abnormal connective tissue which further contract to cause conjunctival “shrinkage.” Extensive subepithelial bands of connective tissue result in symblepharon formation. The functional consequences of scarring in the eye are severe, and patients with advanced conjunctival fibrosis become blind in 30% of cases.4Hardy K.M. Perry H.O. Pingree G.C. Kirby Jr, T.J. Benign mucous membrane pemphigoid.Arch Dermatol. 1971; 104: 467-475Crossref PubMed Scopus (202) Google Scholar Developing effective antifibrotic agents is challenging because the cellular and molecular mechanisms of conjunctival scarring are incompletely understood. Although there is clear evidence that acute inflammation following conjunctival injury in ocular MMP, either iatrogenic due to surgery or due to the autoimmune process, triggers rapid and progressive fibrosis,5Mondino B.J. Brown S.I. Lempert S. Jenkins M.S. The acute manifestations of ocular cicatricial pemphigoid: diagnosis and treatment.Ophthalmology. 1979; 86: 543-555Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 6Mondino B.J. Brown S.I. Ocular cicatricial pemphigoid.Ophthalmology. 1981; 88: 95-100Abstract Full Text PDF PubMed Scopus (171) Google Scholar it has also been observed that conjunctival fibrosis in ocular MMP can still progress despite apparent clinical control of inflammation by treatment with immunosuppressive therapy.7Miserocchi E. Baltatzis S. Roque M.R. Ahmed A.R. Foster C.S. 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 (84) Google Scholar, 8Saw V.P. Dart J.K. Rauz S. Ramsay A. Bunce C. Xing W. Maddison P.G. Phillips M. Immunosuppressive therapy for ocular mucous membrane pemphigoid strategies and outcomes.Ophthalmology. 2008; 115: 253-261Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Histological studies of chronic ocular MMP have shown that even when the conjunctiva appears clinically “white” and uninflamed, there is a significant cellular infiltrate present (“white inflammation”).9Bernauer W. Wright P. Dart J.K. Leonard J.N. 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 (34) Google Scholar, 10Elder M.J. The role of cytokines in chronic progressive conjunctival cicatrisation.Dev Ophthalmol. 1997; 28: 159-175Crossref PubMed Google Scholar Progressive fibrosis, despite immunosuppressive treatment, may be driven by this underlying chronic inflammation and ongoing release of cytokines. Administering higher doses of systemic immunosuppression might possibly control this underlying residual inflammation; however, the majority of mucous membrane pemphigoid patients are elderly and susceptible to toxicity resulting from high-dose immunosuppression.8Saw V.P. Dart J.K. Rauz S. Ramsay A. Bunce C. Xing W. Maddison P.G. Phillips M. Immunosuppressive therapy for ocular mucous membrane pemphigoid strategies and outcomes.Ophthalmology. 2008; 115: 253-261Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Another problem with the administration of immunosuppressive therapy is that there are currently no readily identifiable serological markers11Setterfield J. Shirlaw P.J. Bhogal B.S. Tilling K. Challacombe S.J. Black M.M. Cicatricial pemphigoid: serial titres of circulating IgG and IgA antibasement membrane antibodies correlate with disease activity.Br J Dermatol. 1999; 140: 645-650Crossref PubMed Scopus (44) Google Scholar for disease activity that permit the recognition of an endpoint for the control of subclinical levels of inflammation in clinically controlled disease. Although inflammation typically precedes the development of fibrosis in most fibrotic disorders, some experimental models suggest that fibrosis is not always characterized by persistent inflammation, and that to a degree, the mechanisms regulating fibrosis are distinct from those controlling inflammation.12Stramer B.M. 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 (189) Google Scholar In line with this viewpoint, an alternative theory for the observed progression of fibrosis in ocular MMP is that the fibroblasts have been transformed into an abnormally activated phenotype. Profibrotic phenotypes are observed and maintained in vitro in fibroblasts from patients with scleroderma, pulmonary, renal, and colonic fibrosis.13Ramos C. Montano M. Garcia-Alvarez J. Ruiz V. Uhal B.D. Selman M. Pardo A. