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.

Mini-Factor H Modulates Complement-Dependent IL-6 and IL-10 Release in an Immune Cell Culture (PBMC) Model: Potential Benefits Against Cytokine Storm.

Cytokine storm (CS), an excessive release of proinflammatory cytokines upon overactivation of the innate immune system, came recently to the focus of interest because of its role in the life-threatening consequences of certain immune therapies and viral diseases, including CAR-T cell therapy and Covid-19. Because complement activation with subsequent anaphylatoxin release is in the core of innate immune stimulation, studying the relationship between complement activation and cytokine release in an in vitro CS model holds promise to better understand CS and identify new therapies against it. We used peripheral blood mononuclear cells (PBMCs) cultured in the presence of autologous serum to test the impact of complement activation and inhibition on cytokine release, testing the effects of liposomal amphotericin B (AmBisome), zymosan and bacterial lipopolysaccharide (LPS) as immune activators and heat inactivation of serum, EDTA and mini-factor H (mfH) as complement inhibitors. These activators induced significant rises of complement activation markers C3a, C4a, C5a, Ba, Bb, and sC5b-9 at 45 min of incubation, with or without ~5- to ~2,000-fold rises of IL-1α, IL-1ß, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13 and TNFα at 6 and 18 h later. Inhibition of complement activation by the mentioned three methods had differential inhibition, or even stimulation of certain cytokines, among which effects a limited suppressive effect of mfH on IL-6 secretion and significant stimulation of IL-10 implies anti-CS and anti-inflammatory impacts. These findings suggest the utility of the model for in vitro studies on CS, and the potential clinical use of mfH against CS.

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.

Regulation of regulators: Role of the complement factor H-related proteins.

The complement system, while being an essential and very efficient effector component of innate immunity, may cause damage to the host and result in various inflammatory, autoimmune and infectious diseases or cancer, when it is improperly activated or regulated. Factor H is a serum glycoprotein and the main regulator of the activity of the alternative complement pathway. Factor H, together with its splice variant factor H-like protein 1 (FHL-1), inhibits complement activation at the level of the central complement component C3 and beyond. In humans, there are also five factor H-related (FHR) proteins, whose function is poorly characterized. While data indicate complement inhibiting activity for some of the FHRs, there is increasing evidence that FHRs have an opposite role compared with factor H and FHL-1, namely, they enhance complement activation directly and also by competing with the regulators FH and FHL-1. This review summarizes the current stand and recent data on the roles of factor H family proteins in health and disease, with focus on the function of FHR proteins.

Functional studies of chronic lymphocytic leukemia B cells expressing ß2-integrin type complement receptors CR3 and CR4.

The expression and role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in B cells are not yet explored in contrast to myeloid cells, where these ß2-integrin type receptors are known to participate in various cellular functions, including phagocytosis, adherence and migration. Here we aimed to reveal the expression and role of CR3 and CR4 in human B cells. In B cells of healthy donors CR3 and CR4 are scarcely expressed. However, two patients with chronic lymphocytic leukemia (CLL) characterized by a peculiar immune-phenotype containing both CD5-positive and CD5-negative B cell populations made possible to study these molecules in distinct B cell subsets. We found that CD11b and CD11c were expressed on both CD5-positive and CD5-negative B cells, albeit to different extents. Our data suggest that these receptors are involved in spreading, since this activity of CpG-activated B cells on fibrinogen could be partially blocked by monoclonal antibodies specific for CD11b or CD11c. CpG-stimulation lead to proliferation of both CD5-positive and CD5-negative B cells of the patients with a less pronounced effect on the CD5-positive cells. In contrast to normal B cells, CLL B cells of both patients reacted to CpG-stimulation with robust IL-10 production. The concomitant, suboptimal stimulus via the BCR and TLR9 exerted either a synergistic enhancing effect or resulted in inhibition of proliferation and IL-10 production of patients' B cells. Our data obtained studying B cells of leukemic patients point to the role of CR3 and probably CR4 in the interaction of tumor cells with the microenvironment and suggest the involvement of IL-10 producing B cells in the pathologic process.

Factor H inhibits complement activation induced by liposomal and micellar drugs and the therapeutic antibody rituximab in vitro.

Hypersensitivity reactions to particulate drugs can partly be caused by complement activation and represent a major complication during intravenous application of nanomedicines. Several liposomal and micellar drugs and carriers, and therapeutic antibodies, were shown to activate complement and induce complement activation-related pseudoallergy (CARPA) in model animals. To explore the possible use of the natural complement inhibitor factor H (FH) against CARPA, we examined the effect of FH on complement activation induced by CARPAgenic drugs. Exogenous FH inhibited complement activation induced by the antifungal liposomal Amphotericin-B (AmBisome), the widely used solvent of anticancer drugs Cremophor EL, and the anticancer monoclonal antibody rituximab in vitro. An engineered form of FH (mini-FH) was more potent inhibitor of Ambisome-, Cremophor EL- and rituximab-induced complement activation than FH. The FH-related protein CFHR1 had no inhibitory effect. Our data suggest that FH or its derivatives may be considered in the pharmacological prevention of CARPA. FROM THE CLINICAL EDITOR: Although liposomes and micelles are already in use in the clinical setting as drug carriers, there remains the potential problem of hypersensitivity due to complement activation. In this article, the authors investigated the use of complement inhibitor factor H (FH) on complement activation and showed good efficacy. The results would therefore suggest the potential application of complement inhibitor in the future.

Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells.

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT1-R/cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT2-R- and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at approximately 60 min, were unaffected by a cysLT1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT2-R activation results in a proinflammatory EC phenotype. Because LTD4 and thrombin are likely to be formed concomitantly in vivo, cysLT2-R and PAR-1 may cooperate to augment vascular injury.