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The role of mast cells in experimental bullous pemphigoid
ESDR I JSID I SID Abstracts
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AUTOIMMUNE RESPONSES OF LINEAR IGA DISEASE BOTH T CELLS AND AUfOmODIES RECOGNIZE THE NC16A DOMAIN OF BPI 80. Mong-Shang Lm. Susan I. Swartz, Mmish Gbaria, Janet A. Fairley. George 1. Gnudlce, and Luis A Diaz. Department of Dermatology, Medical College of Wisconsin, and the VA Medtcal Center, Milwaukee, WI, USA. Luwar IgA disease (LAD) is an antibody-mediated cutaneous automumme diseme characterized by IgA autoantibodies against the epidemml basement membrane mne and by subepidmnal blisters It ba prwiowly been shown that IgA autoantibodies of LAD recogmze epidemxd protam of 97, 120, and 180 kDa These results suggest the possible heterogenaty of autoantigen andamoanbbodies involvedm this disease. Recent ammo aad sequence analysis of tie 97 kDa LAD antigen (reputed by Zone et al.) showed multiple peptide stretches that are idmtiEal to BPIBO, a trammembrane hemldesmosomal protein. In tlus study, we provide direct evidence that the BP180 molecule is indeed the target autoantigen of both autoimmune T lvmohocvtes and autoantibo&s from LAD patients IaA auto&bodies from three LAD pat&s i&d by immunoblotting with fusion protans er&npassing the BPI 80 NCl6A domain Interestingly, IgGautamtibodxs from two of these patients also reacted with a segment of NCl6A. Using T cell proliferation assays, we found that T lymphocytes from these LAD patimtx spxi6calIy respaded to BP180 fbs~on proteins, but not to antigens of other unrelated cutaneous diseases. T cells from normal mdwduals or patients with unrelated dermatoses (e g., PV, PF and psonasis) did not proliferate in response to BP180 fusmn proterns, confirming the specdicity of this response T cell lines developed from one LAD patient express TcRa@, CD4, and CD45R0, but not CD8 or CD45RA. mdicahng that the BP180 respondmg T cells represent CD4+ memory T cells Thesefindings suggest that BPIBO-reactwe T cells may participate m the pathogen&s of LAD
THE ROLE OF MAST CELLS IN EXPERIMENTALBULLOUS PEMPHIGOID. Zhi Uu. XjaoveZhou. Gecme J. Giudii. and Luis A. Diaz. Departmentof Dermatology, Medical College of Wisconsin, Milwaukee.WI 53226. Bullout pemphigoid (BP) is an inflammatory subepidermal blistering disease associated with IgG autoimmune responses to the hemidesmosomal proteins, BP180 and BP230. A variety of infiltrating cells have been identified in the perllesionaklesional site in patients with BP. Mast ceil accumulation and degranulation have also been found in these areas. It has been shown that antibodiesto the murine BP180 (mBP180) ectodomain are capable of triggering a subepidennalblistering disease that closely mimics human BP by passive transfer experiments. Subepidenal blistering in experimental BP is dependent on complement activation and neutrophil recruitment. In the present study, we investigatedthe role of mast cells in the immunopathogenesisof this disease model using a mast celldeftdent and control mice. Mast cell-deficientmice (n:lZ) were not susceotible to the oathwenic effects of antimBP180 1% Anti-mBP180 IaG was depositedat Ihe ddrmal-epidermaljunctron of these animals. but there was-no evidenceof inRammatc+y infiltrationor blistedng. Mast cell-sufficientmice pretreated with a mast cell degranulation inhibitor (cmmolyn) became resistant to the pathogenic activity of anti-mBPi80 IgG. Moreover, mast cell-deficient mice could be made susceptible to this IgG-mediated blistering disease by intradermal adminisbatjonofaneub’ophilchemoattractant.intedeukin-8. These findings provide the Rrstdirect evidencethat mast cells by secretinginflammatorymediators to recruit neutroohils to the skin olav an essential role in subevidermal blister formation in experimental BP, and suggest new directions for disease intervention.
