Factor H and Factor H-Related Protein 1 Bind to Human Neutrophils via Complement Receptor 3, Mediate Attachment to Candida albicans, and Enhance Neutrophil Antimicrobial Activity

Complement is a first-line defense system of innate immunity, which promotes pathogen clearance by opsonic, lytic, inflammatory, and immune modulatory activities (1). A growing number of pathogens is shown to bind human complement regulators, likely as part of a strategy to evade the host immune system (2). These bound host proteins include factor H (CFH), a regulator of the alternative complement pathway.

CFH is an abundant plasma glycoprotein that functions as a complement regulator both in body fluids and at cellular surfaces (3). CFH belongs to the factor H protein family that also includes CFH-like protein 1 (CFHL1) and CFH-related proteins (CFHR; CFHR1 to CFHR5). All CFH family proteins are exclusively composed of globular domains termed short consensus repeats (SCRs). CFHRs display a high degree of sequence similarity to CFH and share some functions with CFH, such as interaction with C3b and heparin (4). CFHL1, which is derived from the CFH gene by alternative splicing, consists of SCRs 1–7 of CFH and an additional four amino acid-long unique sequence, and shares the complement regulatory activities with CFH, which are mediated by SCRs 1–4 (5). CFH and CFHL1 bind to several pathogens and are thought to play a role in complement evasion, because they are able to regulate complement in their pathogen-bound form (2, 6–8). CFHR1 does not share SCRs 1–4 with CFH and lacks the complement regulatory activity mediated by these domains (9). Similar to the other CFHR proteins, CFHR1 is poorly characterized. Unlike CFH, CFHR1 seems to regulate the terminal pathway of complement (10). CFHR1 binds to several microbes (11–16), and because of its ability to compete with CFH for binding sites, CFHR1 may reduce complement inhibition at the pathogen surface (13).

In several instances, however, binding of complement regulators is not a likely means to protect the pathogens from complement-mediated lysis, because the lytic membrane attack complex of complement cannot breach bacterial capsules or the fungal cell wall. The opportunistic human pathogenic fungi Candida albicans and Aspergillus fumigatus interact with the host complement system and bind the regulators CFH, CFHL1, and C4b-binding protein (6, 14, 17–19). These regulators may limit complement activation on the fungal surface and reduce the amount of pathogen-bound opsonins, thus influencing opsonophagocytosis. Less attention is paid to the possibility that the pathogen-bound complement regulators function as adhesion molecules, facilitating host-pathogen interactions (18, 20–22).

C. albicans is an opportunistic human-pathogenic yeast that can cause severe infections in immunocompromised individuals, such as oral candidiasis in patients with AIDS, and is a common cause of nosocomial infections (23, 24). The healthy population is commonly colonized with Candida, and the interaction between the immune system and the yeast prevents its dissemination and systemic candidiasis, which is associated with a high mortality rate (23). The innate immune system has a particularly important role in keeping this potential pathogen under control (25–27). Neutrophils represent the major phagocytes that are attracted to the sites of infection and inflammation, execute a potent killing program, and play a central role in antifungal immunity (28, 29). To destroy pathogenic microorganisms efficiently, neutrophils possess a wide range of antimicrobial activities, including physical removal or trapping of the pathogens, production of reactive oxygen species, proteinases, and antimicrobial peptides (30–33). Neutropenic patients and patients with defects in leukocyte phagocytic functions, such as leukocyte adhesion deficiency that is characterized by the absence of β₂-integrins, are susceptible to fungal infections (34, 35).

Binding of CFH to neutrophil granulocytes was demonstrated before (36, 37), with partly contradicting results. Because both human-pathogenic fungi and neutrophils were shown to bind CFH, we addressed the hypothesis that CFH and other CFH family proteins mediate interaction between fungi and neutrophils. In this study, we show that CFH, CFHL1, and CFHR1 in their fungus-bound form facilitate attachment, and CFH and CFHR1 enhance antimicrobial activity of neutrophils.

