Ex Vivo Pathogenicity of Anti–Laminin γ1 Autoantibodies
2013; Elsevier BV; Volume: 184; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2013.10.019
ISSN1525-2191
AutoresFlorina Florea, Claudia Bernards, Marzia Caproni, Jessika Kleindienst, Takashi Hashimoto, Manuel Koch, Cassian Sitaru,
Tópico(s)Urticaria and Related Conditions
ResumoAutoimmunity against laminins has been described in several autoimmune diseases (including mucous membrane pemphigoid, anti–laminin γ1 pemphigoid, and connective tissue diseases), in pregnancy loss, and in infections such as Chagas disease. Except for anti–laminin-332 mucous membrane pemphigoid, adequate evidence has been lacking for the tissue injury potential of laminin-specific antibodies and the pathogenic epitopes. We evaluated the pathogenic potential of antibodies targeting laminin γ1, a major constituent of basement membranes and the main antigen in anti–laminin γ1 pemphigoid. Rabbit antibodies were generated against fragments of the N-terminus and C-terminus of murine laminin γ1, and their ability to disrupt ligand interactions and/or to activate complement and granulocytes was assessed using previously established ex vivo assays. Our findings document a pathogenic potential of antibodies targeting the laminin γ1 N-terminus. These antibodies interfere with the binding of nidogen to laminin and can activate granulocytes and the complement cascade. We detected antibodies with different degrees of reactivity with laminin γ1 N-terminus in patients with anti–laminin γ1 pemphigoid, cutaneous lupus erythematosus, and scleroderma. Our results provide mechanistic insights into the tissue damage associated with laminin autoimmunity and could facilitate development of appropriate diagnostic tools and therapeutic strategies. Autoimmunity against laminins has been described in several autoimmune diseases (including mucous membrane pemphigoid, anti–laminin γ1 pemphigoid, and connective tissue diseases), in pregnancy loss, and in infections such as Chagas disease. Except for anti–laminin-332 mucous membrane pemphigoid, adequate evidence has been lacking for the tissue injury potential of laminin-specific antibodies and the pathogenic epitopes. We evaluated the pathogenic potential of antibodies targeting laminin γ1, a major constituent of basement membranes and the main antigen in anti–laminin γ1 pemphigoid. Rabbit antibodies were generated against fragments of the N-terminus and C-terminus of murine laminin γ1, and their ability to disrupt ligand interactions and/or to activate complement and granulocytes was assessed using previously established ex vivo assays. Our findings document a pathogenic potential of antibodies targeting the laminin γ1 N-terminus. These antibodies interfere with the binding of nidogen to laminin and can activate granulocytes and the complement cascade. We detected antibodies with different degrees of reactivity with laminin γ1 N-terminus in patients with anti–laminin γ1 pemphigoid, cutaneous lupus erythematosus, and scleroderma. Our results provide mechanistic insights into the tissue damage associated with laminin autoimmunity and could facilitate development of appropriate diagnostic tools and therapeutic strategies. Autoimmunity is characterized by a failure or breakdown of self-tolerance mechanisms, leading to a B-cell and/or T-cell–mediated immune response directed against the self that can induce tissue damage and result in autoimmune disease. Among the immunological players involved in autoimmune disease development, the inappropriate production of autoantibodies that mediate tissue injury plays a role in several of the more than 80 described autoimmune diseases.1Sesarman A. Vidarsson G. Sitaru C. The neonatal Fc receptor as therapeutic target in IgG-mediated autoimmune diseases.Cell Mol Life Sci. 2010; 67: 2533-2550Crossref PubMed Scopus (42) Google Scholar The interplay between antibodies and self-antigen, either circulating or tissue-bound, may induce damage by several effector mechanisms, including inflammatory and noninflammatory