0062
0065
COMPLEMENT FORMATION
INDEPENDENT BY PATHOGENIC
PATHWAY MONOCLONAL
FOR
THE
BLISTER
ANTIBODY
BP 180 Toshihiro Tanaka, Norihisa Matsuvoshi and Sadao Imamura. Department of Dermatology, Graduate school of Medicine, Kyoto University, Kyoto, JAPAN Complement independent pathway for the blister formation was demonstrated by the monoclonal antibody (mAb) against BP180 synthetic peptide. In immunofluorescent study, this mAb reacts with a) basement membrane zone ( BMZ ) of mouse skin, b) epidermal site of 1M NaCl split skin, and in immunoblot study, it reacts with c) synthetic peptide by dot blot, d) 180KDa protein with Western blot. Awtes of this mAb were injected into neonatal mice and C5 deficient neonatal mice. Sera from these mice possessed the circulating mAb and these mice skrn revealed the deposition of mAb at BMZ. Hematoxin eosin (HE) staining of the dorsal skin of injected mica revealed subepidermal blister without cell infiltration m&ding polymorpho-nuclear cells in both balblc and C5 deficient neonatal mice. Mice skin injected normal rat IgG revealed no blister formation by HE study. These results indicate the presence of complement Independent pathway for the pathogenesis of bullous pemphigord blrster formation.
AGAINST
0063 DEVELOPMENT AND CHARACTERIZATION OF BP180-SPECIFIC T CELLS FROM PATIENTS WITH BULLOUS PEMPHIGOID Mong-Shag Lin, Susan J Swartq George J. G&dice, and Luis A. Diaz Department of Dermatology, Medical College of Wisconsin, and the VA Medical Center, Milwaukee, WI, USA Bullous pempbigoid (BP) is a cutmeow autoimmune disease associated with subeoidermal blisterinp and autoantibodies aeainst BPl80. a hemidesmosomal glycoprotein localize< to the basement membrane zone. Previously, we have demonstrated that BP autoantibody reactivity is largely restricted to the non-collagenow NC16A domain of BP180, suggesting that this region may be involved in the pathogen& oftbis disease. The purpose of this study was to determine whether BP T cells also respond to antigenic peptides within NC l6A. Using proliferation assays, we demonstrated that T cells from six BP patients specifically respond to a recombinant form of NC16A The T cell responsive epitope was firther defined to a 28 amino acid subfragment of this protein domain. T cells from control groups, such as pempbigus vulgaris (n=lO), pempbigus foliaceus (n=4) and psoriasis patients (n=Z), as well as normal individuals (n=7), do not proliferate in response to BP180 fusion proteins, confirming that the BP18&induced T cell response is disease specific T cell lines and
clonesdevelopedfrom threeBP patients express CD3, CD4, CD45R0, and TcRufP, but not CD8, CD14, CD20, and CD45RA, indicating that they are CD4 memory T cells Initial cytokine profile analyses revealed that these T cells secrete IL-6 but not y-IFN, suggesting they are likely to be ThZ helper T lymphocytes This information will help to tirther understand the autoimmune mechanism underlying the development of BP.
MOLECULAR MODELLMG OF DEFENSIN BOUND TO HLA- DR4. a Kalbacher’. T. Halder’. D. Dress&. S. Stevanovic2. M Braun’. T Bauer’. W-H. Boehncke’. Medical Research Institute’ and Dpt of Immunology*, Univ of Tiibiigen, Dpt of Dermatology’, Univ of Frank&& Germany Defensins are antiicrobial peptides comprising 6 invariant conserved cysteins forming 3 intramolecular disulfite bonds. Recently, a 5-defensin has been isolated from human psoriatic skin Moreover, it was demonstrated that this molecule is also bound to IUA class-11 molecules on human stimulated HaCaT keratinocytes expressing HLA-DR2 and -DR4 molecules We attempted to further characterize the interaction between defensin and HLA class-11 molecules X-ray structural analyses and NMR analyses showed a very rigid molecule due to several S+.beet structures Using matrixassisted laser-desorption-ioniation post source decay (MALDI-PSD) analyses we were able to demonstrate that the structuralIy intact cyclic defensin was indeed present in the HLA class-11 molecules, rather than a processing variant Employing computeraided molecular mode5ing we then attempted to fit the defensin molecule into the binding pocket of HLA-DR2 and -DR4. This was success~id only when @‘rosin at position 22 was used as primary anchor instead of the tyrosin at position 4 (the expected primary anchor) Taken together our observations demonstrate for the first time the binding of an intact peptide rather than a processing variant to HLA class-II. We proposethat defensin interacts with HLA-DR2 and -DR4 by employing a shit&d binding motiv comprising the tyrosin at position 22 rather than the one at position 4.
