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The frequency, isotype, and specificity of antineutrophil cytoplasmic antibodies were investigated in a cross-sectional study of 100 patients with IgA nephropathy and 30 children with Henoch–Schönlein purpura. Two of the patients with IgA nephropathy had high titres of antineutrophil cytoplasmic antibodies which were of IgG isotype and confirmed as antimyeloperoxidase antibodies in solidphase ELISA and inhibition experiments. Antineutrophil cytoplasmic antibodies were not detected in the children with Henoch–Schönlein purpura and none of the patients in either group had IgA antineutrophil cytoplasmic antibodies. A further 20 IgA nephropathy and 10 Henoch–Schönlein purpura patients were studied longitudinally in different clinical phases at 4-monthly intervals over a 2-year period. None of these patients had or developed antineutrophil cytoplasmic antibodies. We conclude that IgA antineutrophil cytoplasmic antibodies are not involved in the vasculitis of Henoch–Schönlein purpura or in the pathogenesis of glomerular injury in IgA nephropathy. The detection of IgG antimyeloperoxidase antibodies in a small minority of IgA nephropathy patients extends the spectrum of diseases associated with autoimmunity to this antigen but is of uncertain significance. [ABSTRACT FROM PUBLISHER]

Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion. [Copyright &y& Elsevier]

The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b-9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target.

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association., (Copyright © 2014 by the American Society of Nephrology.)

Background: Although thrombosis is considered the cardinal feature of the antiphospholipid syndrome, chronic vascular lesions are common, particularly in patients with life-threatening complications. In patients who require transplantation, vascular lesions often recur. The molecular pathways involved in the vasculopathy of the antiphospholipid syndrome are unknown, and adequate therapies are lacking., Methods: We used double immunostaining to evaluate pathway activation in the mammalian target of rapamycin complex (mTORC) and the nature of cell proliferation in the vessels of patients with primary or secondary antiphospholipid syndrome nephropathy. We also evaluated autopsy specimens from persons who had catastrophic antiphospholipid syndrome. The molecular pathways through which antiphospholipid antibodies modulate the mTORC pathway were evaluated in vitro, and potential pharmacologic inhibitors were also tested in vitro. Finally, we studied the effect of sirolimus in kidney-transplant recipients with the antiphospholipid syndrome., Results: The vascular endothelium of proliferating intrarenal vessels from patients with antiphospholipid syndrome nephropathy showed indications of activation of the mTORC pathway. In cultured vascular endothelial cells, IgG antibodies from patients with the antiphospholipid syndrome stimulated mTORC through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Patients with antiphospholipid syndrome nephropathy who required transplantation and were receiving sirolimus had no recurrence of vascular lesions and had decreased vascular proliferation on biopsy as compared with patients with antiphospholipid antibodies who were not receiving sirolimus. Among 10 patients treated with sirolimus, 7 (70%) had a functioning renal allograft 144 months after transplantation versus 3 of 27 untreated patients (11%). Activation of mTORC was also found in the vessels of autopsy specimens from patients with catastrophic antiphospholipid syndrome., Conclusions: Our results suggest that the mTORC pathway is involved in the vascular lesions associated with the antiphospholipid syndrome. (Funded by INSERM and others.).

Vascular endothelial cells (ECs) link hemostasis, thrombosis, and complement. ECs synthesize both the clotting initiator von Willebrand factor (VWF) and the complement regulator factor H (FH). VWF is stored in EC Weibel-Palade bodies (WPBs), but the intracellular location of FH is not well defined. We found that FH colocalizes with VWF in WPBs of human umbilical vein ECs. Moreover, FH bound to VWF with an apparent nanomolar affinity and the complex was present in normal plasma. The binding of VWF to FH enhanced FH cofactor activity toward factor I-mediated downregulation of complement activation. Besides, this interaction inhibited ADAMTS13-mediated proteolysis of VWF and promoted platelet aggregation. Here, we describe a novel interaction between complement and hemostasis. The simultaneous secretion of VWF and FH by activated ECs may promote adhesion of platelets to endothelial injury sites to assure wound healing, simultaneously dampening the proinflammatory effect of complement to limit bystander tissue damage.

Atypical hemolytic uremic syndrome (aHUS) is characterized by genetic and acquired abnormalities of the complement system leading to alternative pathway (AP) overactivation and by glomerular endothelial damage, thrombosis, and mechanical hemolysis. Mutations per se are not sufficient to induce aHUS, and nonspecific primary triggers are required for disease manifestation. We investigated whether hemolysis-derived heme contributes to aHUS pathogenesis. We confirmed that heme activates complement AP in normal human serum, releasing C3a, C5a, and sC5b9. We demonstrated that heme-exposed endothelial cells also activate the AP, resulting in cell-bound C3 and C5b9. This was exacerbated in aHUS by genetic abnormalities associated with AP overactivation. Heme interacted with C3 close to the thioester bond, induced homophilic C3 complexes, and promoted formation of an overactive C3/C5 convertase. Heme induced decreased membrane cofactor protein (MCP) and decay-accelerating factor (DAF) expression on endothelial cells, giving Factor H (FH) a major role in complement regulation. Finally, heme promoted a rapid exocytosis of Weibel-Palade bodies, with membrane expression of P-selectin known to bind C3b and trigger the AP, and the release of the prothrombotic von Willebrand factor. These results strongly suggest that hemolysis-derived heme represents a common secondary hit amplifying endothelial damage and thrombosis in aHUS.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment.

C1q plays a key role in apoptotic cell and immune complex removal. Its absence contributes to the loss of tolerance toward self structures and development of autoimmunity. C1q deficiencies are extremely rare and are associated with complete lack of C1q or with secretion of surrogate C1q fragments. To our knowledge, we report the first case of a functional C1q abnormality, associated with the presence of a normal C1q molecule. Homozygous GlyB63Ser mutation was found in a patient suffering from lupus with neurologic manifestations and multiple infections. The GlyB63Ser C1q bound to Igs, pentraxins, LPSs, and apoptotic cells, similarly to C1q from healthy donors. However, the interaction of C1r(2)C1s(2) and C1 complex formation was abolished, preventing further complement activation and opsonization by C3. The mutation is located between LysB(61) and LysB(65) of C1q, suggested to form the C1r binding site. Our data infer that the binding of C1q to apoptotic cells in humans is insufficient to assure self-tolerance. The opsonization capacity of C4 and C3 fragments has to be intact to fight infections and to prevent autoimmunity.