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Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes*

Open AccessPublished:November 29, 2012DOI:https://doi.org/10.1074/jbc.M112.409664
      Secretion of anti-serpin B13 autoantibodies in young diabetes-prone nonobese diabetic mice is associated with reduced inflammation in pancreatic islets and a slower progression to autoimmune diabetes. Injection of these mice with a monoclonal antibody (mAb) against serpin B13 also leads to fewer inflammatory cells in the islets and more rapid recovery from recent-onset diabetes. The exact mechanism by which anti-serpin activity is protective remains unclear. We found that serpin B13 is expressed in the exocrine component of the mouse pancreas, including the ductal cells. We also found that anti-serpin B13 mAb blocked the inhibitory activity of serpin B13, thereby allowing partial preservation of the function of its target protease. Consistent with the hypothesis that anti-clade B serpin activity blocks the serpin from binding, exposure to exogenous anti-serpin B13 mAb or endogenous anti-serpin B13 autoantibodies resulted in cleavage of the surface molecules CD4 and CD19 in lymphocytes that accumulated in the pancreatic islets and pancreatic lymph nodes but not in the inguinal lymph nodes. This cleavage was inhibited by an E64 protease inhibitor. Consequently, T cells with the truncated form of CD4 secreted reduced levels of interferon-γ. We conclude that anti-serpin antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function and reducing the severity of autoimmune inflammation.
      Background: Humoral immunity against the protease inhibitor serpin B13 is associated with partial protection from type 1 diabetes.
      Results: Anti-serpin B13 antibodies up-regulate the cleavage of CD4 and CD19 molecules in lymphocytes residing in pancreatic islets and lymph nodes.
      Conclusion: Antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function.
      Significance: Enhancement of humoral immunity against serpin B13 should impede the progression of pathologic changes in type 1 diabetes.

      Introduction

      The balance between proteases and their inhibitors is vital to the survival of multicellular organisms (
      • Luke C.J.
      • Pak S.C.
      • Askew Y.S.
      • Naviglia T.L.
      • Askew D.J.
      • Nobar S.M.
      • Vetica A.C.
      • Long O.S.
      • Watkins S.C.
      • Stolz D.B.
      • Barstead R.J.
      • Moulder G.L.
      • Brömme D.
      • Silverman G.A.
      An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury.
      ). Enhanced protease activity impacts negatively on homeostasis by up-regulating the cleavage of native proteins into short peptides and increasing their presentation to autoreactive T cells (
      • Casciola-Rosen L.
      • Andrade F.
      • Ulanet D.
      • Wong W.B.
      • Rosen A.
      Cleavage by granzyme B is strongly predictive of autoantigen status. Implications for initiation of autoimmunity.
      ). Proteases can also influence many other processes, including tissue remodeling and resolution of inflammation (
      • Targoni O.S.
      • Tary-Lehmann M.
      • Lehmann P.V.
      Prevention of murine EAE by oral hydrolytic enzyme treatment.
      ,
      • Wiest-Ladenburger U.
      • Richter W.
      • Moeller P.
      • Boehm B.O.
      Protease treatment delays diabetes onset in diabetes-prone nonobese diabetic (NOD) mice.
      ), suggesting that their activity is not always pathogenic. Given the critical role of protease activity during formation of autoimmune inflammation (
      • Casciola-Rosen L.
      • Andrade F.
      • Ulanet D.
      • Wong W.B.
      • Rosen A.
      Cleavage by granzyme B is strongly predictive of autoantigen status. Implications for initiation of autoimmunity.
      ,
      • Asagiri M.
      • Hirai T.
      • Kunigami T.
      • Kamano S.
      • Gober H.-J.
      • Okamoto K.
      • Nishikawa K.
      • Latz E.
      • Golenbock D.T.
      • Aoki K.
      • Ohya K.
      • Imai Y.
      • Morishita Y.
      • Miyazono K.
      • Kato S.
      • Saftig P.
      • Takayanagi H.
      Cathepsin K-dependent Toll-like receptor 9 signaling revealed in experimental arthritis.
      ,
      • Reinhold D.
      • Bank U.
      • Täger M.
      • Ansorge S.
      • Wrenger S.
      • Thielitz A.
      • Lendeckel U.
      • Faust J.
      • Neubert K.
      • Brocke S.
      DP1V/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implication for experimental encephalomyelitis and multiple sclerosis.
      ), it is not surprising that proteases are modulated by a number of inhibitors, including B clade molecules, also known as ov-serpins (
      • Irving J.A.
      • Pike R.N.
      • Lesk A.M.
      • Whisstock J.C.
      Phylogeny of the serpin superfamily: implications of patterns of amino acid sequences for structure and function.
      ,
      • Silverman G.A.
      • Whisstock J.C.
      • Askew D.J.
      • Pak S.C.
      • Luke C.J.
      • Cataltepe S.
      • Irving J.A.
      • Bird P.I.
      Human clade B serpins (ov-serpins) belong to a cohort of evolutionary dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.
      ). The inhibitory activity of these serpins may in turn be regulated. For example, the cofactor heparin markedly enhances the ability of ov-serpins SCCA-1 and SCCA-2 to neutralize their target protease (
      • Higgins W.J.
      • Fox D.M.
      • Kowalski P.S.
      • Nielsen J.E.
      • Worrall D.M.
      Heparin enhances serpin inhibition of the cysteine protease cathepsin L.
      ).
      Recently, we found that young diabetes-prone nonobese (NOD)
      The abbreviations used are: NOD
      nonobese
      TCR
      T cell receptor
      mAb
      monoclonal antibody
      PE
      phycoerythrin
      PLN
      pancreatic lymph node.
      mice (
      • Anderson M.S.
      • Bluestone J.A.
      The NOD mouse: a model of immune dysregulation.
      ,
      • Solomon M.
      • Sarvetnick N.
      The pathogenesis of diabetes in the NOD mouse.
      ) secrete autoantibodies against a member of the clade B family called serpin B13 (
      • Nakashima T.
      • Pak S.C.
      • Silverman G.A.
      • Spring P.M.
      • Frederick M.J.
      • Clayman G.L.
      Genomic cloning, mapping, structure and promoter analysis of HEADPIN, a serpin which is down-regulated in head and neck cancer cells.
      ,
      • Welss T.
      • Sun J.
      • Irving J.A.
      • Blum R.
      • Smith A.I.
      • Whisstock J.C.
      • Pike R.N.
      • von Mikecz A.
      • Ruzicka T.
      • Bird P.I.
      • Abts H.F.
      Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis.
      ,
      • Jayakumar A.
      • Kang Y.
      • Frederick M.J.
      • Pak S.C.
      • Henderson Y.
      • Holton P.R.
      • Mitsudo K.
      • Silverman G.A.
      • EL-Naggar A.K.
      • Brömme D.
      • Clayman G.L.
      Inhibition of the cysteine proteinases cathepsins K and L by the serpin headpin (SERPIN B13): a kinetic analysis.
      ) and that this response is associated with protection from early-onset autoimmune diabetes (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). Because autoantibodies that interfere with enzyme cascade activities have been described in several pathologic conditions (
      • Spitzer R.E.
      Serum C3 lytic system in patients with glomerulonephritis.
      ,
      • Gawryl M.S.
      • Hoyer L.W.
      Inactivation of factor VIII coagulant activity by two different types of human antibodies.
      ,
      • Alsenz J.
      • Bork K.
      • Loos M.
      Autoantibody-mediated acquired deficiency of C1 inhibitor.
      ,
      • Jackson J.
      • Sim R.B.
      • Whelan A.
      • Feighery C.
      An IgG autoantibody, which inactivates C1-inhibitor.
      ), we aimed to address whether anti-serpin B13 autoantibodies actively protect from autoimmune diabetes by regulating the balance between this serpin and its protease targets. Using a monoclonal antibody against serpin B13 as a model, we found that humoral activity against this serpin partially preserves the function of proteases and causes enhanced cleavage of lymphocyte surface molecules. Our data also show that it is likely that natural anti-serpin autoantibodies act in a fashion similar to that described for monoclonal antibody during months preceding the development of autoimmune diabetes. Ultimately, this response may interfere with the normal function of inflammatory cells in the pancreatic tissue and contribute to slower progression of pathologic changes in autoimmune diabetes.

