Ficolin-2 recognizes DNA and participates in the clearance of dying host cells

Abstract

Ficolin-2 is a serum opsonin, which has been shown to be a pattern recognition molecule in the lectin complement activation pathway. Because innate immune mechanisms are involved in maintaining tissue homeostasis we hypothesized that Ficolin-2 also participate in the clearance of dying host cells. We found that Ficolin-2 binds to late apoptotic cells, as well as to apoptotic bodies and necrotic cells, but not to early apoptotic cells. We demonstrated that Ficolin-2 binds DNA in a calcium dependent manner and that DNA inhibits the binding to late apoptotic and necrotic cells, suggesting that DNA on permeable dying cells is a plausible ligand. Reconstituting serum deficient of Ficolin-2, C1q and mannose-binding lectin with Ficolin-2 augmented deposition of complement C4 on necrotic cells. Opsonization leads to an enhanced attachment/uptake of necrotic cells by macrophages. In conclusion dying host cells expose ligands with the capacity of binding Ficolin-2, which in turn leads to increased attachment and engulfment. Binding of Ficolin-2 to DNA points at nucleic acid exposed by permeable late apoptotic and necrotic cells as one of the ligands for Ficolin-2. Ficolin-2 may therefore be a scavenger molecule participating in the removal of host cells and maintenance of tissue homeostasis.

Introduction

Different serum opsonins including the pentraxins and collectins are believed to orchestrate removal of bacteria and virus from tissues and body fluids prior to establishment of an adaptive immune response. Recently a new group of opsonins, named the Ficolins, have been discovered, which share many features with the collectins. The Ficolins were originally identified as TGF-β1-binding proteins on porcine uterus membranes (Ichijo et al., 1991, Ichijo et al., 1993). Since then three human variants—Ficolin-1 (M-Ficolin), Ficolin-2 (L-Ficolin or Ficolin/P35) and Ficolin-3 (H-Ficolin or Hakata antigen) have been described (Endo et al., 1996, Lu et al., 1996b, Harumiya et al., 1995, Matsushita et al., 1996, Sugimoto et al., 1998). Ficolin-2 and -3 are both synthesized by the liver and are present in serum, whereas Ficolin-1 is membrane-associated and expressed primarily in lung and blood cells (Lu et al., 1996a, Teh et al., 2000). The three human Ficolins share the same general structure comprising monomeric subunits that join in triple-helixes, which oligomerize further into higher order oligomers with a bouquet-like appearance (Ohashi and Erickson, 1998). Each monomeric subunit consists of an N-terminal cysteine rich region followed by a collagen-like domain, a neck region and finally a C-terminal globular fibrinogen-like domain. Ficolin-2 is a carbohydrate-binding lectin with binding specificity towards N-acetyl-d-glucosamine (GlcNAc) but also to N-acetylated groups and may specifically recognize lipotheichoic acid on Gram-positive bacteria, which is mediated through the fibrinogen-like domain (Le et al., 1998, Krarup et al., 2004, Lynch et al., 2004). When Ficolin-2 bind to GlcNAc and N-acetylated groups found in many microbial glycoconjugates, it may function as an opsonin that facilitate clearance of microorganisms by phagocytosis (Matsushita and Fujita, 2002). Furthermore, in addition to being opsonins, Ficolin-2 is capable of activating the complement system through interaction with a set of serine proteases named mannose-binding lectin (MBL) associated serine proteases (MASPs) (Matsushita et al., 2000, Matsushita et al., 2002). Taken together these observations suggest an important role for the Ficolin-2 in innate immunity.

The gene for Ficolin-2 (FCN2) contains eight exons and has been assigned to chromosome 9q34 (Endo et al., 1996). Ficolin-2 is primarily expressed in the liver and subsequently released into the blood stream with a mean serum concentration of 3–4 μg/ml, but with large inter-individual variations, which can be attributed to polymorphisms in the promoter region of the FCN2 gene (Hummelshoj et al., 2005). Moreover, amino acid substituting polymorphisms in the fibrinogen-like domain influence the binding of Ficolin-2 to GlcNAc conjugates.

The pentraxins and collectins have been shown to be pivotal in clearance of apoptotic cells (Roos et al., 2004), recently it was shown that also Ficolin-2 could bind to apoptotic cells (Kuraya et al., 2005). We asked the questions at what stage in the apoptotic process Ficolin-2 binds and which ligands might be involved in the binding. Furthermore, we investigated whether Ficolin-2 could mediate uptake of dying host cells by phagocytes.