Purified human CFH and the goat CFH antiserum were purchased from Complement Technology (Tyler, Texas). The anti-CD16 (clone 3G8), anti-CD11b (clone ICRF44), anti-CD11c (clone B-ly6), anti-CD18 (clone L130) mAbs and isotype controls were obtained from BD Biosciences (Heidelberg, Germany). The rabbit CFHR4 and CFH SCRs 1–4 antisera were described previously (38). HRP-conjugated rabbit anti-goat and swine anti-rabbit IgG, and FITC-conjugated swine anti-rabbit IgG and F(ab′)₂ fragments of goat anti-mouse IgG were obtained from DakoCytomation (Hamburg, Germany). Alexa Fluor 488-conjugated F(ab′)₂ fragments of rabbit anti-goat IgG were purchased from Invitrogen (Karlsruhe, Germany). Recombinant human CFHL1, CFHR1, and CFHR4A, as well as fragments of CFH and CFHR4A (M. Hebecker and M. Józsi, unpublished observations) were expressed in Spodoptera frugiperda cells using the baculovirus expression system as described (39). All proteins used in this study were tested for endotoxin contamination (Lonza, Wuppertal, Germany).

Human neutrophil granulocytes were isolated from peripheral blood by sedimentation on dextran and removal of mononuclear cells by Ficoll-Hypaque (GE Healthcare, Freiburg, Germany) density gradient centrifugation. RBCs were lysed in hypotonic buffer. Purity of isolated neutrophils was analyzed by flow cytometry using anti-CD16 Ab and was over 95%. Cells were resuspended in serum-free X-VIVO 15 medium (Lonza) and cultivated at 37°C in a humidified atmosphere containing 5% CO₂.

HL-60 monocytoid cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and maintained in RPMI 1640 (Lonza) supplemented with 20% FBS (PAA Laboratories, Cölbe, Germany), 1% L-Glutamine and 50 μg/ml gentamicin sulfate (Lonza). HL-60 monocytes (5 × 10⁵ cells/ml) were differentiated along the granulocyte pathway by addition of 0.7% N,N-dimethylformamide (Sigma-Aldrich, Taufkirchen, Germany) for 5 d.

C. albicans strain SC5314 was used for all experiments. Yeasts were stored in a 50% glycerol stock, plated on yeast peptone dextrose agar before cultivation in yeast peptone dextrose medium and incubated at 30°C for 24 h before use. Fungal cells were inactivated in some experiments by incubation with 0.05% thimerosal (Sigma-Aldrich) in Dulbecco’s PBS (DPBS) (Lonza) at 20°C for 90 min to prevent overgrowth in cocultures.

Neutrophils and HL-60 cells (5 × 10⁵ cells) were incubated with 0.4–100 μg/ml CFH, CFH-fragments, CFHL1, CFHR1, CFHR4A, or CFHR4A-fragments as well as with plasma derived from the cell donor in DPBS (Lonza) for 30 min at 20°C. After washing, goat CFH-antiserum (1:1000 in DPBS containing 1% FBS and 0.1% sodium azide) or rabbit CFHR4-antiserum (1:250) was added for 15 min at 4°C, followed by the corresponding secondary Abs for 15 min at 4°C. 10,000 cells were measured using a BD LSRII (BD Biosciences) flow cytometer. Dead cells were excluded from analysis based on propidium iodide staining. Data were analyzed using the FACSDiva (BD Biosciences) and FlowJo softwares (Tree Star, Ashland, OR).

For detection of receptors, cells were incubated with 10 μg/ml mouse anti-human CD11b, CD11c, CD18 or isotype control for 15 min at 4°C, followed by FITC-conjugated F(ab′)₂ fragments of goat anti-mouse IgG (1:50). For blocking experiments, cells were preincubated with 0.4–50 μg/ml anti-CD11b, anti-CD11c, anti-CD18, or control mAb for 15 min at 4°C, and after washing incubated with 25 μg/ml CFH, CFHL1, CFHR1, CFHR4A, or SCRs 15–20 of CFH.