Sll
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AUTOIMMUNE RESPONSES OF LINEAR IGA DISEASE BOTH T CELLS AND AUfOmODIES RECOGNIZE THE NC16A DOMAIN OF BPI 80. Mong-Shang Lm. Susan I. Swartz, Mmish Gbaria, Janet A. Fairley. George 1. Gnudlce, and Luis A Diaz. Department of Dermatology, Medical College of Wisconsin, and the VA Medtcal Center, Milwaukee, WI, USA. Luwar IgA disease (LAD) is an antibody-mediated cutaneous automumme diseme characterized by IgA autoantibodies against the epidemml basement membrane mne and by subepidmnal blisters It ba prwiowly been shown that IgA autoantibodies of LAD recogmze epidemxd protam of 97, 120, and 180 kDa These results suggest the possible heterogenaty of autoantigen andamoanbbodies involvedm this disease. Recent ammo aad sequence analysis of tie 97 kDa LAD antigen (reputed by Zone et al.) showed multiple peptide stretches that are idmtiEal to BPIBO, a trammembrane hemldesmosomal protein. In tlus study, we provide direct evidence that the BP180 molecule is indeed the target autoantigen of both autoimmune T lvmohocvtes and autoantibo&s from LAD patients IaA auto&bodies from three LAD pat&s i&d by immunoblotting with fusion protans er&npassing the BPI 80 NCl6A domain Interestingly, IgGautamtibodxs from two of these patients also reacted with a segment of NCl6A. Using T cell proliferation assays, we found that T lymphocytes from these LAD patimtx spxi6calIy respaded to BP180 fbs~on proteins, but not to antigens of other unrelated cutaneous diseases. T cells from normal mdwduals or patients with unrelated dermatoses (e g., PV, PF and psonasis) did not proliferate in response to BP180 fusmn proterns, confirming the specdicity of this response T cell lines developed from one LAD patient express TcRa@, CD4, and CD45R0, but not CD8 or CD45RA. mdicahng that the BP180 respondmg T cells represent CD4+ memory T cells Thesefindings suggest that BPIBO-reactwe T cells may participate m the pathogen&s of LAD
THE ROLE OF MAST CELLS IN EXPERIMENTALBULLOUS PEMPHIGOID. Zhi Uu. XjaoveZhou. Gecme J. Giudii. and Luis A. Diaz. Departmentof Dermatology, Medical College of Wisconsin, Milwaukee.WI 53226. Bullout pemphigoid (BP) is an inflammatory subepidermal blistering disease associated with IgG autoimmune responses to the hemidesmosomal proteins, BP180 and BP230. A variety of infiltrating cells have been identified in the perllesionaklesional site in patients with BP. Mast ceil accumulation and degranulation have also been found in these areas. It has been shown that antibodiesto the murine BP180 (mBP180) ectodomain are capable of triggering a subepidennalblistering disease that closely mimics human BP by passive transfer experiments. Subepidenal blistering in experimental BP is dependent on complement activation and neutrophil recruitment. In the present study, we investigatedthe role of mast cells in the immunopathogenesisof this disease model using a mast celldeftdent and control mice. Mast cell-deficientmice (n:lZ) were not susceotible to the oathwenic effects of antimBP180 1% Anti-mBP180 IaG was depositedat Ihe ddrmal-epidermaljunctron of these animals. but there was-no evidenceof inRammatc+y infiltrationor blistedng. Mast cell-sufficientmice pretreated with a mast cell degranulation inhibitor (cmmolyn) became resistant to the pathogenic activity of anti-mBPi80 IgG. Moreover, mast cell-deficient mice could be made susceptible to this IgG-mediated blistering disease by intradermal adminisbatjonofaneub’ophilchemoattractant.intedeukin-8. These findings provide the Rrstdirect evidencethat mast cells by secretinginflammatorymediators to recruit neutroohils to the skin olav an essential role in subevidermal blister formation in experimental BP, and suggest new directions for disease intervention.