complement-mediated and Fc-mediated reactions, as well as Fc-independent noninflammatory pathways. Antibodies bound to self-antigens from cell surfaces can trigger tissue damage by Fc-mediated or complement-mediated mechanisms, including induction of cell phagocytosis, cell lysis, and recruitment and activation of inflammatory cells, as is described in autoimmune diseases such as autoimmune hemolytic anemia, rheumatoid arthritis, myasthenia gravis, and bullous pemphigoid. Antibodies may, however, exert direct pathogenic effector functions in an Fc-independent manner, including stimulation of the bound receptor (as in Graves' disease) or disruption of protein–protein interactions through steric hindrance (as in pemphigus). The in vivo pathogenicity of autoantibodies in several autoimmune disorders has been demonstrated by the passive transfer of antigen-specific IgG from patients or immunized animals.1Sesarman A. Vidarsson G. Sitaru C. The neonatal Fc receptor as therapeutic target in IgG-mediated autoimmune diseases.Cell Mol Life Sci. 2010; 67: 2533-2550Crossref PubMed Scopus (42) Google Scholar Ex vivo models have also proved useful in confirming the tissue damage-inducing potential of antibodies in autoimmune diseases, including skin blistering disorders, multiple sclerosis, neuromyelitis optica, and antiphospholipid syndrome.2Sitaru C. Zillikens D. Mechanisms of blister induction by autoantibodies.Exp Dermatol. 2005; 14: 861-875Crossref PubMed Scopus (138) Google Scholar, 3Harrer M.D. von Büdingen H. Stoppini L. Alliod C. Pouly S. Fischer K. Goebels N. Live imaging of remyelination after antibody-mediated demyelination in an ex-vivo model for immune mediated CNS damage.Exp Neurol. 2009; 216: 431-438Crossref PubMed Scopus (23) Google Scholar, 4Zhang H. Bennett J.L. Verkman A.S. Ex vivo spinal cord slice model of neuromyelitis optica reveals novel immunopathogenic mechanisms.Ann Neurol. 2011; 70: 943-954Crossref PubMed Scopus (127) Google Scholar, 5Meroni P.L. Pathogenesis of the antiphospholipid syndrome: an additional example of the mosaic of autoimmunity.J Autoimmun. 2008; 30: 99-103Crossref PubMed Scopus (59) Google Scholar Autoantibodies against laminins have been reported in several human diseases, including anti–laminin-332 mucous membrane pemphigoid,2Sitaru C. Zillikens D. Mechanisms of blister induction by autoantibodies.Exp Dermatol. 2005; 14: 861-875Crossref PubMed Scopus (138) Google Scholar anti–laminin γ1 pemphigoid (previously known as anti-p200 pemphigoid),6Dainichi T. Koga H. Tsuji T. Ishii N. Ohyama B. Ueda A. Natsuaki Y. Karashima T. Nakama T. Yasumoto S. Zillikens D. Hashimoto T. From anti-p200 pemphigoid to anti-laminin gamma1 pemphigoid.J Dermatol. 2010; 37: 231-238Crossref PubMed Scopus (70) Google Scholar lupus erythematosus,7Caproni M. Antiga E. Cardinali C. Del Bianco E. Fabbri P. Antilaminin-1 antibodies in cutaneous lupus erythematosus patients.Lupus. 2009; 18: 858Crossref PubMed Scopus (7) Google Scholar, 8Groth S. Vafia K. Recke A. Dähnrich C. Zillikens D. Stöcker W. Kuhn A. Schmidt E. Antibodies to the c-terminus of laminin γ1 are present in a distinct subgroup of patients with systemic and cutaneous lupus erythematosus.Lupus. 2012; 21: 1482-1483Crossref PubMed Scopus (12) Google Scholar systemic sclerosis and Raynaud phenomena,9Gabrielli A. Montroni M. Rupoli S. Caniglia M.L. DeLustro F. Danieli G. A retrospective study of antibodies against basement membrane antigens (type IV collagen and laminin) in patients with primary and secondary Raynaud's phenomenon.Arthritis Rheum. 1988; 31: 1432-1436Crossref PubMed Scopus (30) Google Scholar cardiomyopathy and myocarditis,10Wolff P.G. Kühl U. Schultheiss H.P. Laminin distribution and autoantibodies to laminin in dilated cardiomyopathy and myocarditis.Am Heart J. 1989; 117: 1303-1309Abstract Full Text PDF PubMed Scopus (93) Google Scholar endometriosis and pregnancy loss,11Inagaki J. Kondo A. Lopez L.R. Shoenfeld Y. Matsuura E. Anti-laminin-1 autoantibodies, pregnancy loss and endometriosis.Clin Dev Immunol. 