Activation at Cell Surfaces Factor H in Regulating Complement Critical

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces

This information is current as Viviana P. Ferreira, Andrew P. Herbert, Henry G. Hocking, of September 25, 2021. Paul N. Barlow and Michael K. Pangburn J Immunol 2006; 177:6308-6316; ; doi: 10.4049/jimmunol.177.9.6308 http://www.jimmunol.org/content/177/9/6308 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Critical Role of the C-Terminal Domains of Factor H in Regulating Complement Activation at Cell Surfaces1

Viviana P. Ferreira,* Andrew P. Herbert,† Henry G. Hocking,† Paul N. Barlow,† and Michael K. Pangburn2*

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19–20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19–20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19–20 (rH 19–20). Direct competition with the N-terminal site is unlikely to explain this because regulation of ﬂuid phase C3b is unaffected by domains 19–20. Finally, we show that addition of Downloaded from isolated rH 19–20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken to- gether, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19–20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue. The Journal of Immunology, 2006, 177: 6308–6316. http://www.jimmunol.org/

typical hemolytic uremic syndrome (aHUS)3 is inherited tor B. In addition to its function as a regulator of alternative pathway either as an autosomal dominant or autosomal recessive activation in plasma, factor H also binds to human endothelial cells A trait and the recessive form has been firmly associated and basement membranes (18), protecting them from complement- with mutations clustering at the C-terminal end of complement mediated attack (19). Moreover, some cancer cell surfaces (20, 21) factor H (1–11). aHUS is characterized by hemolytic anemia, and pathogenic microorganisms (22–27) have developed immune thrombocytopenia, and acute renal failure. Homozygous patients evasion strategies whereby they bind factor H, thus disguising them- typically present the disease in the first few months of life and selves as self cells and escaping elimination by the alternative path- by guest on September 25, 2021 exhibit a very high mortality. Heterozygous individuals may in- way of complement. In a factor H-deficient line of pigs, homozygous termittently exhibit symptoms of varying severity throughout life individuals die soon after birth from complement-mediated acute re- (2, 8–10) depending on mutations in other complement regulators nal failure (28), and factor H-deficient mice develop membranopro- (12, 13). Mutational analyses demonstrated that the majority of the liferative glomerulonephritis, which was shown to be alternative path- mutations associated with aHUS clustered at or indirectly affected way dependent (19). the C-terminal end, specifically domain 20, of complement factor The concentration of factor H in plasma is ϳ500 ␮g/ml. The 20 H (1, 6, 14–17). homologous domains of factor H are each composed of ϳ60 aa Factor H plays a key role in the homeostasis of the complement with 3–8 aa spacers between the domains (29, 30). In the electron system by acting as a cofactor for factor I-mediated cleavage of C3b microscope factor H appears to have the structure of flexible and by accelerating the decay of the alternative pathway C3/C5 con- “beads on a string” (31, 32). The N-terminal four complement vertase C3b,Bb complexes. In the absence of factor H, spontaneous control protein domains (CCP) regulate alternative pathway acti- activation of the alternative pathway of complement occurs in plasma, vation while at least three polyanion binding regions and two ad- which leads to consumption of complement components C3 and fac- ditional C3b binding sites are present in the 16 C-terminal domains (15, 22, 25, 33–40). The three binding sites for C3b have been *Department of Biochemistry, Center for Biomedical Research, University of Texas, mapped to the CCP 1–4, 8–15, and 19–20 (40–42). The site on Health Science Center, Tyler, TX 75708; and †Edinburgh Biomolecular Nuclear Mag- netic Resonance Unit, University of Edinburgh, Edinburgh, United Kingdom CCP 19–20 interacts with C3b, iC3b, and C3d (35, 42, 43). The Received for publication May 3, 2006. Accepted for publication August 21, 2006. three-dimensional structure of domains 19–20 has recently been elucidated by two groups, one using nuclear magnetic resonance The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance spectra (16) and the other, x-ray crystallography (17). Structure- with 18 U.S.C. Section 1734 solely to indicate this fact. function analysis have determined that the C3b-binding site lo- 1 This research was supported by National Institutes of Health Grant DK-35081. cated in the N-terminal four CCP domains possesses decay-accel- 2 Address correspondence and reprint requests to Dr. Michael K. Pangburn, Depart- erating activity for the AP C3/C5 convertase and also serves as a ment of Biochemistry, University of Texas Health Science Center, Tyler, TX 75708. cofactor site for factor I, a serine protease that inactivates C3b (33, E-mail address: [email protected] 35, 36, 44). Inactivation of C3b requires that C3b be in complex 3 Abbreviations used in this paper: aHUS, atypical hemolytic uremic syndrome; CCP, complement control protein domain; rH 19–20, recombinant domains 19–20 of factor with the N-terminal domains of factor H (45). H; VBS, veronal-buffered saline; GVB, gelatin-VBS; GVBE, GVB-EDTA; DGVB, Factor H has at least three binding sites for heparin and other dextrose-GVB; ES,ER, and EH, sheep, rabbit, and human erythrocytes, respectively; polyanions, located in CCP 7, CCP 20, and in the CCP 9–15 re- ESC3b and ERC3b, C3b-coated erythrocyte; NHS, normal human sera; CRP, complement regulatory protein. gion (22, 34, 38–40, 42, 46). The C-terminal site has also been