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression.Am J Respir Cell Mol Biol. 2001; 24: 591-598Crossref PubMed Scopus (312) Google Scholar, 14Schuttert J.B. Liu M.H. Gliem N. Fiedler G.M. Zopf S. Mayer C. Muller G.A. Grunewald R.W. Human renal fibroblasts derived from normal and fibrotic kidneys show differences in increase of extracellular matrix synthesis and cell proliferation upon angiotensin II exposure.Pflugers Arch. 2003; 446: 387-393Crossref PubMed Scopus (20) Google Scholar, 15Trojanowska M. What did we learn by studying scleroderma fibroblasts.Clin Exp Rheumatol. 2004; 22: S59--S63PubMed Google Scholar There is some evidence of phenotypic changes in fibroblasts isolated from mucous membrane pemphigoid patients,16Razzaque M.S. Foster C.S. Ahmed A.R. Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid.Invest Ophthalmol Vis Sci. 2003; 44: 1616-1621Crossref PubMed Scopus (54) Google Scholar, 17Razzaque M.S. Foster C.S. Ahmed A.R. Role of macrophage migration inhibitory factor in conjunctival pathology in ocular cicatricial pemphigoid.Invest Ophthalmol Vis Sci. 2004; 45: 1174-1181Crossref PubMed Scopus (22) Google Scholar, 18Roat M.I. Sossi G. Lo C.Y. Thoft R.A. Hyperproliferation of conjunctival fibroblasts from patients with cicatricial pemphigoid.Arch Ophthalmol. 1989; 107: 1064-1067Crossref PubMed Scopus (34) Google Scholar but whether there are pathological alterations in key aspects of fibroblast behavior, such as motility, contractile function, matrix synthesis, and development of myofibroblast characteristics, has not been investigated. Moreover, whether there are differences in fibroblasts isolated from actively inflamed tissue compared with fibroblasts isolated from clinically uninflamed tissue is unknown. In the present study, we sought to establish whether there are phenotypic differences in fibroblast functional activity between mucous membrane pemphigoid conjunctival fibroblasts and normal conjunctival fibroblasts, and whether fibroblasts isolated from actively inflamed tissue differ from those isolated from clinically uninflamed tissue. Bulbar conjunctival biopsies were obtained from patients with ocular MMP, who were classified according to ocular disease activity as having active disease with acute inflammation (n = 10), or chronic disease without clinically apparent inflammation after treatment with immunosuppressive therapy (n = 7). Healthy, normal bulbar conjunctiva from 11 individuals of similar age (50 to 80 years) undergoing routine cataract surgery was used as controls. Details regarding the patients and normal controls are shown in Table 1. Four-millimeter snip biopsies were taken from the superior bulbar conjunctiva as previously described.10Elder M.J. The role of cytokines in chronic progressive conjunctival cicatrisation.Dev Ophthalmol. 1997; 28: 159-175Crossref PubMed Google ScholarTable 1Details of Patients and ControlsDiagnosisCaseAge (years)SexDIFIIFDisease duration (years)Bulbar inflammation grade (0 to 4)Tauber stage44 (upper stage/lower stage)Topical therapySystemic therapyOther eye pathologyActive MMP154FIgG, IgA, C3 eye and oralIgG 1/100.53IIcIIId/IIcIIIdHypromellose, acetylcysteine, yellow soft paraffinNone280MIgG, IgA eyeIgG 1/100, IgA 1/10103IIbIIIc(2)/IIdIIId(2)CarmelloseMycophenolate + dapsone355MIgG, IgA eyeNegative133IIcIIIc(2)/IIdIIId(1)Dexamethasone, carmelloseMycophenolate + dapsone + deflazocort476FIgG, IgA eyeNegative22.5IIc/IIbIIIa(2)Prednisolone, hypromellose, carmelloseMycophenolate + dapsoneSicca, blepharitis581MIgG, IgA eyeIgG 1/1022.5IIaIIIa(1)/IIbIIIb(2)CarmelloseMycophenolate + dapsone683MIgG oralNegative13IIbIIIa(1)/IIcIIIc(2)Ofloxacin, chloramphenicolNone762FIgG, IgA, C3 eye and oralNegative182IIcIIIc(2)/IIdIIId(2)Timolol, carbomer 980MycophenolateGlaucoma864FIgG, IgA vulva and mouthND12IIa/IIbIIIc(2)PrednisoloneNone983FIgG, IgA oralNC16A ELISA positive73IIa/IIbIIIb(2)Brimonidine, latanoprost, timolol, dorzolamide, carmelloseMycophenolate + doxycyclineGlaucoma1051FIgG, IgA eyeIgG to LAD10.13I/IIIa(1)NoneNoneTreated159FIgG, IgA eyeNegative101IIbIIIb(2)/IIdIIId(2)Carbomer 980Dapsoneuninflamed MMP286FIgG, IgA skinIgG positive, IgG and IgA to LAD-1151IIb/IIcIIIb(2)NoneNone378FIgG, IgA