Characterization of leukotriene D4-induced gene signatures in human endothelial cells

Leukotriene (LTe), d.h. LTB4 und die Cysteinylleukotriene (cysLTe) LTC4, LTD4 und LTE4, sind hochaktive Lipidmediatoren. LTe werden entweder von aktivierten Leukozyten nach inflammatorischen Stimuli endogen aus Arachidonsäure synthetisiert oder durch transzelluläre Metabolisierung des von Leukozyten stammenden LTA4 produziert. LTe sind als Mediatoren inflammatorischer Reaktionen bekannt und werden mit verschiedenen Erkrankungen in Zusammenhang gebracht. LTe haben viele biologische Effekte, von denen die bedeutendsten die chemotaktischen und vasoaktiven Wirkungen sowie der Einfluß auf die Kontraktion glatter Muskelzellen sind. Ihre Funktionen üben CysLTe durch drei CysLT-Rezeptoren (CysLT-Ren) aus, die von Blutleukozyten und Gefäßwandzellen differentiell exprimiert werden. Endothelzellen(ECs) exprimieren selektiv den CysLT2-R, was auf eine Funktion im kardiovaskulären System hindeutet

Factor H-Related Protein 5 Interacts with Pentraxin 3 and the Extracellular Matrix and Modulates Complement Activation.

The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of approximately 2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease

Autoantibodies to complement components in C3 glomerulopathy and atypical hemolytic uremic syndrome.

The alternative pathway of complement is implicated in the pathogenesis of several renal diseases, such as atypical hemolytic uremic syndrome, dense deposit disease and other forms of C3 glomerulopathy. The underlying complement defects include genetic and/or acquired factors, the latter in the form of autoantibodies. Because the autoimmune forms require a specific treatment, in part different from that of the genetic forms, it is important to detect the autoantibodies as soon as possible and understand their characteristics. In this overview, we summarize the types of anti-complement autoantibodies detected in such diseases, i.e. autoantibodies to factor H, factor I, C3b, factor B and those against the C3 convertases (C3 nephritic factor and C4 nephritic factor). We draw attention to newly described autoantibodies and their characteristics, and highlight similarities and differences in the autoimmune forms of these diseases

FHR-1 binds to C-reactive protein and enhances rather than inhibits complement activation

Factor H (FH)-related protein 1 (FHR-1) is one of the five human factor H-related proteins, which share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by co mpetitive inhibition of FH binding to some surfaces and immune proteins. We have recen tly shown that FHR-1 binds to pentraxin 3. Here, our aim was to investigate whether FH R-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction and study the role of FHR- 1 in complement activation and regulation. FHR- 1 did not bind to native, pentameric CRP but it bound strongly to monomeric CRP via its C-term inal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement de -regulation also on this ligand. FHR-1 did not inhibi t regulation of solid phase C3 convertase by FH and did not inhibit terminal complement complex forma tion induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase form ation and thereby enhanced complement activation. FHR-1/CRP interacti ons increased complement activ ation via the classical and alternative pathways on surfaces such as th e extracellular matrix and necrotic cells. Altogether, these results identify CRP as a lig and for FHR-1 and suggest that FHR-1 enhances rather than inhibits complement activation, wh ich may explain the protective effect of FHR-1 deficiency in age-related macular degeneration

Heterogeneity but individual constancy of epitopes, isotypes and avidity of factor H autoantibodies in atypical hemolytic uremic syndrome

Factor H (FH) autoantibodies are present in 6-10% of atypical hemolytic uremic syndrome (aHUS) patients, most of whom have homozygous deficiency of the FH-related protein FHR-1. Although the pathogenic role of the autoantibodies is established, little is known about their molecular characteristics and changes over time. Here, we describe the specificity and other immunological features of anti-FH autoantibodies in the Spanish and Hungarian aHUS cohorts. A total of 19 patients were included and serial samples of 14 of them were available. FH autoantibodies from FHR-1 deficient patients (n=13) mainly recognized FH, its SCR19-20 fragment and FHR-1, but autoantibody specificity in patients who are homo- or heterozygous for the CFHR1 gene (n=6) was heterogeneous. No significant changes apart from total antibody titer were observed during follow-up in each patient. Fine epitope mapping with recombinant FH SCR19-20 containing single amino acid mutations showed significantly reduced binding in 6 out of 14 patients. In most cases, autoantibody binding to residues 1183-1189 and 1210-1215 was impaired, revealing a major common autoantibody epitope. Avidities showed variations between patients, but in most cases the avidity index did not change upon time. Most autoantibodies were IgG3, and all but three presented only with kappa or with lambda light chains. Although the pathogenic role of anti-FH autoantibodies in aHUS is well established, this study shows autoantibody heterogeneity among patients, but no significant variation in their characteristics over time in each patient. The presence of a single light chain in 16 out of 19 patients and the limited number of recognized epitopes suggest a restricted autoantibody response in most patients

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH

FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

Factor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data.A total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR).Eight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters.Our observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS