Both IgG and IgM anti‐pig antibodies induce complement activation and cytotoxicity

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383

285

The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.

Section: Purification Of Human Igg and Igmmentioning

confidence: 99%

Section: Purification Of Human Igg and Igmmentioning

confidence: 99%

Human IgA Activates the Complement System Via the Mannan-Binding Lectin Pathway

Roos

et al. 2001

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383

285

“…Deposition of C3, C4, and C5b-C9 was determined after incubation with different concentrations of normal human serum, as a source of complement, diluted in gelatin veronal buffer (Veronal-buffered saline containing 0.5 mM MgCl 2 , 2 mM CaCl 2 , 0.05% Tween-20, and 0.1% gelatin [pH 7.5]) for 1 h at 37˚C. C3 deposition was determined with a Dig-conjugated mAb anti-human C3 (clone RFK22, Nephrology Department, Leiden University Medical Center) (29), and C4 deposition was determined with a Dig-conjugated mAb C4-4a (anti-human C4d) (30). C5b-9 deposition in the wells was detected using a mAb against a C9 neoantigen (AE-11) coupled to Dig, as described (28).…”

Section: Elisamentioning

confidence: 99%

Complement Activation by Ceramide Transporter Proteins

Bode

Losen

Buurman

et al. 2014

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C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b–9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

“…Complement activation induced by xenoreactivity of human serum to pig cells was assessed essentially as described previously (20). PK15 cells (pig kidney epithelial cell line, obtained from the American Type Culture Collection, Manassas, VA) were cultured in DMEM (Life Technologies, Breda, The Netherlands) supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 g/ml).…”

Section: Assessment Of Complement Deposition On Pk15 Cellsmentioning

confidence: 99%

“…Complement activation was assessed using mouse mAb to detect deposition of activated C4 (C4-4a anti-human C4d (19), from Dr. C. E. Hack, conjugated to dig), activated C3 (RFK22 antihuman C3 (20), conjugated to dig) and the membrane attack complex C5b-9 (unconjugated AE11 anti-C5b-9; kindly provided by Dr. T. E. Mollnes, Nordland Central Hospital, Bodø, Norway). Ab binding was detected using either HRP-conjugated F(abЈ) 2 anti-dig (Boehringer Mannheim) or HRP-conjugated goat anti-mouse Igs (DAKO, Glostrup, Denmark).…”

Section: Elisamentioning

confidence: 99%

Specific Inhibition of the Classical Complement Pathway by C1q-Binding Peptides

Roos¹,

Nauta²,

Broers³

et al. 2001

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Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC50 2–6 μM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.