shown to bind sialic acids (25, 34). Interactions with polyanions VBS. The concentrations of factor H and rH 19–20 were determined at 280 1% are important because the 10-fold higher affinity of factor H for nm using E 1cmof 12.4. C3b on host cells and other nonactivators (47, 48) requires the presence of sialic acid clusters or other polyanions on the surface. Radiolabeling of proteins The polyanion binding site in domain 7 has been strongly associ- Factor H, factor B, and rH 19–20 (50–100 ␮g) were radiolabeled with 500 ated with a disease of the retina, age-related macular degeneration, ␮Ci of 125I for 30 min at 0°C in a glass tube coated with Iodogen (Pierce). 125 suggesting the possibility of tissue specific polyanion recognition After incubation, the free I was removed by centrifugal desalting through a G25 column pre-equilibrated with GVB (57). Specific activities by individual sites of factor H (49–52). While all these sites, as for 125I-labeled proteins ranged from 4 to 7 ␮Ci/␮g. well as others, may be relevant in the recognition of nonactivator structures, the role of the C terminus of native factor H is the best Preparation of C3b-coated cells characterized. It has been shown to recognize polyanionic markers, Deposition of C3b on sheep and rabbit erythrocytes (ES and ER, respec- primarily sialic acid and sulfated polysaccharides such as heparin tively) was accomplished using purified C3, factor B, and factor D as on host cells and tissues, and to mediate the binding of factor H to previously described (58) with the substitution of nickel for magnesium human endothelial cells (4, 18). and nephritic factor (59) to stabilize the C3 convertases on the surface of In the present report, the C-terminal domains 19–20 of factor H the cells. The number of bound C3b molecules was determined to be be- were examined for their ability to discriminate between C3b bound tween 41,000 and 170,000 per cell by radiolabeled factor Bb binding (58). to complement-activating cells lacking polyanionic markers and Binding assays C3b bound to nonactivating cells rich in surface polyanions. The