      DISCUSSION

      We have demonstrated the existence of a novel antibody that recognizes and inhibits the protease inhibitor serpin B13 in the setting of autoimmune diabetes. This antibody leads to marked histological changes in the composition of the inflammatory infiltrate in the pancreatic islets of NOD mice and ultimately contributes to a better clinical outcome (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). In this study, we found that serpin B13 is expressed mainly in the exocrine portion of the pancreas, most notably in the epithelial lining of the pancreatic ducts. This pattern of expression suggests that the protease targets of serpin B13 (e.g. cathepsins L and K) are also expressed in this tissue compartment. Although this possibility was not addressed in this study, it is supported by reports from other studies in which the expression of cathepsin K was detected in the bronchial and bile duct epithelial cells and in the urothelia (
      • Bühling F.
      • Gerber A.
      • Häckel C.
      • Krüger S.
      • Köhnlein T.
      • Brömme D.
      • Reinhold D.
      • Ansorge S.
      • Welte T.
      Expression of cathepsin K in lung epithelial cells.
      ,
      • Haeckel C.
      • Krueger S.
      • Buehling F.
      • Broemme D.
      • Franke K.
      • Schuetze A.
      • Roese I.
      • Roessner A.
      Expression of cathepsin K in the human embryo and fetus.
      ).
      The features of the protective mechanism of anti-serpin antibody are the inhibition of serpin B13 and the consequent maintenance of the limited function of its protease targets, which in turn facilitate the cleavage of cell-surface molecules expressed in lymphocytes, including the extracellular domains of CD4 and CD19. Our conclusion that proteases mediate a serpin B13 antibody-induced shift toward cells expressing low levels of CD4 and CD19 stems from observations that the protease inhibitor E64 prevented this change. On the other hand, the E64 inhibitor failed to decrease the number of CD4low and CD19low expressers in the absence of anti-serpin antibodies (FIGURE 3, FIGURE 4). A possible explanation for this is that, in our experimental system, serpin B13 and E64 may have competed for the same, relatively limited pool of extracellular cysteine proteases. According to this model, NOD mice with very low levels of endogenous anti-serpin autoantibodies would have sufficient levels of functional serpin B13 to block most of the target proteases; thus, the inhibitory effect of E64 would not have been seen under these conditions. Consequently, disengagement of serpin molecules with endogenous autoantibodies (or a mAb) would increase access of E64 to protease targets and allow for a better demonstration of its inhibitory properties.
      It is likely that molecules other than CD4 and CD19 are also cleaved by anti-serpin B13 antibody-enhanced proteases. Consistent with this hypothesis are other studies in which the investigators demonstrated that 1) administration of exogenous hydrolytic enzymes can lead to a partial resolution of inflammation (
      • Targoni O.S.
      • Tary-Lehmann M.
      • Lehmann P.V.
      Prevention of murine EAE by oral hydrolytic enzyme treatment.
      ,
      • Wiest-Ladenburger U.
      • Richter W.
      • Moeller P.
      • Boehm B.O.
      Protease treatment delays diabetes onset in diabetes-prone nonobese diabetic (NOD) mice.
      ), 2) proteolytic enzymes can impair the function of many different cell-surface molecules expressed in inflammatory cells (
      • Roep B.O.
      • van den Engel N.K.
      • van Halteren A.G.
      • Duinkerken G.
      • Martin S.
      Modulation of autoimmunity to β-cell antigens by proteases.
      ), and 3) the expression of CD25 in regulatory T cells is markedly reduced at the site of inflammation (
      • Tang Q.
      • Adams J.Y.
      • Penaranda C.
      • Melli K.
      • Piaggio E.
      • Sgouroudis E.
      • Piccirillo C.A.
      • Salomon B.L.
      • Bluestone J.A.
      Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction.
      ,
      • Lazarski C.A.
      • Hughson A.
      • Sojka D.K.
      • Fowell D.J.
      Regulating Treg cells at sites of inflammation.
      ). Of note, other autoantibodies have been found to be associated with protection from type 1 diabetes (
      • She J.-X.
      • Ellis T.M.
      • Wilson S.B.
      • Wasserfall C.H.
      • Marron M.
      • Reimsneider S.
      • Kent S.C.
      • Hafler D.A.
      • Neuberg D.S.
      • Muir A.
      • Strominger J.L.
      • Atkinson M.A.
      Heterophile antibodies segregate in families and are associated with protection from type 1 diabetes.
      ,
      • Menard V.
      • Jacobs H.
      • Jun H.-S.
      • Yoon J.-W.
