Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.

Immature mouse granulocytic myeloid cells are characterized by production of ficolin-B.

Ficolins activate the lectin pathway of the complement system upon binding to carbohydrate patterns on pathogens. To characterize the producer cells of ficolin-B the expression of mouse ficolin-B, the orthologue of human M-ficolin, was studied in macrophages and dendritic cells during differentiation from bone marrow cells, in primary granulocytes, and during differentiation of granulocytes derived from ER-Hoxb8 cells. Expression of ficolin-B mRNA declined in all myeloid cell types to low levels during terminal differentiation. However, in contrast to macrophages and dendritic cells, ficolin-B expression was enhanced upon activation in granulocytes. High expression of ficolin-B was observed in primary immature neutrophilic CD11b(+) Ly-6C(int) Ly-6G(high) granulocytes when isolated from the bone marrow, in particular during sepsis. Ficolin-B was demonstrated in lysates of primary granulocytes, ER-Hoxb8-derived granulocytes, bone marrow-derived macrophages, and dendritic cells. Native ficolin-B from cell lysates and supernatants of granulocytes activated the lectin pathway as measured by binding to MASP-2 and inducing C4 deposition. Specific staining demonstrated intra-cellular or cell associated ficolin-B protein in activated immature granulocytes deposited in a granular fashion. This study shows that ficolin-B is stored in and set free from immature granulocytic myeloid cells indicating a role in the early infection-induced cellular response of these inflammatory cells.

Functional analysis of mouse ficolin-B and detection in neutrophils.

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.

Ficolin-B marks apoptotic and necrotic cells.

Ficolins are a group of proteins consisting of a fibrinogen-like and a collagen-like domain. They play a role in innate immunity by activating the complement system via the lectin pathway upon binding to carbohydrate patterns on pathogens. Two types of ficolins have been identified in mice, ficolin A and ficolin B (FcnB). We show in this article that recombinant FcnB binds to late apoptotic cells and to apoptotic bodies as well as to necrotic cells but not to early apoptotic cells. This binding was calcium-dependent and could be competitively inhibited by acetylated BSA, a classical binding substrate of FcnB. In addition, DNA inhibited binding of FcnB to apoptotic and necrotic cells, indicating that DNA exposed by dying cells could also be a ligand for FcnB. Thus, FcnB may play a role in the removal of damaged host cells and maintenance of tissue homeostasis.

Essential role of the hprK gene in Ralstonia eutropha H16.

Ralstonia eutropha H16 possesses an incomplete phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) composed of EI, HPr, EIIA(Ntr) (PtsN) and EIIA(Man) (PtsM). We could show that in vitro the incomplete PTS phosphorylation cascade is partially functional. HPr becomes phosphorylated by PEP and EI, and transfers the phosphoryl group to EIIA(Ntr), but only extremely slowly to EIIA(Man). Components of this system have previously been shown to regulate the metabolism of polyhydroxybutyrate. Downstream from ptsN this organism contains an hprK gene, which codes for a homologue of HPr kinase/phosphorylase. We show that this enzyme phosphorylates HPr using ATP as phosphoryl donor. Interestingly, hprK appeared to be essential in R. eutropha because this gene could not be deleted in the wild-type strain, but could be deleted in mutants lacking ptsH or ptsI. This suggests that an increase in the HPr and/or P approximate His-HPr concentrations might be responsible for the growth defect. To test this hypothesis, various ptsH alleles were introduced into the ptsH hprK double mutant. Complementation of this mutant was possible only with the ptsH(His15Ala) allele, but not with the wild-type or ptsH(Ser46Ala) alleles. We conclude that elevated amounts of His-15-phosphorylated HPr, formed in the hprK mutant, are responsible for its growth defect.