Stefanie Strobel

Leptospiral Immunoglobulin-like Proteins Interact With Human Complement Regulators Factor H, FHL-1, FHR-1, and C4BP

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Anti-factor B autoantibody in dense deposit disease.

Dense deposit disease (DDD), also known as membranoproliferative glomerulonephritis type II, is a rare kidney disorder that is associated with dysregulation of the alternative pathway of complement. Autoantibodies against the C3bBb convertase termed C3 nephritic factor are common in DDD patients. Here we report an autoantibody that binds to complement factor B in a DDD patient who was negative for C3 nephritic factor. This anti-factor B autoantibody recognized an epitope within the Bb fragment and was able to bind to the C3bBb convertase. Upon binding, the anti-factor B autoantibody stabilized the convertase against both intrinsic and factor H-mediated extrinsic decay and thus enhanced C3 consumption. Functional analyses demonstrated that, in contrast to C3 nephritic factor, the anti-factor B autoantibody inhibited complement-mediated lysis in vitro due to inhibition of the C5 convertase and the terminal complement pathway. Analysis of C5a plasma levels indicated that not all C5 convertases are inhibited by the autoantibodies in the patient in vivo. Antigen array experiments confirmed the presence of anti-factor B autoantibodies and also revealed complement activating anti-C1q antibodies in the patients plasma. In summary, the present report describes a new autoantibody in DDD that binds to factor B and to the alternative pathway C3 convertase and alters the kinetics of complement activation and regulation.

Factor H-related protein 1 neutralizes anti-factor H autoantibodies in autoimmune hemolytic uremic syndrome

The autoimmune form of atypical hemolytic uremic syndrome (HUS) is characterized by circulating autoantibodies against the complement regulator factor H, and is often associated with deficiency of the factor H-related proteins CFHR1 and CFHR3. Here we studied whether anti-factor H autoantibodies crossreact with CFHR1, and determined functional consequences of this. In ELISA, anti-factor H immunoglobulin G (IgG) autoantibodies from 24 atypical HUS patients bound to the short consensus repeat 20 domain of factor H, 21 antibodies also recognized CFHR1, but none CFHR3. Three patients also had anti-factor H IgA autoantibodies crossreacting with CFHR1. Analysis of the IgG fractions in CFHR1-deficient patients found that CFHR1-IgG complexes were formed during plasma exchange treatment, indicating that autoantibodies recognize CFHR1 in vivo. Recombinant CFHR1 prevented hemolysis of sheep erythrocytes caused by patient plasma containing anti-factor H IgG, but it did not inhibit red cell lysis caused by a factor H mutation (W1183 L) in the short consensus repeat 20 domain. Thus, exogenous CFHR1 provided during plasma exchange therapy may neutralize anti-factor H autoantibodies and help in the treatment of autoimmune atypical HUS.

Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3

Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS.

Autoimmune forms of thrombotic micorangiopathy and membranoproliferative glomerulonephritis: Indications for a disease spectrum and common pathogenic principles

There is increasing evidence that Thrombotic Thrombocytopenic Purpura (TTP), atypical Hemolytic Uremic Syndrome (aHUS) and Membranoproliferative Glomerulonephritis (MPGN), especially subtype II (also termed Dense Deposit Disease) represent a spectrum of related disorders. Thrombi are common for all three disorders, develop in different microvascular beds and appear relevant for organ dysfunction. TTP not only develops primarily at neurological sites, but also in the kidney and aHUS develops primarily in the kidneys. In TTP thrombi formation occurs subsequently to the release of multimers of von Willebrand Factor (vWF) and in HUS (both typical and atypical) to endothelial cell damage (via toxins or complement dysregulation). In MPGN thrombus formation occurs in the kidney, however, the cause for thrombi development is less clear. In addition autoimmune forms, in which acquired inhibitors in form of autoantibodies are de novo generated, exist for all three disorders. However, the autoantibodies are directed against different antigens. In TTP against ADAMTS 13, the vWF cleaving protease and in the DEAP-HUS (Deficient for CFHR1 and CFHR3 proteins and autoantibody positive) group against the major complement regulator Factor H. Autoantibodies in MPGN are termed C3 Nephritic Factor (C3NeF) and are directed against a neoepitope of the complement C3 convertase C3bBb. Apparently C3NeF stabilizes this convertase and this results in C3 amplification and complement activation. Based on the existence of acquired immune inhibitors and the shared thrombus formation in TTP, aHUS (DEAP-HUS) and MPGN we here address the hypothesis if the three autoimmune forms represent a spectrum of related diseases and share a common pathogenic principle.

Anti-factor H antibody affecting factor H cofactor activity in a patient with dense deposit disease

Complement alternative pathway dysregulation seems to be the pathophysiological basis of Dense Deposit Disease (DDD). Here, we describe a monoclonal anti-factor H (FH) autoantibody in a woman diagnosed with DDD with a monoclonal gammapathy. Enzyme-linked immunosorbent assays evidenced the presence of anti-FH antibodies in the patients serum and showed that they were associated with the monoclonal IgG-λ fraction. These autoantibodies recognize the N-terminal region of FH and interfere with its regulatory function. In summary, in the DDD patient described here, the activation of complement alternative pathway was favoured by the presence of anti-FH autoantibodies that recognize the regulatory region of this protein and impede its function and which could ultimately cause the glomerulopathy.