Neutrophils (5 × 10⁶ cells) were incubated in gelatin veronal buffer (GVB; Sigma-Aldrich) with 50% normal human plasma (NHP) in the absence or presence of 10 mM EDTA for 25 min at 37°C. After extensive washing, the cells were lysed in DPBS containing 1% Triton X-100 and Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany) for 1 h on ice. Cell lysates were centrifuged at 10,000 × g for 10 min at 4°C. The supernatants were separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with Roti-Block (Roth) supplemented with 2.5% BSA (AppliChem, Darmstadt, Germany) overnight at 4°C, the blot was developed using goat CFH-antiserum (1:1000) or rabbit CFH SCRs 1–4-antiserum (1:500) followed by the corresponding HRP-conjugated secondary Abs, and specific bands were visualized by ECL (AppliChem).

C. albicans yeasts (5 × 10⁶ cells) were incubated in 50% NHP in 50 mM Tris buffer containing 150 mM NaCl, pH 7.2, for 25 min at 37°C. Bound proteins were eluted with 3 M potassium thiocyanate followed by buffer change using Vivaspin500 (Sartorius Stedim Biotech, Göttingen, Germany). The elute fractions were analyzed for CFH family proteins as described for the neutrophils.

Inactivated C. albicans yeasts (5 × 10⁵ cells per well) were immobilized on microtiter plates (Corning Life Sciences, Amsterdam, the Netherlands) in carbonate-bicarbonate buffer (Sigma-Aldrich) at 4°C overnight. After washing, nonspecific binding sites were blocked with PBS containing 2.5% BSA and Roti-Block for 3 h at 20°C. CFH, CFHL1, CFHR1, CFHR4A, and HSA (Sigma-Aldrich) were added at 10 μg/ml for 1.5 h at 20°C. After extensive washing, C. albicans cells were incubated with goat CFH-antiserum (1:1000) or rabbit CFHR4-antiserum (1:1000) followed by the corresponding HRP-conjugated secondary Ab. To visualize binding, TMB PLUS substrate (Kem-En-TEC Diagnostics, Taastrup, Denmark) was added, and the reaction was stopped with 2 M H₂SO₄. The absorbance was measured at 450 nm in an ELISA plate reader (Multiskan Ascent; Thermo Labsystems, Langenselbold, Germany).

Yeasts were immobilized as described for the ELISA. Three different approaches were used for ligand incubation: 1) 25 μg/ml CFH, CFHL1, CFHR1, CFHR4A, and HSA; 2) 50% NHP, 50% CFH-depleted NHP without or with 2.5-7.5 μg CFH, CFHL1, CFHR1, CFHR4A, and HSA; or 3) 50% NHP containing 100 μg/ml F(ab′)₂ fragments of rabbit IgG (Sigma-Aldrich) or of rabbit anti-CFH IgG, all diluted in GVB, were added for 1 h at 37°C. After extensive washing, 4 × 10⁵ neutrophils in X-VIVO 15 medium were added to the wells and allowed to adhere for 1 h at 37°C. In some experiments, neutrophils were preincubated with the used proteins in DPBS for 30 min at 20°C, before being added to immobilized yeasts. Nonadherent cells were removed by extensive washing. Adherent cells were quantified using the CyQuant Cell Proliferation Assay kit (Invitrogen) according to the manufacturer’s instructions in a fluorescence reader (Safire²; TECAN, Crailsheim, Germany) with excitation and emission filters of 480 nm and 520 nm. Data are presented as relative fluorescence intensity values.

For depletion of CFH, CFHL1, and CFHR proteins from NHP, Protein A Sepharose 4 Fast Flow (GE Healthcare) was incubated with CFH-antiserum at 4°C overnight and, after washing, plasma derived from the cell donor was added for 60 min at 4°C in two cycles. Efficiency of CFH-depletion was monitored by ELISA. For generation of F(ab′)₂ fragments, 4 mg/ml purified IgG was cleaved by incubation with 80 μg pepsin per mg Ab in 100 mM sodium citrate at 37°C for 22 h. Undigested IgG and the F(ab′)₂-fragments were separated using a HiTrap Protein G HP column (GE Healthcare).

