Elsevier

Immunobiology

Volume 217, Issue 10, October 2012, Pages 982-985
Immunobiology

Functional analysis of mouse ficolin-B and detection in neutrophils

https://doi.org/10.1016/j.imbio.2012.01.013Get rights and content

Abstract

Ficolins and mannan-binding lectin recognize pathogen-associated molecular patterns and initiate the lectin pathway of complement activation via the associated serine proteases. In contrast to human ficolins and mouse ficolin-A, mouse ficolin-B has been considered incapable of complement activation. Dose-dependent binding of recombinant ficolin-B to immobilized GlcNAc, acetylated BSA, acetylated LDL, and fetuin was detected with ficolin-B-specific monoclonal antibodies. Recombinant ficolin-B bound to immobilized acetylated bovine serum albumin interacted with recombinant human mannan-binding lectin-associated serine protease-2, which led to C4 cleavage, thus demonstrating the capability of ficolin-B to activate the lectin pathway. Ficolin-B-specific monoclonal antibodies identified natural ficolin-B protein in lysates of mouse granulocytes isolated from the bone marrow. These results identify mouse ficolin-B as a functional member of the ficolin family activating complement via the lectin pathway.

Introduction

Ficolins are a family of pattern recognition molecules, which are multimeric proteins consisting of a collagen-like region and a fibrinogen-like domain (Matsushita et al. 1996). The globular C-terminal fibrinogen-like domain binds acetylated groups on various molecules, thus also acetylated sugars when presented in a suitable pattern on microbial surfaces (Matsushita and Fujita, 2002a, Krarup et al., 2004, Frederiksen et al., 2005). Three ficolins are found in humans: H-ficolin is produced by hepatocytes and alveolar type II cells, L-ficolin is produced by hepatocytes, and M-ficolin is produced by monocytes and neutrophilic granulocytes. All are found in serum with reported concentrations of about 20, 5 and 1 μg/ml, respectively (Moller-Kristensen et al., 2003, Sallenbach et al., 2011).

In mice, rats and pigs only 2 ficolins are found. Ficolin-A, appears to be a functional equivalent to human L-ficolin and ficolin-B of human M-ficolin (reviewed in (Endo et al. 2011)). Ficolin-A is present in serum and is produced by Kupffer cells and splenic macrophages (Liu et al. 2005a). The human M-ficolin is named that way because of its association with monocytes (Teh et al. 2000). Presence of M-ficolin in granules of neutrophils and monocytes was described (Liu et al. 2005b) while previously it was thought to be a membrane molecule (Teh et al., 2000, Frederiksen et al., 2005). Mouse ficolin-B has not been detected in plasma or serum but, as for M-ficolin, it is expressed by cells of the myeloid lineage (Liu et al. 2005a). Ficolin-B protein has been stained in lysosomal structures of peritoneal exudate macrophages (Runza et al. 2006).

With the exception of mouse ficolin-B, all ficolins tested (including rat ficolin-B (Girija et al. 2011)) have been shown to form complexes with mannose-binding lectin (MBL)-associated serine proteases (MASPs) and activate the complement system via the lectin pathway similar to MBL (Matsushita and Fujita, 2002a, Matsushita et al., 2002b, Endo et al., 2005, Endo et al., 2011). The failure of mouse ficolin-B to activate complement was attributed to a single amino acid mutation in the sequence of the MASP-2 binding site, which is located in the collagen region, preventing functional interaction with MASP-2 (Girija et al. 2011).

In this report, we analyzed the functional properties of recombinant mouse ficolin-B both in respect to its binding specificities and to its complement activation capacity. Concurring with the literature, mouse ficolin-B was found to interact with acetylated compounds such as GlcNAc, acetylated BSA, acetylated LDL, and fetuin demonstrating similar characteristics to the other ficolin family members. Functional interaction with recombinant human MASP-2 and subsequent C4 cleavage was documented, demonstrating its capacity to initiate the lectin pathway of complement activation. The demonstration of ficolin-B protein in lysate of primary granulocytes points to a specialized function of mouse ficolin-B in the innate immune system.

Section snippets

Antibodies and reagents

Recombinant mouse ficolin-B was expressed in Drosophila S2 cells and purified as reported earlier (Runza et al. 2006). Recombinant mouse ficolin-A was expressed and purified using the same strategy and protocols as for ficolin-B. Recombinant M-ficolin was produced in HEK293 cells (Frederiksen et al. 2005). The monoclonal rat antibody against recombinant ficolin-B (1A4, IgG2b) was generated and characterized in our laboratory as described recently (Schmid et al. 2011). The monoclonal antibody

Binding specificities of recombinant ficolin-B

Binding studies with ficolin-B expressed in insect cells were performed by ELISA using a ficolin-B-specific monoclonal antibody. Dose- and calcium-dependent binding of recombinant ficolin-B to immobilized Ac-BSA has been described recently (Schmid et al. 2011). Recombinant ficolin-B also bound to GlcNAc- and Ac-LDL-coated plates in a calcium dependent manner (Fig. 1A and B), which is in full agreement with the common understanding of M-ficolin binding to acetylated structures (Frederiksen et

Discussion

The anti-ficolin-B monoclonal antibody 1A4, used in this study, was generated by immunization of rats with recombinant ficolin-B expressed in Drosophila cells and was shown to specifically recognize mouse ficolin-B (Schmid et al. 2011). Using this ficolin-B-specific antibody natural ficolin-B was identified in the lysates of bone marrow-derived granulocytes. Previously, ficolin-B protein was demonstrated in lysosomal structures of activated peritoneal exudate macrophages by the use of a rabbit

Acknowledgements

The work has been supported by a grant of the DFG to DNM (Ma760/18-1).

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      Mouse FcnB mRNA was strongly expressed in bone marrow and spleen by Gr-1-positive myeloid cells (Ohashi and Erickson, 1998; Liu et al., 2005a). The protein was located in lysosomes of activated macrophages where it co-localized with the lysosomal marker Lamp-1 (Runza et al., 2006), in cell lysates of bone marrow cells (Endo et al., 2012a; Hunold et al., 2012), and recently, similar to M-ficolin, was also found in low concentrations in serum (Endo et al., 2012b). In contrast to all other known ficolins, mouse FcnB was considered to fail in forming complexes with MASP-2 due to a single amino acid change in the putative MASP-binding site and, therefore, seemed incapable of activating complement (Girija et al., 2011; Endo et al., 2005).

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