oralND101IIbIIIb(2)/IIdIIId(2)NoneNone484FIgG, IgA eyeND20IIb/IIcIIIc(2)NoneMycophenolate566FIgG, IgA eyeND20IIb/IIcIIIb(2)NoneDapsone676MIgG, IgA eyeNegative61.5IIdIIId(2)/IIbIIIb(2)Chloramphenicol, hypromellose, retinoic acid, acetylcysteineCyclophosphamide760FNegativePositive31I/IIaIIIa(1)CarmelloseCyclophosphamide + dapsoneNormal150F—0—NoneNoneCataractcontrol276M—0—NoneNoneCataract365M—0—NoneNoneCataract470F—0—NoneNoneCataract565M—0—NoneNoneCataract675F—0—NoneNoneCataract773F—0—NoneNoneCataract857M—0—NoneNoneCataract965M—0—NoneNoneCataract1079F—0—NoneNoneCataract1162M—0—NoneNoneCataractDIF, direct immunofluorescence; IIF, indirect immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay results; MMP, mucous membrane pemphigoid; F, female; M, male; IgG, immunoglobulin G; IgA, immunoglobulin A; ELISA, enzyme-linked immunosorbent assay; LAD-1, Linear IgA Disease-1 protein, which is a cleavage product of bullous pemphigoid BP180 protein; ND, not done.* Values in the IIF column include the results from immunoblotting and the ELISA. Open table in a new tab DIF, direct immunofluorescence; IIF, indirect immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay results; MMP, mucous membrane pemphigoid; F, female; M, male; IgG, immunoglobulin G; IgA, immunoglobulin A; ELISA, enzyme-linked immunosorbent assay; LAD-1, Linear IgA Disease-1 protein, which is a cleavage product of bullous pemphigoid BP180 protein; ND, not done. * Values in the IIF column include the results from immunoblotting and the ELISA. The diagnosis of MMP was based on clinical presentation with progressive conjunctival cicatrization and positive direct immunofluorescence microscopy of the conjunctiva or other tissues (oral mucosa, genital mucosa, and skin) showing linear deposition of IgG and/or IgA and/or C3 at the BMZ (Figure 1), and/or positive circulating anti-epithelial BMZ autoantibodies.19Thorne J.E. Anhalt G.J. Jabs D.A. Mucous membrane pemphigoid and pseudopemphigoid.Ophthalmology. 2004; 111: 45-52Abstract Full Text Full Text PDF PubMed Scopus (154) Google ScholarSerological testing for anti-epithelial BMZ autoantibodies was carried out by i) indirect immunofluorescence microscopy on human salt-split skin (for IgG and IgA), ii) enzyme-linked immunosorbent assay using the immunodominant NC16A domain of BP180 (type XVII collagen) as target antigen (MBL, Nagoya, Japan), iii) immunoblotting with the linear IgA dermatoses antigen 1 (LAD-1) representing the soluble ectodomain of BP180 generated from the concentrated supernatant of cultured human keratinocytes,20Schmidt E. Skrobek C. Kromminga A. Hashimoto T. Messer G. Brocker E.B. Yancey K.B. Zillikens D. Cicatricial pemphigoid: igA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180.Br J Dermatol. 2001; 145: 778-783Crossref PubMed Scopus (103) Google Scholar iv) immunoblotting with the extracellular matrix of cultured human keratinocytes for anti-laminin 332 reactivity,21Lazarova Z. Sitaru C. Zillikens D. Yancey K.B. Comparative analysis of methods for detection of anti-laminin 5 autoantibodies in patients with anti-epiligrin cicatricial pemphigoid.J Am Acad Dermatol. 2004; 51: 886-892Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar v) immunblotting with extract from human dermis for antibodies to the p200 antigens and type VII collagen,22Zillikens D. Kawahara Y. Ishiko A. Shimizu H. Mayer J. Rank C.V. Liu Z. Giudice G.J. Tran H.H. Marinkovich M.P. Brocker E.B. Hashimoto T. A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone.J Invest Dermatol. 1996; 106: 1333-1338Crossref PubMed Scopus (149) Google Scholar and vi) immunoblotting with the recombinant NC1 domain of type VII collagen, the immunodominant region in epidermolysis bullosa acquisita. Compared with indirect immunofluorescence microscopy, which detects circulating autoantibodies in about half the MMP sera, the combined testing for anti-NC16A and anti–LAD-1 autoantibodies has been shown previously to increase sensitivity to more than 80% in MMP sera.20Schmidt E. Skrobek C. Kromminga A. Hashimoto T. Messer G. Brocker E.B. Yancey K.B. Zillikens D. Cicatricial pemphigoid: igA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180.Br J Dermatol. 