Adhesion to and phagocytosis of living C. albicans by neutrophils was analyzed by flow cytometry. C. albicans yeasts (5 × 10⁶ cells) were incubated with 50 μg/ml CFH proteins in 50 mM Tris buffer containing 150 mM NaCl, pH 7.2, for 25 min at 37°C. After washing, fungal cells were stained with 10 μg/ml FITC (Sigma-Aldrich) for 30 min at 4°C. With this method, Candida cells remained in the yeast form and did not form germ tubes. After extensive washing, 8 × 10⁵ neutrophils were added to the yeasts in a 1:1 ratio in X-VIVO 15 medium and coincubated at 37°C for 60 min. The relative fluorescence intensity of living neutrophils resulting from adhered and phagocytosed labeled yeasts was measured by flow cytometry.

Cell migration assays were performed in serum-free X-VIVO 15 medium using Costar 24-transwell plates (Corning Life Sciences) with 3-μm pore polycarbonate membranes. Viable C. albicans yeast cells (1 × 10⁶) opsonized with CFH proteins or with human serum albumin (HSA) as described above were added to the lower chamber. Neutrophils were stained with 4 μM Calcein-AM (Sigma-Aldrich) for 45 min at 37°C. After washing, 1 × 10⁶ neutrophils were added to the top chamber for 60 min at 37°C, then 25 mM EDTA was added to the lower chamber to release neutrophils adhering to the bottom of the membrane and the bottom of the well. The relative fluorescence intensity of migrated neutrophils was measured using a fluorescence reader with excitation and emission filters of 495 nm and 515 nm.

Inactivated C. albicans yeast cells (5 × 10⁶ cells) were preincubated in GVB with or without 50 μg/ml CFH proteins for 25 min at 37°C. After extensive washing, 1 × 10⁶ neutrophils were coincubated with 1 × 10⁶ C. albicans yeasts in X-VIVO 15 medium at 37°C for 90 min. After centrifugation, the cells were incubated in X-VIVO 15 medium containing 10 μM dihydrorhodamine (Sigma-Aldrich) for 15 min at 37°C and then lysed in DPBS supplemented with 1% Triton X-100. The fluorescence signal of the oxidized dihydrorhodamine was measured in a fluorescence reader with excitation and emission filters of 485 nm and 538 nm.

For detection of lactoferrin, supernatants of cocultures were taken after 90 min and immobilized in a 1:10 dilution in carbonate-bicarbonate buffer on microtiter plates (Corning) at 4°C overnight. After washing, nonspecific binding sites were blocked with DPBS containing 2.5% BSA and Roti-Block for 3 h at 20°C. Lactoferrin was detected using anti-lactoferrin mAb (HyTest; Turku, Finland; 1:1000) followed by the corresponding HRP-conjugated secondary Ab.

Viable C. albicans yeasts were preincubated with the analyzed proteins as described for the phagocytosis assays. After extensive washing, 1 × 10⁶ neutrophil granulocytes were incubated with the yeasts in a 1:1 ratio in X-VIVO 15 medium for 60 min at 37°C_. As control for 100% survival, 1 × 10⁶ C. albicans cells were incubated in medium only. CFUs were counted using dilution series of all samples, plated on Sabouraud dextrose agar, after incubation for 24 h at 30°C.

The between-group differences were analyzed for significance by more than three comparisons of twofold to threefold determinations using a two-way ANOVA, depending on the number of conditions. A p value ≤ 0.05 was considered statistically significant (**p ≤ 0.01, 99% significance; *p ≤ 0.05, 95% significance).

