The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

Objectives: The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody-mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods: We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D. Results: We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane-bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody-mediated diseases. Conclusion: The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement-mediated killing.

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants.

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

Reference Intervals of Factor H and Factor H-Related Proteins in Healthy Children.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.

Substitution of Mannan-Binding Lectin (MBL)-Deficient Serum With Recombinant MBL Results in the Formation of New MBL/MBL-Associated Serine Protease Complexes.

The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon in vivo pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.

The High Prevalence of Functional Complement Defects Induced by Chemotherapy.

INTRODUCTION: To date, oncology patients are more dependent on non-cellular host defense against pathogens due to intensive (chemo)therapy-related bone marrow suppression. Since data on complement functionality in oncology patients are limited, we aimed to investigate the innate complement function in relation to the type of malignancy and therapy in a longitudinal cohort of patients. METHODS: A large single-center, prospective non-intervention study was conducted, in which blood samples were taken from patients before, during, and after treatment with chemotherapy and/or subsequent admittance for (febrile) neutropenia. RESULTS/FINDINGS: Analysis of 48 patients showed a high percentage of defects in complement activity of the alternative pathway (19.1%), the classical pathway (4.3%), or both (42.6%). Post hoc analysis of six different treatment protocols with more than three patients each showed distinct effects of specific therapies. Whereas patients treated according to the Ewing, EpSSG-rhabdomyosarcoma, or SIOP CNS germ cell tumor protocol showed no defects, patients treated according to the ALL-11 (leukemia), the EURAMOS I (osteosarcoma), or the ACNS (medulloblastoma) protocols showed an almost universal reduction in complement function. Although we could not explain the reduced complement functionality under all conditions, a strong effect was observed following high-dose methotrexate or ifosfamide. CONCLUSION: Acquired complement defects were commonly observed in more than 50% of oncology patients, some of which associated with certain chemotherapeutic drugs. Additional studies are needed to determine the clinical and therapeutic context of complement defects and their possible effect on treatment outcome or the increased risk of infection.

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

Plasma-derived mannose-binding lectin shows a direct interaction with C1-inhibitor.

MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0µg/ml MBL functionality was not efficiently restored upon ex vivo testing. PdMBL showed C4-converting activity by itself, indicating the presence of MASPs. Upon incubation of pdMBL with MBL-deficient sera this C4-converting activity was significantly reduced. Depletion of the MASPs from pdMBL, paradoxically, restored the C4-converting activity. Subsequent depletion or inhibition of C1-inh, the major inhibitor of the lectin pathway, in the recipient serum restored the C4-converting activity as well. Complexes between MBL/MASPs and C1-inh (MMC-complexes) were detected after ex vivo substitution of MBL-deficient serum with pdMBL. These MMC-complexes could also be detected in the sera of the patients included in the MBL-substitution study shortly after pdMBL infusion. Altogether, we concluded that active MBL-MASP complexes in pdMBL directly interact with C1-inh in the recipient, leading to the formation of a multimolecular complex between C1-inh and MBL/MASPs, in contrast to the classical pathway where C1r and C1s are dissociated from C1q by C1-inh. Because of the presence of activated MASPs in the current pdMBL products efficient MBL-mediated host protection cannot be expected because of the neutralizing capacity by C1-inh.

IL-21-stimulated human plasmacytoid dendritic cells secrete granzyme B, which impairs their capacity to induce T-cell proliferation.

Plasmacytoid dendritic cells (pDCs) play a crucial role during innate immunity by secreting bulk amounts of type I interferons (IFNs) in response to Toll-like receptor (TLR)-mediated pathogen recognition. In addition, pDCs can also contribute to adaptive immunity by activation of antigen-specific T cells. Furthermore, it is well established that pDCs contribute to the pathogenesis of autoimmune diseases, including lupus. Interleukin-21 (IL-21) is a cytokine produced by activated CD4(+) T and natural killer T (NKT) cells and has a pleiotropic role in immunity by controlling myeloid DC-, NKT-, T-, and B-cell functions. It has remained elusive whether IL-21 affects pDCs. Here we investigate the role of IL-21 in human pDC activation and function and observe that IL-21 activates signal transducer and activator of transcription 3 in line with the finding that pDCs express the IL-21 receptor. Although IL-21 did not affect TLR-induced type I IFNs, IL-6, and TNF-α nor expression of major-histocompatibility-complex class II or costimulatory molecules, IL-21 markedly increased expression of the serine protease granzyme B (GrB). We demonstrate that GrB induction was, in part, responsible for IL-21-mediated downmodulation of CD4(+) T-cell proliferation induced by TLR preactivated pDCs. Collectively, our data provide evidence that pDCs are important cells to consider when investigating the role of IL-21 in immunity or pathogenesis.