      • Kim S.W.
      Anti-GAD monoclonal antibody delays the onset of diabetes mellitus in NOD mice.
      ,
      • Koczwara K.
      • Bonifacio E.
      • Ziegler A.-G.
      Transmission of maternal islet antibodies and risk of autoimmune diabetes in offspring of mothers with type 1 diabetes.
      ), although the exact molecular events following their mode of action have not been determined.
      It should be noted that anti-clade B serpin autoantibodies may slow down development of the autoimmune form of diabetes by other mechanisms. For example, an anti-serpin autoantibody-mediated up-regulation of proteases may lead to pancreatic islet tissue injury, followed by the compensatory regeneration of islet tissue, de novo formation of islets, or both (
      • Miralles F.
      • Battelino T.
      • Czernichow P.
      • Scharfmann R.
      TGF-β plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.
      ). Support for this hypothesis has been provided through the observations that cathepsin K (
      • Boyce B.F.
      • Xing L.
      • Yao Z.
      • Shakespeare W.C.
      • Wang Y.
      • Metcalf 3rd, C.A.
      • Sundaramoorthi R.
      • Dalgarno D.C.
      • Iuliucci J.D.
      • Sawyer T.K.
      Future anti-catabolic therapeutic targets in bone disease.
      ) and cathepsin L (
      • Afonso S.
      • Romagnano L.
      • Babiarz B.
      The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation.
      ,
      • Robker R.L.
      Progesterone-related genes in the ovulation process: ADAMTS-1 and cathepsin L proteases.
      ) play important roles in tissue remodeling and that the expression of transcription factors associated with the differentiation of pancreatic islets is increased in NOD mice treated with anti-serpin B13 mAb (data not shown). Another possibility for protective mechanisms that are delivered by anti-serpin antibodies may involve 1) induction of neonatal β-cell apoptosis and immunological tolerance to molecules released from dying cells (
      • Hugues S.
      • Mougneau E.
      • Ferlin W.
      • Jeske D.
      • Hofman P.
      • Homann D.
      • Beaudoin L.
      • Schrike C.
      • Von Herrath M.
      • Lehuen A.
      • Glaichenhaus N.
      Tolerance to islet antigens and prevention from diabetes induced by limited apoptosis of pancreatic β cells.
      ) and 2) generation of the biologically active form of transforming growth factor-β, which is responsible for generating regulatory T cells with anti-inflammatory properties (
      • Pesu M.
      • Watford W.T.
      • Wei L.
      • Xu L.
      • Fuss I.
      • Strober W.
      • Andersson J.
      • Shevach E.M.
      • Quezado M.
      • Bouladoux N.
      • Roebroek A.
      • Belkaid Y.
      • Creemers J.
      • O'Shea J.J.
      TGF T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance.
      ).
      In addition to the evidence presented in this study that the preservation of proteases in the pancreas helps to keep inflammation in check and in another study that the cathepsin L gene belongs to a group of the 100 “protective genes” in NOD mice (
      • Fu W.
      • Wojtkiewicz G.
      • Weissleder R.
      • Benoist C.
      • Mathis D.
      Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging.
      ), it has been demonstrated that down-regulation of proteases using chemical inhibitors (
      • Ishimaru N.
      • Arakaki R.
      • Katunuma N.
      • Hayashi Y.
      Critical role of cathepsin-inhibitors for autoantigen processing and autoimmunity.
      ,
      • Yamada A.
      • Ishimaru N.
      • Arakaki R.
      • Katunuma N.
      • Hayashi Y.
      Cathepsin L inhibition prevents murine autoimmune diabetes via suppression of CD8+ T cell activity.
      ), genetic knock-out techniques (
      • Maehr R.
      • Mintern J.D.
      • Herman A.E.
      • Lennon-Duménil A.-M.
      • Mathis D.
      • Benoist C.
      • Ploegh H.L.
      Cathepsin L is essential for onset of autoimmune diabetes in NOD mice.
      ,
      • Hsing L.C.
      • Kirk E.A.
      • McMillen T.S.
      • Hsiao S.H.
      • Caldwell M.
      • Houston B.
      • Rudensky A.Y.
      • LeBoeuf R.C.
      Roles of cathepsin S, L, and B in insulitis and diabetes in the NOD mouse.
      ), or natural serpin inhibitors (
      • Lu Y.