To assess the potential role of CFH and CFH family proteins in neutrophil adhesion, CFH binding to the cells was characterized. Human neutrophil granulocytes were incubated with increasing amounts of purified human CFH or NHP as a more physiologic source of CFH, and CFH binding was measured by flow cytometry. In both cases, CFH bound to the cells in a dose-dependent manner (Fig. 1A, 1B). Because the CFH antiserum used for detection also recognizes the closely related CFHR proteins and CFHL1 in plasma, we analyzed whether these related proteins represent neutrophil ligands. Neutrophils were incubated in buffer only, in NHP or in NHP containing EDTA; after washing, the cell lysates were analyzed by Western blotting. The CFH-antiserum revealed prominent binding of CFH and the two CFHR1 isoforms (Fig. 1C). Two protein bands with apparent m.w. of ∼60 kDa and ∼85 kDa likely correspond to CFHR5 and CFHR4A, respectively (40, 41). Binding of native CFHR4A was confirmed using CFHR4 antiserum (data not shown). Furthermore, an antiserum raised against SCRs 1–4 of CFH revealed CFHL1 binding to neutrophils from plasma (Fig. 1D). CFH, but not the other CFH family proteins that have a much lower concentration in plasma than CFH (4), was detectable on freshly isolated neutrophils (Fig. 1C, lane 1), indicating that CFH binds to neutrophils in the circulation in vivo. Thus, in addition to CFH, at least three additional members of the factor H protein family (i.e., CFHL1, CFHR1, CFHR4A), bind to human neutrophils. The difference in the binding intensities between NHP and NHP-EDTA samples indicate a role of complement-derived opsonins and/or divalent cations for CFH binding. Both mechanisms are likely to play a role in binding of CFH family proteins to neutrophils under physiologic conditions, because both purified C3b and the presence of Ca²⁺ and Mg²⁺ resulted in enhanced CFH binding (data not shown).

To determine which regions of CFH are responsible for binding to neutrophils, recombinant CFH fragments were used in flow cytometric experiments. The CFH fragments SCRs 1–7 and SCRs 15–20 bound strongly to neutrophils. The N-terminal fragments SCRs 1–4, SCRs 1–5 and SCRs 1–6 bound to a lesser extent, and SCRs 8–11, SCRs 11–15 and SCRs 15–18 showed weak binding (Fig. 2A). These results suggest that the main cell binding sites within CFH are located in SCR7 and in SCRs 19-20, while a minor site may be present within SCRs 1-4. Closely related domains to these binding sites are present in two factor H family proteins identified as neutrophil ligands by Western blot. The CFHL1 protein shares SCRs 1-7 with CFH, and the three C-terminal domains SCRs 3–5 of CFHR1 are almost identical with SCRs 18–20 of CFH (Fig. 2B). Both recombinant CFHL1 and CFHR1 bound to neutrophils in a dose-dependent manner (Fig. 2C), confirming the results with the native proteins. In addition, CFHR4A, which is more distantly related to CFH, bound to neutrophils as a recombinant protein (Fig. 2D). Analysis of various fragments of CFHR4A indicated a binding site within SCRs 5–7 (Fig. 2B, 2D).

Two previous studies reported controversial results regarding CFH binding to complement receptor (CR) type 3 on neutrophils (36, 37). To analyze whether CR3 (CD11b/CD18) and the related CR4 (CD11c/CD18) are involved in binding CFH family proteins, neutrophils were preincubated with monoclonal Abs against CD18, CD11b, and CD11c. Binding of CFH family proteins was analyzed by flow cytometry. Preincubation of the cells with anti-CD18 and anti-CD11b resulted in a dose-dependent inhibition of CFH binding to neutrophils, whereas anti-CD11c had only a weaker effect (Fig. 3A). Thus, the major CFH receptor on neutrophils is CR3, but CR4 may also play a role owing to the common CD18 chain. Binding of CFH SCRs 15–20, CFHL1, and CFHR1 to neutrophil granulocytes could also be specifically inhibited with anti-CD18 and anti-CD11b mAbs (Fig. 3B). Similar to CFH, the binding of CFHL1, but not of CFHR1, could be partially inhibited by anti-CD11c mAb. In contrast to this, none of the Abs inhibited binding of recombinant CFHR4A to neutrophils (Fig. 3B), indicating that the CFHR4A receptor is distinct from CR3 and CR4.