results show that a small recombinant protein composed of only C3b-bearing cells (ESC3b and ERC3b) had between 100,000 and 120,000 Downloaded from domains 19–20 of factor H (rH 19–20) is capable of discriminat- molecules of C3b per cell. Varying numbers of cells were incubated for 20 min at 22°C with 10–20 ng of radiolabeled human factor H or rH 19–20 ing between host-like cells and complement-activating cells. We in half ionic strength buffer (DGVB) in a total volume of 100 ␮l. Bound also examined the ability of rH 19–20 to block control of the and free radiolabeled proteins were separated by layering 80 ␮lofthe complement activation process on nonactivating cells (sheep and mixture on top of 20% sucrose in DGVB, centrifuging for 2 min at human erythrocytes). It selectively inhibited the control activities 10,000 ϫ g, and cutting the tube to separate the pellet and supernatant (48). Background binding was measured using ES and ER cells without C3b on of factor H on these cells without affecting the fluid phase com- http://www.jimmunol.org/ their surface. For binding inhibition assays, cells bearing 1 ␮g C3b were plement control functions of the full-length regulator. The pres- incubated with ϳ20 ng of radioiodinated human factor H and 0–20.7 ␮M ence of the rH 19–20 fragment in normal human serum led to (0–300 ␮g/ml) of nonlabeled rH 19–20, or 0–0.75 ␮M of rH 1–3, rH aggressive complement-mediated lysis of normally nonactivating 6–15, rH 1–6, and rH 19–20, in 100 ␮l of DGVB. Alternatively, the cells ϳ 125 ␮ sheep erythrocytes as well as moderate lysis of normal human were incubated with 20 ng I-rH 19–20 and 0–1.35 M nonlabeled rH 19–20 or rH 1–7. After 20 min at 22°C, the samples were processed as erythrocytes, which have the membrane-bound complement regu- described above. The percentage of factor H bound under these conditions, lators CD35, CD55, and CD59. Our results highlight the impor- in the absence of rH 19–20, was ϳ40%. tance of the interaction between the C-terminal end of factor H and C3b on surfaces bearing polyanions and may explain why aHUS C3/C5 convertase decay acceleration assays patients with mutations in these C-terminal domains can maintain by guest on September 25, 2021 Decay accelerating activity expressed by factor H was measured by deter- complement homeostasis in plasma while their complement sys- mining its ability to accelerate the natural release of 125I-labeled Bb from tem attacks normal cells and tissues, with the kidney being most cell bound C3b,Bb. The C3b,Bb complexes were formed by incubating ϫ 7 ␮ ϳ ␮ 125 ␮ susceptible. 4.2 10 ESC3b with 0.8 g( 1 Ci) of I-factor B and 0.5 gof ␮ factor D in 75 l of GVB containing 1 mM NiCl2 at 22°C for 3 min. Formation of the C3 convertase was stopped by the addition of 145 ␮lof Materials and Methods GVBE. The cells (10 ␮l) were added immediately to reaction mixtures Reagents containing varying amounts of factor H or to 0.67 nM factor H (concentration required to release 50% of the 125I-Bb) in the presence of various Buffers used were sodium PBS, 10 mM sodium phosphate, 140 mM NaCl, concentrations of rH 19–20 (0–27.6 ␮M), in 40 ␮l of PBS. After 10 min 0.02% NaN (pH 7.4); veronal-buffered saline (VBS), 5 mM veronal, 145 at 22°C the cells were sedimented rapidly (2 min, 10,000 ϫ g) through 250 3 ␮ mM NaCl, 0.02% NaN3 (pH 7.3); gelatin-VBS (GVB), VBS containing l of 20% sucrose in GVBE in a Microfuge tube. The bottoms of the tubes 0.1% gelatin; GVB-EDTA (GVBE), GVB containing 10 mM EDTA; dex- were cut off, and the radioactivity in the cell pellet and the supernatant were trose-GVB (DGVB), half physiological ionic strength buffer prepared by measured to determine the percent Bb remaining bound. diluting GVB 2-fold with 5% dextrose in water; and MgEGTA, 0.1 M MgCl2, 0.1 M EGTA (pH 7.3). Factor H cofactor activity for factor I: cell surface cofactor activity Purified proteins Cofactor activity assays measured the ability of human factor H to behave Complement proteins factor H (45), C3 (53, 54), factor B (55), factor D as a cofactor for factor I in the digestion of cell-bound 125I-C3b to C3bi, in (56), and factor I (45) were all purified from normal human plasma as the presence of various concentrations of rH 19–20. The 125I-C3b-coated ␮ ϫ 8 described in the references cited. A fragment consisting of residues 1107– cells were formed by incubating 100 l of GVB containing 5 10 ESC3b, ␮ 1231 of factor H (domains 19–20, i.e., rH 19–20) was cloned, expressed, 10 g of factor B, 50 ng of factor D, 0.25 mM NiCl2, for 5 min at 22°C. and purified by us as described previously (16). Briefly, Pichia pastoris EDTA was added to a concentration of 16 mM, immediately followed by strain KM71H was transformed with the expression vector pPICZ␣ (In- 30 ␮g (350 ␮Ci) of 125I-C3, and incubation for 30 min at 37°C. The vitrogen Life Technologies), and expression of the protein was directed to 125I-C3b-loaded cells were washed thoroughly in GVB and 10 ␮l of cells the secretory pathway by placing the coding sequence upstream from the (ϳ20,000 cpm) were mixed with 14 ng of factor I and various concentra- Saccharomyces cerevisiae ␣-factor secretion sequence. Recombinant pro- tions of factor H in GVB, for 10 min at 37°C. Reactions were placed on ice, tein was purified to homogeneity from culture supernatant using cation 200 ␮l of GVB with 5 ␮g of trypsin was added to each reaction, and the exchange chromatography (16). Samples of this protein were analyzed by samples were incubated for 5 min at 37°C. Only C3b converted to iC3b by mass spectrometry, and the results were consistent with the predicted mo- factors H and I was cleaved by trypsin to release 125I-C3c into the super- lecular mass. The solution structure of this protein was also determined by natant. The cells were sedimented by centrifugation (2 min, 10,000 ϫ g), nuclear magnetic resonance (16). A fragment consisting of factor H do- and the distribution of the radioactivity between the cell pellet and the mains 1–3 was cloned, expressed, and purified as above. Fragments con- supernatant was determined. Inhibition of factor H cofactor activity by rH sisting of factor H domains 1–7, domains 1–6, and domains 6–15 were 19–20 was measured using an amount of factor H that caused 50% C3b cloned into an insect cell expression system and were expressed and pu- conversion to iC3b (50% maximal C3c release) and varying concentrations rified as described previously (43). All proteins were stored at Ϫ75°C in of rH 19–20. 6310 SURFACE-SPECIFIC REGULATION OF COMPLEMENT ACTIVATION