2004; 11: 261-266Crossref PubMed Scopus (20) Google Scholar and Chagas disease.12Szarfman A. Terranova V.P. Rennard S.I. Foidart J.M. de Fatima Lima M. Scheinman J.I. Martin G.R. Antibodies to laminin in Chagas' disease.J Exp Med. 1982; 155: 1161-1171Crossref PubMed Scopus (130) Google Scholar Laminin γ1 is an extracellular protein present in all basement membranes as a constitutive chain of almost all laminin isoforms. It has a critical role in embryogenesis; its absence in mice results in lack of basement membrane formation and death at the peri-implantation stage of embryonic development.13Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation.J Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (421) Google Scholar Through its short arm located at the N-terminus, laminin γ1 binds nidogen, which cross-links the two meshworks of laminins and collagen IV within the basement membrane.14Mayer U. Nischt R. Pöschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. A single EGF-like motif of laminin is responsible for high affinity nidogen binding.EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (247) Google Scholar Laminin γ1 and γ2 chains are critically involved in laminin recognition by integrins through their C-terminal regions.15Ido H. Nakamura A. Kobayashi R. Ito S. Li S. Futaki S. Sekiguchi K. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.J Biol Chem. 2007; 282: 11144-11154Crossref PubMed Scopus (78) Google Scholar Integrins mediate the adhesive interactions of laminin with cells, which are fundamental for tissue development, differentiation, and function. In the skin, laminin γ1 is present as a constituent of laminin-311, -321, and -511, which can bind to integrins α3β1, α6β4, and α6β1.16Margadant C. Charafeddine R.A. Sonnenberg A. Unique and redundant functions of integrins in the epidermis.FASEB J. 2010; 24: 4133-4152Crossref PubMed Scopus (121) Google Scholar It has been recently shown that serum of anti–laminin γ1 pemphigoid patients reacts mainly with the C-terminus of laminin γ1, but epitopes located outside this fragment may also be targeted.6Dainichi T. Koga H. Tsuji T. Ishii N. Ohyama B. Ueda A. Natsuaki Y. Karashima T. Nakama T. Yasumoto S. Zillikens D. Hashimoto T. From anti-p200 pemphigoid to anti-laminin gamma1 pemphigoid.J Dermatol. 2010; 37: 231-238Crossref PubMed Scopus (70) Google Scholar, 17Groth S. Recke A. Vafia K. Ludwig R.J. Hashimoto T. Zillikens D. Schmidt E. Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid.Br J Dermatol. 2011; 164: 76-82Crossref PubMed Scopus (64) Google Scholar, 18Vafia K. Groth S. Beckmann T. Hirose M. Dworschak J. Recke A. Ludwig R. Hashimoto T. Zillikens D. Schmidt E. Pathogenicity of autoantibodies in anti-p200 pemphigoid.PLoS One. 2012; 7: e41769Crossref PubMed Scopus (37) Google Scholar Anti–laminin γ1 pemphigoid is an autoimmune subepidermal blistering disease characterized by circulating and tissue-bound antibodies and a neutrophil-rich inflammatory infiltrate.6Dainichi T. Koga H. Tsuji T. Ishii N. Ohyama B. Ueda A. Natsuaki Y. Karashima T. Nakama T. Yasumoto S. Zillikens D. Hashimoto T. From anti-p200 pemphigoid to anti-laminin gamma1 pemphigoid.J Dermatol. 2010; 37: 231-238Crossref PubMed Scopus (70) Google Scholar Interestingly, despite the widespread distribution of the autoantigen, the pathology remains organ-specific. The pathogenic potential of antibodies targeting laminin-111 is still poorly understood. Immunization with the autoantigen and passive transfer of specific antibodies in animal models had little effect or (depending on the experimental setting) induced limited renal disease, myositis, abortion, or reproductive failure.19Abrahamson D.R. Caulfield J.P. Proteinuria and structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin g.J Exp Med. 1982; 156: 128-145Crossref PubMed Scopus (67) Google Scholar, 20Murphy-Ullrich J.E. Oberley T.D. Immune-mediated injury to basement membranes in mice immunized with murine laminin.Clin Immunol Immunopathol. 