2001; 145: 778-783Crossref PubMed Scopus (103) Google Scholar No patients with pseudopemphigoid (diagnosed by the presence of conjunctival cicatrization, negative direct immunofluorescence microscopy, negative circulating anti-epithelial autoantibodies, and the presence of an alternative cause for cicatrization, eg, drug-induced conjunctival cicatrization, atopic conjunctivitis, rosacea, lichen planus)19Thorne J.E. Anhalt G.J. Jabs D.A. Mucous membrane pemphigoid and pseudopemphigoid.Ophthalmology. 2004; 111: 45-52Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar were included. Drug-induced conjunctival cicatrization was diagnosed if there was a history of use of topical agents previously reported to cause pseudopemphigoid (eg, β-blockers, pilocarpine) and progressive cicatrization in the eye in which the topical agent was used.23Broadway D. Drug-induced conjunctival cicatrisation.Dev Ophthalmol. 1997; 28: 86-101Crossref PubMed Scopus (6) Google Scholar Patients 7 and 9 initially presented with both oral and ocular lesions that were biopsy positive for MMP; they subsequently developed glaucoma, for which they received topical medication. The possibility cannot be excluded that, in these two patients, chronic, low-grade conjunctival inflammation and/or direct tissue damage following chronic administration of topical agents may have added to the conjunctival cicatrization mediated by MMP.23Broadway D. Drug-induced conjunctival cicatrisation.Dev Ophthalmol. 1997; 28: 86-101Crossref PubMed Scopus (6) Google Scholar Indirect immunofluorescence microscopy using human salt-split skin showed the presence of circulating anti-epithelial BMZ antibodies in 5/17 (29%) MMP patients (3/10 active MMP patients, 2/7 uninflamed MMP patients). IgG and/or IgA to LAD-1 was detected in 2/17 (12%) MMP patients, and IgG to BP180 NC16A was present in 1/17 (6%) MMP sera. No circulating autoantibodies against laminin 332 or type VII collagen were detected in any MMP patient. The immunosuppressive regimen used to control inflammation was that which we have described previously.8Saw V.P. Dart J.K. Rauz S. Ramsay A. Bunce C. Xing W. Maddison P.G. Phillips M. Immunosuppressive therapy for ocular mucous membrane pemphigoid strategies and outcomes.Ophthalmology. 2008; 115: 253-261Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar All patients and controls were white. Institutional research and ethics committee approval was granted, and informed consent was obtained from all patients and normal controls participating in the study. Fibroblasts were grown from the MMP and normal conjunctival biopsies as previously described.24Khaw P.T. Ward S. Porter A. Grierson I. Hitchings R.A. Rice N.S. 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, Scotland, 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. From 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, Gillingham, Dorset, UK) was used where possible in all experiments in which the effect of the addition of a cytokine or growth factor was being evaluated. Testing under serum-free conditions eliminates the possibility that observed responses could be due to the activity of unspecified cytokines and growth factors present in serum-containing medium. 3T3 cells (a fibroblast cell line) were used for initial calibration experiments that compared the negative-control SFM with the positive-control FCM. Fibroblast cell division was analyzed by flow cytometry using the fluorescent probe CFSE (carboxyfluorescein diacetate succinimidyl ester) (Vybrant CFDA SE Cell Tracer Kit, Invitrogen). Fibroblasts deprived of serum for 24 hours were detached with trypsin, and the cell pellet was incubated with 1 μmol/L CFSE in serum-free medium at 37°C for 10 minutes, then ice-cold FCM was added to stop the reaction. The cell pellet was resuspended in ice-cold FCM for 30 minutes to allow free CFSE to come out of the cells, then the cells were resuspended at 2 × 105 cells/ml in FCM. At time 0, one quarter of these cells were immediately acquired for flow cytometry (10,000 events…etc) these cells (time 0) were immediately acquired for flow cytometry (10,000 events; FACSCalibur; BD Biosciences, Oxford, UK). The remaining cells were seeded into six-well culture plates at a density of 4 × 105 cells per well in FCM for 72 hours (time 96 hours). After 96 hours, the cells were detached with trypsin and 10,000 events acquired for flow cytometry. 