0062
0065
COMPLEMENT FORMATION
INDEPENDENT BY PATHOGENIC
PATHWAY MONOCLONAL
FOR
THE
BLISTER
ANTIBODY
BP 180 Toshihiro Tanaka, Norihisa Matsuvoshi and Sadao Imamura. Department of Dermatology, Graduate school of Medicine, Kyoto University, Kyoto, JAPAN Complement independent pathway for the blister formation was demonstrated by the monoclonal antibody (mAb) against BP180 synthetic peptide. In immunofluorescent study, this mAb reacts with a) basement membrane zone ( BMZ ) of mouse skin, b) epidermal site of 1M NaCl split skin, and in immunoblot study, it reacts with c) synthetic peptide by dot blot, d) 180KDa protein with Western blot. Awtes of this mAb were injected into neonatal mice and C5 deficient neonatal mice. Sera from these mice possessed the circulating mAb and these mice skrn revealed the deposition of mAb at BMZ. Hematoxin eosin (HE) staining of the dorsal skin of injected mica revealed subepidermal blister without cell infiltration m&ding polymorpho-nuclear cells in both balblc and C5 deficient neonatal mice. Mice skin injected normal rat IgG revealed no blister formation by HE study. These results indicate the presence of complement Independent pathway for the pathogenesis of bullous pemphigord blrster formation.
AGAINST
0063 DEVELOPMENT AND CHARACTERIZATION OF BP180-SPECIFIC T CELLS FROM PATIENTS WITH BULLOUS PEMPHIGOID Mong-Shag Lin, Susan J Swartq George J. G&dice, and Luis A. Diaz Department of Dermatology, Medical College of Wisconsin, and the VA Medical Center, Milwaukee, WI, USA Bullous pempbigoid (BP) is a cutmeow autoimmune disease associated with subeoidermal blisterinp and autoantibodies aeainst BPl80. a hemidesmosomal glycoprotein localize< to the basement membrane zone. Previously, we have demonstrated that BP autoantibody reactivity is largely restricted to the non-collagenow NC16A domain of BP180, suggesting that this region may be involved in the pathogen& oftbis disease. The purpose of this study was to determine whether BP T cells also respond to antigenic peptides within NC l6A. Using proliferation assays, we demonstrated that T cells from six BP patients specifically respond to a recombinant form of NC16A The T cell responsive epitope was firther defined to a 28 amino acid subfragment of this protein domain. T cells from control groups, such as pempbigus vulgaris (n=lO), pempbigus foliaceus (n=4) and psoriasis patients (n=Z), as well as normal individuals (n=7), do not proliferate in response to BP180 fusion proteins, confirming that the BP18&induced T cell response is disease specific T cell lines and
clonesdevelopedfrom threeBP patients express CD3, CD4, CD45R0, and TcRufP, but not CD8, CD14, CD20, and CD45RA, indicating that they are CD4 memory T cells Initial cytokine profile analyses revealed that these T cells secrete IL-6 but not y-IFN, suggesting they are likely to be ThZ helper T lymphocytes This information will help to tirther understand the autoimmune mechanism underlying the development of BP.
MOLECULAR MODELLMG OF DEFENSIN BOUND TO HLA- DR4. a Kalbacher’. T. Halder’. D. Dress&. S. Stevanovic2. M Braun’. T Bauer’. W-H. Boehncke’. Medical Research Institute’ and Dpt of Immunology*, Univ of Tiibiigen, Dpt of Dermatology’, Univ of Frank&& Germany Defensins are antiicrobial peptides comprising 6 invariant conserved cysteins forming 3 intramolecular disulfite bonds. Recently, a 5-defensin has been isolated from human psoriatic skin Moreover, it was demonstrated that this molecule is also bound to IUA class-11 molecules on human stimulated HaCaT keratinocytes expressing HLA-DR2 and -DR4 molecules We attempted to further characterize the interaction between defensin and HLA class-11 molecules X-ray structural analyses and NMR analyses showed a very rigid molecule due to several S+.beet structures Using matrixassisted laser-desorption-ioniation post source decay (MALDI-PSD) analyses we were able to demonstrate that the structuralIy intact cyclic defensin was indeed present in the HLA class-11 molecules, rather than a processing variant Employing computeraided molecular mode5ing we then attempted to fit the defensin molecule into the binding pocket of HLA-DR2 and -DR4. This was success~id only when @‘rosin at position 22 was used as primary anchor instead of the tyrosin at position 4 (the expected primary anchor) Taken together our observations demonstrate for the first time the binding of an intact peptide rather than a processing variant to HLA class-II. We proposethat defensin interacts with HLA-DR2 and -DR4 by employing a shit&d binding motiv comprising the tyrosin at position 22 rather than the one at position 4.