HL-60 cells were used to further analyze the role of CR3 as a neutrophil CFH receptor and to exclude the potential effect of serum-derived C3 fragments on neutrophils. Upon differentiation from HL-60 monocytes to neutrophil-like cells, CD18, CD11b, and CD11c were upregulated on HL-60 granulocytes (Fig. 3C). HL-60 granulocytes showed a stronger, dose-dependent binding of CFH than did HL-60 monocytes (Fig. 3D). Likewise, recombinant CFHL1 and CFHR1 showed stronger binding to HL-60 granulocytes than to HL-60 monocytes, but CFHR4A did not bind to HL-60 cells (data not shown).

To address the physiologic role of CFH/neutrophil interaction, CFH family proteins as adhesion ligands were analyzed. C. albicans was used as a pathogen model and was previously shown to bind CFH and CFHL1 (6). Serum adsorption experiments in combination with Western blotting, microtiter plate binding assays, and immunofluorescence studies also demonstrated binding of native and recombinant CFHL1, CFHR1, and CFHR4A to C. albicans (Fig. 4 and data not shown).

In cellular adhesion assays, C. albicans yeasts were incubated with purified human CFH and recombinant CFHL1, CFHR1, and CFHR4A, and attachment of neutrophils was analyzed. CFH, CFHL1, and CFHR1 significantly enhanced neutrophil adhesion to yeasts, whereas the effect of CFHR4A was minor (Fig. 5A). However, when neutrophils were preincubated with CFH family proteins before addition to yeasts, both CFH and CFHR1 increased adhesion, but CFHL1 in these assays had no significant effect (Fig. 5B). This finding is likely explained by the dual binding sites on CFH and CFHR1 and by the different accessibility of the interacting domains of these proteins in their cell-bound form.

The role of these proteins under more physiologic conditions was analyzed using NHP as a source of CFH family proteins. When C. albicans yeasts were incubated with NHP, an enhanced adhesion of neutrophils to the fungal cells was observed, which was significantly reduced in NHP depleted of CFH, CFHL1, and CFHR1. Addition of purified CFH or recombinant CFHL1 and CFHR1 to NHP depleted of CFH family proteins restored neutrophil adhesion (Fig. 5C) in a dose-dependent manner (Fig. 5D). Furthermore, addition of F(ab′)₂ fragments of anti-CFH IgG, but not of control IgG, to NHP caused a reduced adhesion of neutrophils to C. albicans (Fig. 5D). CFHR4A had no significant effect in these assays.

We next tested the capacity of CFH family proteins to support cell migration. When viable C. albicans cells preincubated with CFH and CFHR1 were added to the lower chamber of transwells, a significantly increased number of neutrophils migrated through the membrane inserts as compared with CFHL1, CFHR4A, and HSA, which had no effect on cell migration (Fig. 6). In addition, fluorescence-labeled yeasts preincubated with CFH family proteins were coincubated with neutrophils. The adhered and phagocytosed fungal cells were measured by flow cytometry. CFH, CFHL1, and CFHR1 significantly enhanced yeast adhesion and phagocytosis by neutrophils (Fig. 6).

To assess whether enhanced adhesion mediated by CFH family proteins also lead to functional effects on neutrophils, the release of the antimicrobial protein lactoferrin was measured from coculture supernatants by ELISA. Preincubation of C. albicans yeasts with CFH and CFHR1 resulted in higher lactoferrin release from neutrophils as compared with unstimulated and control treated cells (Fig. 7A). CFHL1 and CFHR4A had no significant effect on lactoferrin release under these assay conditions.

Reactive oxygen species (ROS) are often generated by human cells to use oxidative stress on microbes. CFH and CFHR1 bound on C. albicans enhanced ROS production in human neutrophils, as measured by a fluorescence assay (Fig. 7B). Thus, the two proteins CFH and CFHR1 that had the strongest effect on neutrophil adhesion to yeasts also had enhancing effects on antimicrobial responses of neutrophils. Recombinant CFHL1 and CFHR4A did not influence neutrophil ROS production.

