Functional Analysis of Ficolin-3 Mediated Complement Activation

Estrid Hein,Mikkel-Ole Skjoedt,Tina Hummelshøj,Christian Honoré,Peter Garred,Lea Munthe-Fog,David M Ojcius

doi:10.1371/journal.pone.0015443

Estrid Hein, Mikkel-Ole Skjoedt + Show 5 more

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https://doi.org/10.1371/journal.pone.0015443

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Journal: PloS one	Publication Date: Nov 10, 2010
Citations: 98	License type: CC BY 4.0

Affiliation: University of Copenhagen, Rigshospitalet

Abstract

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.

Highlights

The complement system is an integral part of the innate immune system that protects the host against invading pathogens
We have recently described a patient with a history of severe recurrent infections that was homozygous for the FCN3+1637delC mutation and had no detectable Ficolin-3 in serum [22], suggesting that complete lack of Ficolin-3 is a novel immunodeficiency associated with disease
In order to assess the ability of acBSA to function as a ligand for human Ficolins, we applied recombinant Ficolins in known concentrations to microtiter wells coated with acBSA and assessed the binding of the individual Ficolins using monoclonal antibodies

Summary

Introduction

The complement system is an integral part of the innate immune system that protects the host against invading pathogens. Three distinct pathways constitute the complement system; the classical pathway, the alternative pathway and the lectin pathway [1]. The C1 complex initiates the classical pathway upon recognition of immune complexes and dying host cells [2]. The alternative pathway is spontaneously activated by C3 hydrolysis, but it has been reported that properdin, a stabilizer of the alternative pathway convertase [3], is capable of initiating the complement cascade [4]. Upon recognition of pathogen-associated molecular patterns or altered self by MBL and the Ficolins, the associated proteases cleave C4 and C2, hereby activating the complement cascade which leads to the formation of the TCC [5]

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