Factor H cofactor activity for factor I: ﬂuid phase cofactor activity This assay measured the ability of human factor H to behave as a cofactor for factor I in the digestion of ﬂuid phase C3b to iC3b, in the presence of various concentrations of rH 19–20. This assay was also used to verify the cofactor activity of the rH 1–7. Five micrograms of C3b (1.8 ␮M) was incubated with 2 ␮g (1.4 ␮M) of factor I and 5 ng (2.1 nM) of factor H, or 2.7 ng (3.3 nM) of rH 1–7, in the presence of up to 17.9 ␮M (260 ␮g/ml) rH 19–20, in 16 ␮l of total volume. C3b cleavage was analyzed by 10% SDS-PAGE. Reactions with or without factor H, rH 1–7, or rH 19–20 were included as controls. Hemolytic assays

Lysis of ES or human erythrocytes (EH) was measured by mixing, on ice, GVB, normal human serum (NHS; 40% ﬁnal), and 0.1 M MgEGTA (5 mM ﬁnal concentration) in the presence of 0–24 ␮M (0–360 ␮g/ml) rH 19–20. 6 Cells (5 ϫ 10 ) were added and the mix (24 ␮l total) was immediately FIGURE 1. Discrimination between activators and nonactivators by rH transferred to a 37°C water bath and incubated for 20 min. To rule out any 19–20 alone. Various concentrations of cells bearing the indicated amounts effect that anti-EH Abs in the NHS could have on the lytic assays, control ϳ ␮ of C3b were incubated with 20 ng of radioiodinated rH 19–20 (A)or experiments were performed in which 400 l NHS was preadsorbed with ␮ ϫ 9 ϫ 6 factor H (B)in100 l of DGVB. After 20 min at 22°C, the bound and free 2 10 EH for1hat4°C, before the lytic assays. EH (5 10 ) were also preincubated with 7 ␮g/ml anti-CD59 mAb (clone MEM43; Abcam) or 10 radiolabel were separated by centrifugation of cells through 20% sucrose. Downloaded from ‚ छ ␮g/ml anti-CD55 polyclonal Abs previously prepared by us (60) for 20 min Background binding to the appropriate cell lacking C3b (ER, ;orES, ) at 4°C, then mixed with NHS and rH 19–20, as above. rH 6–15 was at the appropriate cell concentration was used as a control and was Ͻ0.5% included as a control in these assays. To determine the extent of hemolysis, in all cases. Results are representative of at least three separate experi- 100 ␮l of cold GVBE was added, the samples were centrifuged, and the ments, and the data shown are the mean of duplicate observations. OD of supernatant was determined at 414 nm. The percent lysis was de-