      • Tang M.
      • Wasserfall C.
      • Kou Z.
      • Campbell-Thompson M.
      • Gardemann T.
      • Crawford J.
      • Atkinson M.
      • Song S.
      α1-Antitrypsin gene therapy modulates cellular immunity and efficiently prevents type 1 diabetes in nonobese diabetic mice.
      ,
      • Koulmanda M.
      • Bhasin M.
      • Hoffman L.
      • Fan Z.
      • Qipo A.
      • Shi H.
      • Bonner-Weir S.
      • Putheti P.
      • Degauque N.
      • Libermann T.A.
      • Auchincloss Jr., H.
      • Flier J.S.
      • Strom T.B.
      Curative and β cell regenerative effects of α1-antitrypsin treatment in autoimmune diabetic NOD mice.
      ) can block inflammation in the pancreatic islets of NOD mice. We would like to argue that by using a mAb against serpin B13 and the E64 inhibitor, which has limited cell permeability, we were able to explore primarily the role of extracellular proteases in the regulation of inflammation, despite the fact that protease targets of serpin B13 (e.g. cathepsins L and K) are located mainly in the lysosomal and endosomal vesicles. There is ample evidence that these cathepsins can be secreted to process proteins in the extracellular matrix, where they can promote tissue remodeling (
      • Reiser J.
      • Adair B.
      • Reinheckel T.
      Specialized roles for cysteine cathepsins in health and disease.
      ,
      • Dickinson D.P.
      Cysteine peptidases of mammal: their biological roles and potential effects in the oral cavity and other tissues in health and disease.
      ). Cathepsin L can be active at a close-to-neutral pH, and both hypoxia and acidification that are associated with inflammation can increase the stability of proteases. By contrast, the proteases manipulated in other studies may have been both extracellular and intracellular (
      • Ishimaru N.
      • Arakaki R.
      • Katunuma N.
      • Hayashi Y.
      Critical role of cathepsin-inhibitors for autoantigen processing and autoimmunity.
      ,
      • Yamada A.
      • Ishimaru N.
      • Arakaki R.
      • Katunuma N.
      • Hayashi Y.
      Cathepsin L inhibition prevents murine autoimmune diabetes via suppression of CD8+ T cell activity.
      ), and the manipulation of those proteases may have affected multiple tissues over extended periods of time (
      • Maehr R.
      • Mintern J.D.
      • Herman A.E.
      • Lennon-Duménil A.-M.
      • Mathis D.
      • Benoist C.
      • Ploegh H.L.
      Cathepsin L is essential for onset of autoimmune diabetes in NOD mice.
      ,
      • Hsing L.C.
      • Kirk E.A.
      • McMillen T.S.
      • Hsiao S.H.
      • Caldwell M.
      • Houston B.
      • Rudensky A.Y.
      • LeBoeuf R.C.
      Roles of cathepsin S, L, and B in insulitis and diabetes in the NOD mouse.
      ). As for α1-antitrypsin (
      • Lu Y.
      • Tang M.
      • Wasserfall C.
      • Kou Z.
      • Campbell-Thompson M.
      • Gardemann T.
      • Crawford J.
      • Atkinson M.
      • Song S.
      α1-Antitrypsin gene therapy modulates cellular immunity and efficiently prevents type 1 diabetes in nonobese diabetic mice.
      ,
      • Koulmanda M.
      • Bhasin M.
      • Hoffman L.
      • Fan Z.
      • Qipo A.
      • Shi H.
      • Bonner-Weir S.
      • Putheti P.
      • Degauque N.
      • Libermann T.A.
      • Auchincloss Jr., H.
      • Flier J.S.
      • Strom T.B.
      Curative and β cell regenerative effects of α1-antitrypsin treatment in autoimmune diabetic NOD mice.
      ), this serpin neutralizes proteases other than those regulated by serpin B13; thus, its anti-diabetic effect may reflect the inhibition of additional proteases.
      In conclusion, anti-clade B serpin antibodies induced under inflammatory conditions can fine-tune the balance between proteases and their inhibitors in damaged tissue and may thus contribute to homeostatic events that subdue the early stages of inflammation. If so, the protocols designed to enhance humoral immunity against clade B serpins should impede the progression of pathologic changes that occur in autoimmune diabetes and in other forms of inflammatory disease.

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