The cytokines TNF-α, IL-6, IL-8, and IL-10 were measured from coculture supernatants by ELISA. There was no significant release of TNF-α, IL-6, and IL-10 on coincubation of neutrophils with C. albicans, and CFH family proteins did not enhance the amounts of these cytokines. The release of IL-8, a cytokine involved in neutrophil recruitment, was triggered by coincubation with yeasts, but this effect was not influenced by CFH, CFHL1, CFHR1, or CFHR4A (data not shown). CFH alone (i.e., without yeasts) also had no effect on IL-8 release.

To analyze whether the enhanced neutrophil adhesion and antimicrobial activity in the presence of CFH and CFHR1 have a direct effect on killing of fungal cells, C. albicans yeasts were preincubated with CFH family proteins, NHP or HSA, and the number of surviving yeasts were determined after coincubation with neutrophils. CFH, CFHR1, and NHP led to a significantly reduced survival rate, whereas CFHR4A and HSA had no effect on fungal killing (Table I).

Upon entering the body, microorganisms come into contact with body fluids and activate the complement system, which deposits opsonins onto their surface. Because CFH and CFH family proteins interact with the major complement-derived opsonin C3b, opsonized pathogens may also be covered with CFH, CFHL1, and CFHRs. In addition, some pathogens express CFH binding molecules on their surface and bind CFH and/or CFHL1 and CFHR1 in the absence of C3b, as has been demonstrated for Streptococcus pyogenes, Borrelia burgdorferi, Leptospira interrogans, Pseudomonas aeruginosa, and C. albicans (7, 11–13, 42, 43). Carrying surface-bound CFH family proteins may help the microbes in complement evasion. The various pathogens that bind CFH are often evaluated in serum sensitivity assays that measure the capacity of human serum to lyse these microbes, and in several cases enhanced resistance against serum killing upon CFH binding was indeed demonstrated (8, 16, 44–46). These tests, however, do not take into consideration the cellular components of blood and tissues. Furthermore, several pathogens have a capsule or thick cell wall and therefore cannot directly be lysed by complement.

Complement proteins bound on the surface of microbes can be exploited by the host as adhesive molecules to facilitate contact with immune cells. Several studies indicate the existence of specific CFH receptors on human cells, although the data are inconclusive (47–51). No receptor, except for CR3 on neutrophils, was identified at the molecular level. In addition, CFHL1 but not CFH was described to mediate adhesion of fibroblasts via an Arg-Gly-Asp motif in SCR4 (52). These data indicate cell type-specific differences in expression of CFH/CFHL1 receptors and in ability to bind CFH and/or CFHL1.

Binding of CFH to neutrophils was first characterized by Avery and Gordon (36), who showed that divalent cations influence CFH binding, a result consistent with our data. In addition, an N-terminal tryptic fragment of CFH was shown to bind to neutrophils. However, no role of the Arg-Gly-Asp motif within CFH and of CR3 on neutrophils was found. In contrast to this, DiScipio et al. (37) identified CR3 as a CFH receptor on neutrophils, using CFH immobilized on plastic surfaces for neutrophil adherence. Our data confirm the role of CR3 as the major CFH receptor on neutrophils (Fig. 3). CR4 shares the CD18 chain with CR3 and, because anti-CD18 inhibited CFH binding to neutrophils, CR4 may also play a role in binding CFH. Nevertheless, expression levels of CR4 on freshly isolated neutrophils were lower compared with those of CR3 (data not shown), in agreement with literature data (53).

Importantly, we identify the factor H-family proteins CFHL1 and CFHR1 as novel ligands of neutrophils that bind to the cells via CR3. The binding of CFHL1, CFHR1, and recombinant CFH fragments indicate two major binding sites within CFH, one residing in or near SCR7 and one at the C-terminus (SCRs 19–20) of the molecule. Although an additional site might be located in SCRs 1–6 (Fig. 2), our results do not support a CR3-binding site in SCR5, as suggested by Avery and Gordon (36), because SCRs 1–4 showed similar binding in our assays. Based on these identified binding sites, CFHL1 likely binds to neutrophils mainly via SCR7, and CFHR1 might use two binding sites—a C-terminal site almost identical with SCRs 19–20 of CFH and an N-terminal SCR7-homolog domain.