termined by subtracting the A414 in the absence of serum, and dividing by

the maximum possible A414 determined by water lysis of the erythrocytes. http://www.jimmunol.org/ Detection of C3b deposition Fig. 2A shows that rH 19–20 inhibits, in a dose-dependent manner, the binding of full-length factor H to C3b-coated host-like cells C8-depleted sera was prepared by immunoadsorption of serum on anti-C8 (E ). Inhibition by the two-domain fragment exhibited an IC of Sepharose. Deposition of C3b on E was measured by mixing, on ice, S 50 H ␮ GVB, C8-depleted serum (40% final), and 0.1 MgEGTA (5 mM final con- 0.3 M. Fig. 2B shows that the recombinant subfragments of fac- ␮ ϫ 6 centration) in the presence of 12 M rH 19–20. EH (5 10 ) preincubated tor H that contain other C3b or heparin binding domains (rH 1–6 with or without anti-CD55 polyclonal Ab, as described above, were added and rH 6–15) partially inhibit the binding of intact factor H, al- and the mixture transferred to a 37°C water bath for 20 min. Cells were though not to the same extent as rH 19–20. rH 1–3, which does not washed with GVB, and C3b deposition was detected with FITC-conjugated goat F(abЈ) anti-human C3 (Cappel). Cells were analyzed by flow cytom- contain the full N-terminal C3b-binding site, has no effect at the 2 by guest on September 25, 2021 etry using BD Biosciences FACScan equipped with Cell Quest Pro soft- concentrations tested. Unlabeled rH 19–20 was effective (Fig. 3) ␮ ware, and a minimum of 10,000 events were acquired. against itself, exhibiting an IC50 of 0.2 M. In contrast, no inhibition of rH 19–20 binding was seen using rH 1–7 up to 1.5 ␮M, Results the maximum concentration attainable with this purified recombi- Discrimination between activators and nonactivators by CCP nant protein. When tested in a fluid phase cofactor assay and com- domains 19–20 alone pared with equimolar concentrations of factor H, rH 1–7 was at Factor H confers on the human alternative pathway of complement least 80% active (results not shown). the ability to discriminate between activating cells that possess

minimal surface polyanions (e.g., ER, bacteria, fungi, etc.) and

nonactivating host and host-like cells (e.g., ES). ES and most human cells and tissues possess high levels of sialic acid-containing polysaccharides as well as other polyanions. Work done in the late 1970s demonstrated that removal of sialic acid leads to activation of the human alternative pathway (48, 61). These early studies also showed that factor H bound 5- to 10-fold better to C3b on cells bearing polyanions than to cells without polyanions as shown in Fig. 1B. Fig. 1A demonstrates that rH 19–20, without the aid of the rest of factor H, exhibits a quantitatively similar ability to distinguish between complement-activating and nonactivating cells. Both factor H and rH 19–20 bind with 7-fold higher afﬁnity to C3b-bearing cells that possess high levels of sialic acid-containing FIGURE 2. Binding of radiolabeled factor H to C3b on E C3b cells in polysaccharides (ESC3b) than to cells (ERC3b) possessing mini- S 11 mal surface sialic acid. This can be seen in Fig. 1 as the 7-fold the presence of factor H recombinant domains. A, Cells bearing 20 ϫ 10 ϳ difference in C3b concentrations required to bind similar amounts C3b were incubated with 20 ng of radioiodinated human factor H and 0–20.7 ␮M (0–300 ␮g/ml) of nonlabeled rH 19–20, in 100 ␮l of DGVB. of factor H. After 20 min at 22°C, the bound and free radiolabel were separated by Domains 19–20 inhibit binding of full-length factor H to centrifugation of cells through 20% sucrose. The results are graphed rel- cell-bound C3b ative to binding observed with labeled factor H in the absence of rH 19–20. B, Same as above, except the C3b-coated cells were incubated with radio- A competitive binding assay was performed to determine the con- iodinated factor H and 0–0.75 ␮M nonlabeled rH 19–20, rH 1–3, rH 6–15, tribution of the C-terminal C3b/polyanion binding site of factor H or rH 1–6. Results are representative of three separate experiments, and the to the ability of full-length factor H to bind to nonactivating cells. data show the mean of duplicate observations. The Journal of Immunology 6311