1984; 31: 33-43Crossref PubMed Scopus (18) Google Scholar, 21Nakano J. Yoshimura T. Okita M. Motomura M. Kamei S. Matsuo H. Eguchi K. Laminin-induced autoimmune myositis in rats.J Neuropathol Exp Neurol. 2005; 64: 790-796Crossref PubMed Scopus (10) Google Scholar, 22Matalon S.T. Blank M. Matsuura E. Inagaki J. Nomizu M. Levi Y. Koike T. Shere Y. Ornoy A. Shoenfeld Y. Immunization of naïve mice with mouse laminin-1 affected pregnancy outcome in a mouse model.Am J Reprod Immunol. 2003; 50: 159-165Crossref PubMed Scopus (20) Google Scholar, 23Weeks B.S. Klein N.W. Kleinman H. Frederickson T. Sackett G.P. Laminin immunized monkeys develop sera toxic to cultured rat embryos and fail to reproduce.Teratology. 1989; 40: 47-57Crossref PubMed Scopus (31) Google Scholar Because a skin-specific effect had not yet been demonstrated for autoantibodies against laminin γ1,18 in the present study we addressed the ex vivo pathogenic potential of laminin γ1–specific antibodies. For this purpose, rabbit antibodies were generated against N- and C-terminal fragments of mouse laminin γ1. Subsequently, their direct effect on nidogen and integrin interactions was investigated in a cell adhesion assay and a solid-phase ligand inhibition and disruption assay. Furthermore, the ability of the newly generated antibodies to activate the complement system and/or inflammatory cells was assessed in a complement fixation test and antibody-induced granulocyte-dependent dermal–epidermal separation assay or by measurement of reactive oxygen species (ROS) production. Our findings show that anti–laminin γ1 antibodies, especially those targeting the N-terminus, have pathogenic potential either through direct inhibition and/or disruption of the nidogen 1 binding or by Fc-mediated activation of granulocytes and complement cascade. Interestingly, autoantibodies against laminin γ1 N-terminus were detected not only in cases of anti–laminin γ1 pemphigoid, but also in cutaneous lupus erythematosus (CLE) and scleroderma. Recombinant human laminin-511 was purchased from BioLamina (Stockholm, Sweden), natural mouse laminin-111 from Life Technologies–Invitrogen (Darmstadt, Germany; Carlsbad, CA), and recombinant human α6β4 from R&D Systems (Minneapolis, MN). Human laminin γ1 N-terminus was expressed in a mammalian system as described previously.24Patel T.R. Morris G.A. Zwolanek D. Keene D.R. Li J. Harding S.E. Koch M. Stetefeld J. Nano-structure of the laminin γ-1 short arm reveals an extended and curved multidomain assembly.Matrix Biol. 2010; 29: 565-572Crossref PubMed Scopus (30) Google Scholar The murine laminin γ1 fragments and nidogen 1 were recombinantly expressed in a mammalian or a bacterial system, as described below. The DNA sequence data for murine laminin γ1 was retrieved from GenBank (http://www.ncbi.nlm.nih.gov/nuccore, accession number NM_010683) using the accession number NM_010683. The cDNA sequence encoding mouse laminin γ1 N-terminal fragment [mLnγ1-Nterm, amino acids (aa) 30–988] was synthesized from total RNA (SuperScript III kit; Life Technologies–Invitrogen) using primers as presented in Table 1 and cloned via NheI/BamHI restriction sites into a modified pCEP-Pu expression vector containing a thrombin cleavage site next to a 2×StrepII/FLAG tag at the C-terminal end of the protein sequence.25Koch M. Schulze J. Hansen U. Ashwodt T. Keene D.R. Brunken W.J. Burgeson R.E. Bruckner P. Bruckner-Tuderman L. A novel marker of tissue junctions, collagen XXII.J Biol Chem. 2004; 279: 22514-22521Crossref PubMed Scopus (138) Google Scholar 293-EBNA cells were transfected with the expression vector, and the supernatant was collected and supplemented with 1 mmol/L phenylmethylsulfonyl fluoride (Sigma-Aldrich, Munich, Germany; St. Louis, MO). After filtration, the supernatant was passed through a Strep-Tactin Sepharose column (IBA, Göttingen, Germany), and the recombinant protein was eluted with buffer [100 mmol/L Tris, 150 mmol/L NaCl (pH 7.4)] containing 2.5 mmol/L d-desthiobiotin (Sigma-Aldrich). For the removal of the 2×StrepII/FLAG tag, the purified glycosylated protein (Supplemental