The α_Mβ₂ integrin (CD11b/CD18 or CR3) has long been implicated in binding yeasts. CR3 is a receptor for iC3b, an opsonin present on pathogens, and mediates phagocytosis of iC3b-covered particles (54, 55). In addition, CR3 can bind to nonopsonized yeasts, because it is also a receptor for the fungal cell wall component β-glucan (56–58) and for pH-regulated Ag 1 on C. albicans (59). CFH (37) and the two CFH family proteins CFHL1 and CFHR1 are also identified as CR3 ligands (Fig. 3). Thus, CR3 is a multifunctional receptor for neutrophil interactions with C. albicans and may bind several different ligands on opsonized yeasts.

In this study, we analyzed the functional consequences of the binding of CFH family proteins to neutrophils and yeasts in the context of host-pathogen interaction. CFH, CFHL1, and CFHR1 enhanced adhesion of neutrophils to yeast cells and increased phagocytosis when bound on C. albicans. Furthermore, CFH and CFHR1 supported cell migration (Figs. 5, 6). Whereas immobilized CFH alone had no significant effect on ROS and lactoferrin production by neutrophils (data not shown), both ROS and lactoferrin release were enhanced when C. albicans was covered with CFH or with CFHR1 (Fig. 7). Thus, by promoting physical contact with the pathogen and increasing antimicrobial activity, CFH and CFHR1 facilitate the antifungal response of neutrophils (Table I). In a previous study (37), the proinflammatory stimuli TNF-α and C5a augmented CFH-mediated neutrophil adhesion and enhanced neutrophil activation on CFH-covered wells, such as the production of hydrogen peroxide and superoxide anion, and the release of lactoferrin. In the present study, an additional stimulus is likely provided by pattern recognition receptors engaged with the pathogen.

We also analyzed whether the cytokine release from neutrophils is modulated by CFH family proteins. Exposure to C. albicans triggered the release of IL-8, which is important in neutrophil attraction to inflammation sites, but not of TNF-α, IL-6, and IL-10, which is in agreement with a previous report (60). CFH, however, did not influence the amounts of IL-8 in the cell culture supernatants.

In contrast to CFH, the CFHR proteins are poorly characterized to date (4). Based on their similar structure and homologous domains with various degrees of sequence identities to each other, CFHR proteins have both redundant and unique functions. Data presented in this study, show that the host can exploit CFHR1 as an adhesion ligand for neutrophils, and that CFHR1 enhances ROS and lactoferrin release as well as fungal killing by neutrophils. These results identify a new function for CFHR1 to bind to and activate host cells.

CFHR4A, although bound to both neutrophils and yeasts, had no significant effect in the functional assays. In line with the lack of its capacity to potentiate neutrophil antimicrobial responses, CFHR4A did not bind to CR3 on neutrophils (Fig. 3). CFHR4 was shown to bind to some pathogens (8, 61) in addition to C. albicans (Fig. 4). Because CFHR4 has a synergistic effect and enhances CFH cofactor activity (61), it is possible that CFHR4 contributes to complement evasion of pathogens.

In conclusion, CFHL1, CFHR1, and CFHR4A are identified as novel ligands for neutrophils, and CFHR1 and CFHR4A for C. albicans. Although these proteins may downregulate complement at the microbial surface, we identify CFH, CFHL1, and CFHR1 as three related proteins that enhance neutrophil adhesion via CD11b/CD18. Furthermore, the results indicate that CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils and lead to a more efficient killing of the pathogen. These data contribute to our understanding of the complex and dynamic process of the interaction of the innate immune system and complement in particular with C. albicans.

We thank Stephanie Förster, Andrea Hartmann, and Mario Hebecker for production of recombinant proteins; Uta Wohlfeld for help with C. albicans; Rita Cebulla for blood withdrawal; Sebastian Losse for help with statistical analyses; and Antje Albrecht and Bernhard Hube for helpful discussions.

Disclosures The authors have no financial conflicts of interest.