FIGURE 3. rH 19–20 does not compete with rH 1–7 for binding to C3b FIGURE 5. rH 19–20 inhibits the factor H-mediated cofactor activity ϫ 11 ϳ for factor I on the surface of E C3b. Cofactor activity assays measured the on ESC3b cells. Cells bearing 20 10 C3b were incubated with 20 ng S of radioiodinated rH 19–20 and 0–1.35 ␮M nonlabeled rH 19–20 or rH ability of human factor H to behave as a cofactor for factor I in the diges- 125 1–7, in 100 ␮l of DGVB. After 20 min at 22°C, the bound and free ra- tion of cell-bound I-C3b to C3bi, in the presence of various concentra- 125 diolabel were separated by centrifugation of cells through 20% sucrose. tions of rH 19–20. The I-C3b was deposited on ES as described in 125 The results are graphed relative to binding observed with labeled rH 19–20 Materials and Methods. The I-C3b-loaded cells were washed in GVB in the absence of unlabeled protein. and 10 ␮l of cells (ϳ20,000 cpm) were mixed with 14 ng of factor I and Downloaded from varying amounts of human factor H (A) and incubated for 10 min at 37°C. In B, 24 nM factor H was used in a similar assay in the presence of 0–27.6 ␮ ␮ ␮ rH 19–20 inhibits both decay acceleration and cofactor activity M (0–400 g/ml) rH 19–20. Reactions were placed on ice, and 200 l ␮ of factor H on nonactivating cells of GVB with 5 g of trypsin was added to each reaction and incubated 5 min at 37°C to cleave iC3b and release C3c. The cells were sedimented by The N-terminal domains 1–4 of factor H are responsible for its centrifugation (2 min, 10,000 ϫ g), and the distribution of the radioactivity complement regulatory activities in the fluid phase and on the cell between the cell pellet and the supernatant was determined. A total of 24 http://www.jimmunol.org/ surface. The dependency of these functional activities on the pres- nM factor H gave 50% cofactor activity, in the absence of rH 19–20 (arrow ence of C-terminal domains has been investigated previously using in A), and the results in B were graphed relative to this activity. Results are deletion mutants of factor H (62), and has been observed with representative of two separate experiments, and the data show the mean of inherited mutations in these domains (7), but site-specific compe- duplicate observations. tition has not been reported. Thus, we decided to test the ability of rH 19–20 to inhibit the complement regulatory activities of full- Decay acceleration assays measured the ability of factor H to length factor H. Isolated rH 19–20 specifically inhibited, in a dose- increase the rate of dissociation of the two subunits of the alter- dependent manner, factor H-mediated decay acceleration of the native pathway C3/C5 convertase (C3b,Bb). The dose dependence by guest on September 25, 2021 C3/C5 convertases (Fig. 4B) and factor I cofactor activity (Fig. 5B) of this factor H activity is shown in Fig. 4A. The release of radio- on cell surfaces. labeled Bb from surface-bound C3b was measured and a concentration of factor H was chosen that decayed half the enzymes remaining after a 10-min incubation. This concentration of factor H was then used for decay acceleration assays in the presence of various concentrations of rH 19–20 (Fig. 4B). At 28 ␮MrH19– 20, the accelerated decay due to factor H was strongly inhibited and controls showed that rH 19–20 alone did not significantly