Figure S1) was incubated with 1 U bovine thrombin (Sigma-Aldrich) per milligram protein overnight, at room temperature [(in Tris-buffered saline with 5 mmol/L CaCl2 (pH 8.2)]. Afterward, the protein was applied on a Strep-Tactin Sepharose column and the digested protein was collected in the flow-through.Table 1Primer Sequences for PCR Amplification of cDNA Fragments of Murine Laminin γ1 and Nidogen 1FragmentRestriction sitePrimer sequencemLnγ1-NtermNheIForward: 5′-CACGCTAGCAGCCATGGACGAGTGCGCGGAT-3′BamHIReverse: 5′-TAGGATTCCCGACGTGTGGGCCTAGGAAC-3′mLnγ1-Cterm-1BamHIForward: 5′-GATCGGATCCTTCTACAATCGGTCCTGGCCTG-3′SalIReverse: 5′-GATCGTCGACTCAAATCTCCAGCGCAGTTTGA-3′mLnγ1-Cterm-2BamHIForward 5′-GATCGGATCCGCAGGAGAAAATCAAACTGCGC-3′SalIReverse: 5′-GATCGTCGACTCAGGCGTCAGCGGCAGCATTG-3′mLnγ1-Cterm-3BamHIForward: 5′-GATCGGATCCAACCGGACCATAGCTGAAGCC-3′SalIReverse: 5′-GATCGTCGACTCAGGGCTTCTCGATGGACGG-3′Nidogen 1NheIForward: 5′-AAAGCTAGCCTGAATCGCCAGGAGCTCTTCCC-3′NotIReverse: 5′-CTGACGTAACTTGCCTTTACTCGCCGGCGTTAT-3′ Open table in a new tab The cDNA sequences coding for the three overlapping fragments of murine laminin γ1 C-terminus (aa 1018–1607) were cloned into prokaryotic vectors and expressed in Escherichia coli as glutathione S-transferase (GST)–fusion proteins according to protocols described previously.26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar Synthesis of the primers used in PCR (Table 1), as well as cloning of the cDNA encoding the fragments into pUC57 vector, was performed at GenScript (Piscataway, NJ). The cDNA sequences were subcloned into the linearized pGEX-6P-1 vector (Amersham; GE Healthcare, Freiburg, Germany; Little Chalfont, UK) using the BamHI/SalI cutting site, resulting in the recombinant vectors pGEX-LAMC1-Cterm-1, pGEX-LAMC1-Cterm-2, and pGEX-LAMC1-Cterm-3. The correct ligation and in-frame insertion of various DNA fragments were confirmed by DNA sequence analysis performed by GATC Biotech (Konstanz, Germany). Recombinant GST-fusion proteins were expressed in E. coli strain BL21 and purified by glutathione agarose affinity chromatography, as described previously.26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar The DNA sequence data for murine nidogen 1 was retrieved from GenBank (http://www.ncbi.nlm.nih.gov/nuccore, accession number NP_035047.2). The full-length murine nidogen 1 cDNA sequence obtained by reverse transcription from total RNA (SuperScript III kit, Life Technologies–Invitrogen) was cloned into a modified pCEP-Pu vector containing a thrombin cleavage site next to a 2×StrepII/FLAG tag at the N-terminal end of the protein sequence and expressed in 293-EBNA cells. Primer sequences are presented in Table 1. Protein concentrations were determined by spectrophotometry at 280 nm (NanoDrop 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA). Recombinant 2×StrepII/FLAG–mLnγ1-Nterm, GST-tagged C-terminal fragments (GST-mLnγ1-Cterm-1, GST-mLnγ1-Cterm-2, GST-mLnγ1-Cterm-3), and 2×StrepII/FLAG–nidogen 1 were fractionated by SDS-PAGE, transferred onto nitrocellulose, and analyzed by immunoblotting.25Koch M. Schulze J. Hansen U. Ashwodt T. Keene D.R. Brunken W.J. Burgeson R.E. Bruckner P. Bruckner-Tuderman L. A novel marker of tissue junctions, collagen XXII.J Biol Chem. 2004; 279: 22514-22521Crossref PubMed Scopus (138) Google Scholar, 26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar For detection of the GST-tagged fragments, after blocking, the membrane was incubated for 2 hours at room temperature with goat polyclonal antibody specific to GST (GE Healthcare) diluted 3000-fold. Subsequently, the bound goat IgG was detected with 5000-fold diluted horseradish peroxidase (HRP)–labeled antibody (Promega, Mannheim, Germany; Madison, WI) and diaminobenzidine. GST alone was used as control. For detection of recombinant 2×StrepII/FLAG-fusion nidogen 1, the membrane was incubated with murine monoclonal antibody specific to StrepII tag (IBA) diluted 2000-fold, for 1 hour at room temperature. Next, the murine antibody was detected with a 3000-fold diluted