The human factor H protein family - an update.

Complement is an ancient and complex network of the immune system and, as such, it plays vital physiological roles, but it is also involved in numerous pathological processes. The proper regulation of the complement system is important to allow its sufficient and targeted activity without deleterious side-effects. Factor H is a major complement regulator, and together with its splice variant factor H-like protein 1 and the five human factor H-related (FHR) proteins, they have been linked to various diseases. The role of factor H in inhibiting complement activation is well studied, but the function of the FHRs is less characterized. Current evidence supports the main role of the FHRs as enhancers of complement activation and opsonization, i.e., counter-balancing the inhibitory effect of factor H. FHRs emerge as soluble pattern recognition molecules and positive regulators of the complement system. In addition, factor H and some of the FHR proteins were shown to modulate the activity of immune cells, a non-canonical function outside the complement cascade. Recent efforts have intensified to study factor H and the FHRs and develop new tools for the distinction, quantification and functional characterization of members of this protein family. Here, we provide an update and overview on the versatile roles of factor H family proteins, what we know about their biological functions in healthy conditions and in diseases.

Complement Factor H Family Proteins Modulate Monocyte and Neutrophil Granulocyte Functions.

Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1ß and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.

Interaction of the Factor H Family Proteins FHR-1 and FHR-5 With DNA and Dead Cells: Implications for the Regulation of Complement Activation and Opsonization.

Complement plays an essential role in the opsonophagocytic clearance of apoptotic/necrotic cells. Dysregulation of this process may lead to inflammatory and autoimmune diseases. Factor H (FH), a major soluble complement inhibitor, binds to dead cells and inhibits excessive complement activation on their surface, preventing lysis, and the release of intracellular material, including DNA. The FH-related (FHR) proteins share common ligands with FH, due to their homology with this complement regulator, but they lack the domains that mediate the complement inhibitory activity of FH. Because their roles in complement regulation is controversial and incompletely understood, we studied the interaction of FHR-1 and FHR-5 with DNA and dead cells and investigated whether they influence the regulatory role of FH and the complement activation on DNA and dead cells. FH, FHR-1, and FHR-5 bound to both plasmid DNA and human genomic DNA, where both FHR proteins inhibited FH-DNA interaction. The FH cofactor activity was inhibited by FHR-1 and FHR-5 due to the reduced binding of FH to DNA in the presence of the FHRs. Both FHRs caused increased complement activation on DNA. FHR-1 and FHR-5 bound to late apoptotic and necrotic cells and recruited monomeric C-reactive protein and pentraxin 3, and vice versa. Interactions of the FHRs with pentraxins resulted in enhanced activation of both the classical and the alternative complement pathways on dead cells when exposed to human serum. Altogether, our results demonstrate that FHR-1 and FHR-5 are competitive inhibitors of FH on DNA; moreover, FHR-pentraxin interactions promote opsonization of dead cells.

Sex hormone-binding globulin provides a novel entry pathway for estradiol and influences subsequent signaling in lymphocytes via membrane receptor.

The complex effects of estradiol on non-reproductive tissues/cells, including lymphoid tissues and immunocytes, have increasingly been explored. However, the role of sex hormone binding globulin (SHBG) in the regulation of these genomic and non-genomic actions of estradiol is controversial. Moreover, the expression of SHBG and its internalization by potential receptors, as well as the influence of SHBG on estradiol uptake and signaling in lymphocytes has remained unexplored. Here, we found that human and mouse T cells expressed SHBG intrinsically. In addition, B lymphoid cell lines as well as both primary B and T lymphocytes bound and internalized external SHBG, and the amount of plasma membrane-bound SHBG decreased in B cells of pregnant compared to non-pregnant women. As potential mediators of this process, SHBG receptor candidates expressed by lymphocytes were identified in silico, including estrogen receptor (ER) alpha. Furthermore, cell surface-bound SHBG was detected in close proximity to membrane ERs while highly colocalizing with lipid rafts. The SHBG-membrane ER interaction was found functional since SHBG promoted estradiol uptake by lymphocytes and subsequently influenced Erk1/2 phosphorylation. In conclusion, the SHBG-SHBG receptor-membrane ER complex participates in the rapid estradiol signaling in lymphocytes, and this pathway may be altered in B cells in pregnant women.

Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands.

Complement factor H modulates the activation of human neutrophil granulocytes and the generation of neutrophil extracellular traps.

Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal ß-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus ß-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation.

Secreted aspartic protease 2 of Candida albicans inactivates factor H and the macrophage factor H-receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18).

The opportunistic pathogenic yeast Candida albicans employs several mechanisms to interfere with the human complement system. This includes the acquisition of host complement regulators, the release of molecules that scavenge complement proteins or block cellular receptors, and the secretion of proteases that inactivate complement components. Secreted aspartic protease 2 (Sap2) was previously shown to cleave C3b, C4b and C5. C. albicans also recruits the complement inhibitor factor H (FH), but yeast-bound FH can enhance the antifungal activity of human neutrophils via binding to complement receptor type 3 (CR3). In this study, we characterized FH binding to human monocyte-derived macrophages. Inhibition studies with antibodies and siRNA targeting CR3 (CD11b/CD18) and CR4 (CD11c/CD18), as well as analysis of colocalization of FH with these integrins indicated that both function as FH receptors on macrophages. Preincubation of C. albicans yeast cells with FH induced increased production of IL-1ß and IL-6 in macrophages. Furthermore, FH enhanced zymosan-induced production of these cytokines. C. albicans Sap2 cleaved FH, diminishing its complement regulatory activity, and Sap2-treatment resulted in less detectable CR3 and CR4 on macrophages. These data show that FH enhances the activation of human macrophages when bound on C. albicans. However, the fungus can inactivate both FH and its receptors on macrophages by secreting Sap2, which may represent an additional means for C. albicans to evade the host innate immune system.

A dynamic network of estrogen receptors in murine lymphocytes: fine-tuning the immune response.

The actual level of circulating estrogen (17ß-estradiol, E2) has a serious impact on regulation of diverse immune cell functions, where their classical cytoplasmic receptors, ERα and ERß, act as nuclear transcriptional regulators of multiple target genes. There is growing evidence, however, for rapid, "non-nuclear" regulatory effects of E2 on lymphocytes. Such effects are likely mediated by putative membrane-associated receptor(s) (mER), but the mechanistic details and the involved signaling pathways still remained largely unknown because of their complexity. Here, we show that in lymphocytes, mERs can signalize themselves, and upon ligation, they are able to coordinate translocation of other E2Rs to the PM. Our data firmly imply existence of a complex, dynamic network of at least seven ER forms in murine lymphocytes: cytoplasmic and membrane-linked forms of ERα, ERß, or GPR30 and a mER that can receive extracellular E2 signals. The latter mERs are likely palmitoylated, as they are enriched in lipid-raft microdomains, and their E2 binding is also cholesterol dependent. The data also support that ligation of mERs can induce rapid regulatory signals to lymphocytes and then internalize and let the E2 liberate in lysosomes. In addition, they can dynamically control the cell-surface linkage of other cytoplasmic ERs. As demonstrated by the differential effects of mER or cytoplasmic ER ligation on the proliferation of activated T and B lymphocytes, such a dynamic E2R network can be considered as a tool to manage accommodation/fine-tuning of lymphocytes to rapidly changing hormone levels.

B cell receptor-induced Ca2+ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes.

B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC-mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin-based membrane protrusion is strongly linked to the dynamics of a cytoplasmic-free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin-severing protein cofilin, linking BCR-induced calcium signaling to the actin dynamics.