increase or decrease decay. The IC50 of rH 19–20 for decay acceleration was 0.7 ␮M. Factor I cofactor assays measured the ability of the N terminus of factor H to bind to C3b and promote cleavage and inactivation of C3b by the serine protease factor I. The product of this reaction is iC3b, a protein extremely sensitive to trypsin, whereas C3b is relatively resistant. Cells coated with radiolabeled C3b were incubated with increasing concentrations of factor H in the presence of FIGURE 4. rH 19–20 inhibits the factor H-mediated decay acceleration excess factor I for 10 min, then the reactions were diluted in GVB of the alternative pathway C3/C5 convertase on ES. Decay acceleration assays measured the ability of human factor H to release 125I-Bb from containing trypsin. Fig. 5A shows the dependence of C3c release cell-bound C3b,Bb, in the presence of various concentrations of recombi- on the concentration of factor H. A concentration of factor H was nant rH 19–20. The C3b,125I-Bb complexes were formed as described in chosen that caused 50% C3c release, and the effect of increasing Materials and Methods. The 125I-Bb-loaded cells (10 ␮l) were added im- concentrations of rH 19–20 on this reaction was determined (Fig. ␮ mediately to reaction mixtures (40 l) containing varying amounts of hu- 5B). The IC50 of rH 19–20 for factor I cofactor activity of factor man factor H (A), or 0.67 nM factor H (100 ng/ml) in the presence of H was 1.9 ␮M. 0–27.6 ␮M (0–400 ␮g/ml) rH 19–20 (B), in PBS. After 10 min at 22°C the cells were sedimented rapidly by centrifugation through 20% sucrose in Measurement of factor H-mediated ﬂuid phase complement GVBE. The distribution of the radioactivity between the cell pellet, and the control activities in the presence of rH 19–20 supernatant was determined. Factor H at 0.67 nM induced 50% decay acceleration (% Bb bound) in the absence of rH 19–20 (arrow in A), and To determine whether the inhibitory effects of rH 19–20 on factor the results in B were graphed relative to this initial decay. Results are H functions were limited to cell surface bound C3b, we measured representative of at least three separate experiments, and the data show the the effect of rH 19–20 on the factor I-mediated cleavage of soluble mean of duplicate observations. C3b (Fig. 6). In this system, as shown for surface-bound C3b 6312 SURFACE-SPECIFIC REGULATION OF COMPLEMENT ACTIVATION

of factor H to control complement activation on what are normally nonactivating surfaces.

Fig. 7A shows that incubation of ES or EH in the presence of 40% NHS and MgEGTA resulted in no lysis in the absence of rH 19–20 (see the origin of Fig. 7A). Thus, in the absence of rH

19–20, spontaneous activation on ES and EH was controlled, indicating normal host cell recognition. However, in the presence of increasing concentration of rH 19–20, lysis of both cell types was ␮ observed. Lysis of ES was aggressive, with an IC50 of 0.9 MrH 19–20, whereas lysis of EH only reached 20% hemolysis with 27-fold higher concentrations of inhibitor (Fig. 7A). NHS was also

preadsorbed before use in these lytic assays with EH to eliminate any possible anti-EH Abs that could affect complement activation. FIGURE 6. rH 19–20 does not affect factor H-mediated fluid phase fac- Adsorbed and nonadsorbed NHS yielded identical results. It is tor I cofactor activity. Cofactor activity assays measured the ability of noteworthy that significant lysis of E occurred upon loss of pro- human factor H to behave as a cofactor for factor I in the digestion of fluid H tection by factor H, even though E possess the membrane bound phase C3b to C3bi, in the presence of various concentrations of recombi- H nant rH 19–20. C3b (1.8 ␮M) was incubated with factor I (1.4 ␮M) and regulators CD35, CD55, and CD59 (63, 64). This result suggests with factor H (2 nM), in the presence of varying concentrations of rH that factor H plays a substantial role in red cell survival in blood. 19–20 (as indicated below the lanes). C3b cleavage was analyzed by 10% To compare the regulatory efficacy of factor H, CD55 and CD59 Downloaded from

SDS-PAGE. Reactions with or without factor H or rH 19–20 were included in protecting the surface of EH, each were inhibited individually as controls as indicated. Cofactor activity is apparent from the loss of, or and in combination (Fig. 7B). No lysis was observed in the absence the reduced intensity of, the ␣Ј-chain of C3b and the appearance of the of any inhibitor (EH at zero input of rH 19–20). No lysis was ␣Ј-chain cleavage fragments (C3bi) at 68 and 43 kDa. Results are repre- observed with saturating doses of anti-CD55 Ab, 10% lysis was sentative of three separate experiments. observed with saturating doses of anti-CD59 Ab, and 16% of the

cells lysed upon inhibition of factor H by 12 ␮M rH 19–20. Loss http://www.jimmunol.org/ of the protection by factor H appeared to be at least as important above, soluble C3b forms a complex with factor I and factor H that to red cell survival as the other regulators on these cells. Interest- results in cleavage of the C3b ␣-chain to form the iC3b ␣-chain ingly, while the effect of completely inhibiting either of the two fragments of 68 and 43 kDa (compare Fig. 6, lanes 1 and 3). The cell-bound complement regulatory proteins (CRP) caused little or presence of rH 19–20 (0–17.9 ␮M) did not inhibit factor H/factor no lysis in this assay (origin of Fig. 7B), inhibition of either cell- I-mediated cleavage (Fig. 6, lanes 3–8). Lanes 1 and 2 were neg- bound regulator combined with the loss of factor H cell surface ␮ ative controls for the cleavage of C3b in the absence of factor H control resulted in aggressive lysis with an IC50 of 0.9 M. When