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, Lutterworth, Leicestershire, UK), which fit into wells in a tissue culture plate, were used to assess fibroblast migration. Cells were seeded into the inserts at a density of 8000 per insert in 200 μl of SFM and allowed to attach to the upper surface of the membrane for 4 hours. A volume of 700 μl of test medium, consisting of either i) SFM or ii) 10% serum-containing FCM, was added to the well in the tissue culture plate, so that the test medium was in contact with the undersurface of the membrane in the culture insert. 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, stained with Mayer's hematoxylin (Dako, Ely, Cambridgeshire, UK) for 30 minutes, and then rinsed in tap water. Settled cells on the upper surface of the membrane in the culture inserts were removed using cotton swabs. The number of migrated cells on the membrane per 10× objective field (average of five fields) was counted using an inverted phase contrast microscope and cell counting software (Image J public domain Java image processing program; http://rsb.info.nih.gov/ij/, last accessed July 22, 2006). To assess matrix contraction, free-floating, relaxed collagen gel lattice models were used. Three-dimensional, fibroblast-populated, type I rat tail collagen (5 mg/ml; Sigma-Aldrich) lattices were prepared with 16.7 × 105 cells/ml of lattice mixture as previously described.25Mazure 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 i) SFM, ii) SFM containing 10 ng/ml interleukin-13 (recombinant human IL-13; R&D Systems, Abingdon, UK), iii) SFM containing 10 ng/ml tumor necrosis factor-α (recombinant human TNFα; R&D Systems), or iv) FCM containing 10% gelatinase-free fetal calf serum (Gibco, Invitrogen, Paisley, UK). The optimal concentration of IL-13 and TNFα used was determined from previous results.26Saw V.P. Offiah I. Dart R.J. Galatowicz G. Dart J.K. Daniels J.T. Calder V.L. Conjunctival interleukin-13 expression in mucous membrane pemphigoid and functional effects of interleukin-13 on conjunctival fibroblasts in vitro.Am J Pathol. 2009; 175: 2406-2415Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 27Saw V.P. Dart R.J. Galatowicz G. Daniels J.T. Dart J.K. Calder V.L. Tumor necrosis factor-alpha in ocular mucous membrane pemphigoid and its effect on conjunctival fibroblasts.Invest Ophthalmol Vis Sci. 2009; 50: 5310-5317Crossref PubMed Scopus (27) Google Scholar Gelatin Sepharose beads (Gelatin Sepharose 4B; Amersham Biosciences, Little Chalfont, 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). Conditioned medium collected from contracting lattices was analyzed for matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-10, MMP-13, tissue inhibitor of matrix metalloproteinase-1 (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. MMP and TIMP levels in baseline SFM and FCM were subtracted from the conditioned medium results. Conditioned medium collected from contracting lattices was also analyzed for secretion of the C-terminal propeptide of type I collagen using an enzyme-linked immunosorbent assay (Quidel Corp, San Diego, CA) carried out according to the manufacturer's instructions. Confluent fibroblasts in six-well culture plates were serum starved for 24 hours, then stimulated for 72 hours with a panel of cytokines in 10% heat-inactivated serum-containing FCM. The cytokines selected were those previously reported to be present in ocular MMP, including transforming growth factor-β (TGFβ) 50 ng/ml,16Razzaque M.S. Foster C.S. Ahmed A.R. Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid.Invest Ophthalmol Vis Sci. 2003; 44: 1616-1621Crossref PubMed Scopus (54) Google Scholar IL-4 20 ng/ml,28Caproni M. Calzolari A. Giomi B. Santucci M. Ficarra G. Fabbri P. IL-4. IL-5, TGF-beta1 and IFN-gamma mRNAs detected by a new in situ amplification system in cicatricial pemphigoid.Exp Dermatol. 2002; 11: 421-427Crossref PubMed Scopus (8) Google Scholar interferon-γ (IFNγ) 200 ng/ml,28Caproni M. Calzolari A. Giomi B. Santucci M. Ficarra G.
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