Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coliinduced "oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death. complement | coagulation | sepsis | Escherichia coli | organ failure

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“…RA101295 fully inhibited deposition of C5b-9 induced via classic, alternative, or lectin pathways ( Fig. 1 C-E) (24).…”

Section: Resultsmentioning

confidence: 90%

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis

Keshari

Silasi‐Mansat

Popescu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…NHS was collected from nine healthy volunteers, all of whom were normal in classical pathway, lectin pathway, and AP activity, as detected by the Wieslab total complement system screen assay (Euro Diagnostica) (52). Sera were pooled and stored in aliquots at -70°C.…”

Section: Methodsmentioning

confidence: 99%

Properdin binding to complement activating surfaces depends on initial C3b deposition

Harboe

Johnson

Nymo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

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“…The sera used as a complement source were diluted in either gelatin veronal buffer containing Ca 2ϩ and Mg 2ϩ (GVBϩϩ) Complement assays. Functional activity of the 3 complement activation routes was analyzed by enzyme-linked immunosorbent assay using IgM as the ligand for the classical pathway, LPS as the ligand for the alternative pathway, and mannan as the ligand for the lectin pathway (31). MaxiSorp plates (Nunc, Naperville, IL) were coated with human IgM at 2 g/ml, with LPS at 10 g/ml, or with mannan at 100 g/ml in coating buffer (100 mM Na 2 CO 3 /NaHCO 3 [pH 9.6]) overnight at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Anti–cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways

Trouw¹,

Haisma²,

Levarht³

et al. 2009

Arthritis & Rheumatism

217

149

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Objective. It has been suggested that anticitrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). To exert their pathologic effects, ACPAs must recruit immune effector mechanisms such as activation of the complement system. Mouse models of RA have shown that, surprisingly, arthritogenic antibodies activate the alternative pathway of complement rather than the expected classical pathway. This study was undertaken to investigate whether human anti-cyclic citrullinated peptide (anti-CCP) antibodies activate the complement system in vitro and, if so, which pathways of complement activation are used.Methods. We set up novel assays to analyze complement activation by anti-CCP antibodies, using cyclic citrullinated peptide-coated plates, specific buffers, and normal and complement-deficient sera as a source of complement.Results. Anti-CCP antibodies activated complement in a dose-dependent manner via the classical pathway of complement, and, surprisingly, via the alternative pathway of complement. The lectin pathway was not activated by anti-CCP antibodies. Complement activation proceeded in vitro up to the formation of the membrane attack complex, indicating that all activation steps, including the release of C5a, took place.Conclusion. Our findings indicate that anti-CCP antibodies activate the complement system in vitro via the classical and alternative pathways but not via the lectin pathway. These findings are relevant for the design of interventions aimed at inhibition of complement-mediated damage in RA.

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Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA

Cited by 252 publications

References 26 publications

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis

Properdin binding to complement activating surfaces depends on initial C3b deposition

Anti–cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways

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