HRP-coupled antibody (Dako, Hamburg, Germany; Carpinteria, CA), and the signal was visualized with chemiluminescence substrate. The recombinant 2×StrepII/FLAG-fusion mLnγ1-Nterm and the tag-free mLnγ1-Nterm were detected using the 1000-fold diluted serum of rabbits immunized with mLnγ1-Nterm, for 1 hour at room temperature, followed by incubation with the 3000-fold diluted HRP-coupled antibody specific to rabbit IgG (Dako) and then signal detection by chemiluminescence. Rabbit polyclonal antibodies were produced against the recombinant mLnγ1-Nterm fragment and the GST-tagged mLnγ1-Cterm-1, -2, and -3 fragments according to established protocols.27Sitaru C. Mihai S. Otto C. Chiriac M.T. Hausser I. Dotterweich B. Saito H. Rose C. Ishiko A. Zillikens D. Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type vii collagen.J Clin Invest. 2005; 115: 870-878Crossref PubMed Scopus (210) Google Scholar Rabbit polyclonal antibodies were also generated against the recombinant GST-fusion protein containing sequences of murine collagen VII (GST-mCVII-1, aa 1-300 and GST-mCVII-2, aa 281-594)26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar and used as controls in several experiments, as specified. IgG from rabbit serum was purified by protein G Sepharose 4 fast flow affinity column chromatography (GE Healthcare), as described previously.27Sitaru C. Mihai S. Otto C. Chiriac M.T. Hausser I. Dotterweich B. Saito H. Rose C. Ishiko A. Zillikens D. Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type vii collagen.J Clin Invest. 2005; 115: 870-878Crossref PubMed Scopus (210) Google Scholar The eluted fractions were concentrated by ultrafiltration using Amicon Ultra-15 (15 mL, 30 kDa) centrifugal filter units (EMD Millipore, Billerica, MA) in a centrifuge at 3600 × g and 4°C, 45 minutes per cycle. Protein concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Serum samples were obtained from patients with anti–laminin γ1 pemphigoid (n = 7), CLE (discoid CLE, n = 13; subacute CLE, n = 2; acute CLE, n = 4), dermatomyositis (n = 9), systemic sclerosis, (n = 10); and localized scleroderma, (n = 14). Anti–laminin γ1 pemphigoid patients were characterized by i) blisters on the skin; ii) linear deposits of IgG at the dermal–epidermal junction (DEJ), as observed under direct immunofluorescence (IF) microscopy; iii) circulating IgG binding the dermal side of the 1 mol/L NaCl-split skin, as observed under indirect IF microscopy; and iv) immunoblot reactivity with the 200-kDa protein from dermal extracts. Normal control serum samples were obtained from healthy donors (n = 20). Informed consent was obtained from patients whose material was used in the study, in adherence with Helsinki principles. The ability of the antibodies to activate the complement cascade was assessed in vitro by a complement fixation test, according to described protocols.26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar In brief, cryosections (6 μm thick) of normal mouse skin were incubated with normal rabbit serum, with rabbit serum raised against mLnγ1-Nterm (diluted 1:10) or against GST-mLnγ1-Cterm-1, -2, or -3 (diluted 1:2), or with purified rabbit IgG antibodies targeting the C-terminal fragments of laminin γ1 (diluted 1:10), followed by incubation with serum from a healthy human donor fivefold diluted in gelatin Veronal buffer (Sigma-Aldrich) as a source of complement. Rabbit polyclonal antibody targeting GST-mCVII-2 (diluted 1:10) was used as control. Bound human complement C3 was subsequently detected with 100-fold diluted fluorescein isothiocyanate–labeled antibody (Cappel; MP Biomedicals, Eschwege, Germany; Santa Ana, CA). Sections were counterstained with 1000-fold diluted DAPI (Sigma-Aldrich). The blister-inducing potential of antibodies targeting different fragments of murine laminin γ1 was determined by an ex vivo assay of antibody-induced granulocyte-dependent dermal–epidermal separation, as described previously.26Csorba K. Sesarman A. Oswald E. Feldrihan V. Fritsch A. Hashimoto T. Sitaru C. Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.Cell Mol Life Sci. 2010; 67: 1343-1351Crossref PubMed Scopus (21) Google Scholar In brief, cryosections of murine skin were incubated with rabbit serum against mLnγ1-Nterm (diluted 1:5), GST-mLnγ1-Cterm-1, -2, or -3 (diluted 1:2) or with purified rabbit IgG specific to mLnγ1-Nterm (diluted 1:10), GST-mLnγ1-Cterm-1, -2, or -3 (diluted 1:5). Rabbit polyclonal antibody specific to GST-mCVII-1 (diluted 1:5) was used as control. Leukocytes isolated from healthy human donors were added to cryosections and incubated at 37°C for 3 hours. The extent of dermal–epidermal separation was assessed under a microscope and quantified as the percentage of the split area at the DEJ relative to the total length of the DEJ. Recombinant mLnγ1-Nterm [5 μg/mL in 0.1 mol/L carbonate buffer (pH 9.3)] was coated onto 96-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany) overnight, at 4°C. The wells were then blocked with 1% bovine serum albumin (BSA) (fraction V; Sigma-Aldrich) in PBS and subsequently incubated with serial dilutions of recombinant mouse 2×StrepII/FLAG-fusion nidogen 1. The amount of bound nidogen was detected with 5000-fold diluted HRP-tagged mouse antibody against StrepII tag (AbD Serotec, Kidlington, UK; Raleigh, NC). For enzymatic reaction, 50 μL of orthophenylenediamine (Sigma-Aldrich) solution in water and 0.1% (v/v) H2O2 was added to each well. The reaction was stopped after 10 minutes with 50 μL/well of 0.5 mol/L H2SO4, and absorbance at 490 nm was read using a microplate reader (Labsystems Multiskan Multisoft; Thermo Fisher Scientific). For the inhibition assay, mLnγ1-Nterm–coated wells were first incubated with serial dilutions of rabbit serum reactive with mLnγ1-Nterm, patient's serum, or normal rabbit or human serum for 1 hour at room temperature. Subsequently the recombinant 2×StrepII/FLAG-tagged nidogen 1, 3.5 nmol/L in blocking buffer was added to the wells and the plate was incubated for 2 hours at room temperature. After washing, the bound nidogen was detected with 5000-fold diluted HRP-tagged antibody against StrepII tag (AbD Serotec). For the disruption of protein binding assay, wells were first incubated with nidogen 1, followed by incubation with serial dilutions of rabbit serum against mLnγ1-Nterm or normal rabbit serum; subsequently, the nidogen 1 was detected. Inhibition and disruption were calculated as percentages, relative to uninhibited controls. For integrin binding inhibition assay, published protocols were followed, with minor modification.28Nishiuchi R. Takagi J. Hayashi M. Ido H. Yagi Y. Sanzen N. Tsuji T. Yamada M. Sekiguchi K. Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.Matrix Biol. 2006; 25: 189-197Crossref PubMed Scopus (271) Google Scholar In brief, 10 nmol/L recombinant human laminin-511 was coated onto 96-well microtiter plates in 0.1 mol/L carbonate buffer (pH 9.3) overnight, at 4°C. Wells were washed with PBS, blocked with 1% BSA in PBS, and then incubated with a 25-fold diluted mix of rabbit serum specific to GST-mLnγ1-Cterm-1, -2, or -3 or with normal rabbit serum in blocking buffer. After washing with PBS containing either 1 mmol/L MnCl2 and 2 mmol/L MgCl2 or 10 mmol/L EDTA, 80 nmol/L recombinant human integrin α6β4 (R&D Systems) in blocking buffer was added in the presence of 2 mmol/L MgCl2 and 1 mmol/L MnCl2 or of 10 mmol/L EDTA and incubated for 2 hours at room temperature. After washing, the bound integrin α6β4 was detected by a 3000-fold diluted HRP-labeled rabbit polyclonal anti-His antibody (Novus Biologicals, Littleton, CO). Readings obtained in the presence of 10 mmol/L EDTA were subtracted. The binding of integrin was calculated as percentage of the value obtained in the absence of rabbit serum. HaCaT immortalized human keratinocytes (Dr. Petra Boucamp, DKFZ, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum, 4 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biochrom; Merck Millipore, Billerica, MA). Cell adhesion assay was performed a
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