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.

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.

Comparison of surface recognition and C3b binding properties of mouse and human complement factor H.

Factor H (FH) is a central complement regulator both in plasma and on certain cellular and acellular surfaces that are in contact with plasma. Although FH deficiency has been shown to lead to similar diseases in man and mice (membranoproliferative glomerulonephritis or dense deposit disease) little is known about the similarity between the human and murine FH functions. We here characterize the interactions of murine FH (mFH) with C3b, glycosaminoglycans, and endothelial cells and compare these interactions with those of human FH (hFH). To achieve this we purified mFH and murine C3 from plasma, prepared murine C3b, and expressed recombinant mFH constructs containing domains 1-5 and 18-20 (mFH1-5 and mFH18-20). For comparisons, hFH, human C3b, and recombinant hFH1-5 and hFH18-20 were used. We demonstrate that mFH and mFH1-5 do act as cofactors for factor I-mediated cleavage of human C3b. Surface plasmon resonance analysis showed binding of mFH18-20 to murine C3b and weak binding to human C3b. The mFH18-20 construct bound to heparin in a manner comparable to hFH18-20. It was demonstrated by flow cytometry that mFH and mFH18-20 bind to human endothelial cells in a similar manner to hFH and hFH18-20. Taken together, locations of the key functions of mFH, i.e. complement regulation and surface recognition, are comparable to hFH. Recently, mutations in the carboxy-terminal end of hFH have been found to be associated with atypical hemolytic uremic syndrome (aHUS). Based on the results in this report it is conceptually attractive to establish a murine model for aHUS.

A case of a diffuse large B-cell lymphoma of plasmablastic type associated with the t(2;5)(p23;q35) chromosome translocation.

Anaplastic large cell lymphomas are associated with the t(2;5)(p23;q35) chromosome translocation in 40% to 60% of cases, leading to a new chimeric gene NPM-ALK. NPM-ALK positive lymphomas are generally reported to be of either T cell or null phenotype. In this report, we describe a diffuse large B-cell lymphoma associated with the classic t(2;5) translocation and both nuclear and cytoplasmic expression of ALK. The tumor consisted of medium-sized to large immunoblasts and plasmablasts that on immunohistology were negative for CD30, CD20, and CD79a but showed monotypic cytoplasmic expression of lambda light chains. Clonality analysis confirmed B-cell lineage of the tumor cells. The t(2;5)(p23;q35) chromosome translocation was demonstrated as part of a complex karyotypic alteration by classic banding and spectral karyotyping (SKY) analyses. Reverse transcription polymerase chain reaction confirmed rearrangement of NPM and ALK genes. This case exemplifies that the t(2;5) can, albeit rarely, occur in large B-cell lymphomas and is not entirely limited to anaplastic large cell lymphomas of T or null cell phenotypes.

Tumor-infiltrating T cells with similar antigen binding sites in different mucosa-associated lymphoid tissue type B cell lymphomas.

B cell lymphomas of mucosa-associated lymphoid tissue (MALT)-type arise on the background of chronic inflammation due to either autoimmunity or infection at various sites of the organism. Characteristically the tumor-infiltrating T cells found in large numbers in MALT-type lymphomas are predominantly of CD4(+) phenotype and may have impact on tumor pathogenesis. To assess whether the chromosomal translocation t(11;18)(q21; q21) that is specific for at least a subset of MALT-type lymphomas may have an impact on tumor environment, we investigated the antigen binding sites of tumor-infiltrating T cells from one t(11;18)-positive tumor of the thyroid and one t(11;18)-negative tumor of the stomach. MHC Allelotyping was performed and revealed common alleles in HLA-DR and HLA-DQ loci. Cloning and sequencing of the complementary determining region 3 in V(beta2) chains of T cell receptors demonstrated the use of identical J(beta)segments in both patients, suggestive for recognition of the same antigen. We therefore suggest that tumor-infiltrating CD4(+) T cells are tightly integrated in MALT-type lymphoma immunoarchitecture irrespective of genetic background and localization of the tumors.

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

Two factor H-related proteins from the mouse: expression analysis and functional characterization.

Complement factor H-related (FHR) proteins display structural and functional similarities to each other and to the complement regulator factor H (FH). FHRs have been identified in various species, including human, rat, and the fish barred sand bass. As mice provide a useful model system to study the physiological role of FHRs in vivo, we aimed at characterizing murine FHR proteins. Two putative FHRs of approximately 100 and 38 kDa were detected in mouse plasma using FH-specific antiserum. In a liver cDNA library, three murine FHR-encoding transcripts were identified. Two clones code for related FHR proteins termed FHR-C and FHR-C_v1, which in secreted form are composed of 14 and 13 short consensus repeat (SCR) domains, homologous to SCRs 6-17 and 19-20 of FH. The third transcript, FHR-B, is derived from a separate gene and codes for a secreted protein composed of five SCR domains. FHR-B displays homology to SCRs 5-7 and 19-20 of FH. Expression of FHR-B in various tissues was analyzed by real-time polymerase chain reaction and was identified at high levels in liver, kidney and heart. In liver, FHR-B transcript level was even higher than that of FH. In addition, FHR-B was expressed as a recombinant 37-kDa protein, and this recombinant FHR-B interacted with the ligands heparin and human C3b. Using mouse plasma, the native presumptive FHR proteins were also analyzed in binding assays. In summary, we identify two FHR proteins in mice and for the first time characterize a murine FHR as a heparin- and C3b-binding protein.

Binding of complement factor H to endothelial cells is mediated by the carboxy-terminal glycosaminoglycan binding site.

Factor H (FH), the major fluid phase regulator of the alternative complement pathway, mediates protection of plasma-exposed host structures. It has recently been shown that short consensus repeats 19 to 20 of FH are mutational hot spots associated with atypical hemolytic uremic syndrome (aHUS), a disease with endothelial cell damage. Domain 20 of FH contains binding sites for heparin, C3b, and the cleavage product C3d. To study the role of these binding sites in target recognition, we performed site-directed mutagenesis in domain 20 and assayed the resulting recombinant proteins. The mutant FH15-20A (substitutions R1203E, R1206E, and R1210S) bound neither heparin nor endothelial cells. Similarly, an aHUS-derived mutant FH protein (E1172Stop, lacking domain 20) failed to bind endothelial cells and showed impaired binding to heparin. Binding of FH to endothelial cells was inhibited by heparin and a specific monoclonal antibody that inhibited heparin but not C3d binding, demonstrating that the heparin site on domains 19 to 20 mediates interaction of FH to endothelial cells. Binding of FH15-20 to heparin was inhibited by several cell surface- and basement membrane-associated glycosaminoglycans, suggesting that binding site specificity is not restricted to heparin. Thus, defective heparin/glycosaminoglycan-binding site on domains 19 to 20 of FH most probably mediates complement-induced endothelial cell damage in aHUS.

Pig complement regulator factor H: molecular cloning and functional characterization.

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1-4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15-20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1-7 and SCR 15-20 proteins, but not the SCR 1-4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.