with or without rH 19–20, which can be seen at the bottom of lane rH 6–15 was used in this system, at the IC50 concentration of rH 2 at a molecular mass of 14,000 Da. Identical results (data not 19–20 (0.7–1 ␮M), it yielded no measurable effect (data not by guest on September 25, 2021 shown) were obtained in experiments using rH 1–7 as the cofactor shown). molecule, in the presence of the same concentrations of rH 19–20. Fig. 7C shows that deposition of C3b is readily detectable when Complement homeostasis requires factor H for control of spon- inhibiting factor H complement control on the surface of human taneous fluid phase activation of the alternative pathway amplifi- erythrocytes with rH 19–20. The effect of inhibiting CD55, which cation feedback system (62). The absence of factor H, or inhibition unlike CD59 participates in the regulation of C3b, is only detect- of its function, will result in activation. Because of the results able when factor H is also inhibited. shown in Figs. 2–5, we measured the effect of rH 19–20 on the fluid phase stability of the alternative pathway. Incubation of 40% Discussion normal human serum at 37°C with 3.5 ␮M rH 19–20 (over three The results presented in this study lead to three conclusions. First,

times the average IC50 concentration), for up to 1 h, resulted in the two C-terminal domains of factor H (rH 19–20) are able to Ͻ10% loss of alternative pathway function. This compares with distinguish between activators and nonactivators, without the aid complete consumption of both C3 and factor B in Ͻ60sinthe of the rest of the N-terminal 18 domains of the factor H molecule. absence of factor H function. The results indicate that fluid phase Second, rH 19–20, by efficiently competing with factor H for bind- control of the amplification cascade by factor H is largely unaf- ing to cell-bound C3b, is able to inhibit regulatory activities of the fected by rH 19–20. This result and that shown in Fig. 6 suggest N-terminal site directed toward cell-bound C3b, while not affect- that the inhibitory effects of rH 19–20 are largely confined to cell ing control of fluid-phase C3b. Third, factor H through recognition surfaces. of host cell surfaces by its domains 19–20, is a critical component of the complex system of control factors used by host cells and rH 19–20 promotes complement activation on the surface of tissues to protect their surfaces from complement-mediated attack. host-like cells This last conclusion derives from the observation that addition of The human alternative pathway of complement spontaneously ac- rH 19–20 to normal human serum leads to aggressive comple-

tivates on and lyses ER, which possess minimal surface polyan- ment-mediated lysis of nonactivating sheep erythrocytes, and even ions. ES, in contrast, possess high levels of sialic acid-containing to partial lysis of human erythrocytes, which have membrane- polysaccharides as do most human cells and tissues. Sheep eryth- bound regulators of complement. When either CD55 or CD59 was

rocytes are used as the standard nonactivator for human comple- blocked on EH, aggressive lysis was prevented by factor H. This ment analysis and as shown in Fig. 1B factor H binds more efﬁ- ﬁnding demonstrates the importance of the factor H contribution to

ciently to ESC3b than to ERC3b cells. Because the fragment rH regulatory redundancy at host cell surfaces. 19–20 behaves similarly (Fig. 1A) and because it does not inhibit Discrimination between potential pathogens and the host is a factor H-mediated homeostasis in the ﬂuid phase, we examined fundamental function of any system of innate immunity. Early whether cell-speciﬁc binding of rH 19–20 might inhibit the ability studies showed that regulation of alternative pathway activation on The Journal of Immunology 6313

targets was quantitatively different from that on host cells (61). This was later shown to be due to the presence of sialic acids or other polyanions on nonactivating cells (47, 48) and this was subsequently shown to be due to polyanion recognition by factor H (37). Clinical evidence has appeared in recent years indicating that alleles and mutations that affect polyanion recognition domains in factor H result in pathology involving complement activation. In- herited HUS focused attention on the C-terminal domains bearing C3d and sialic acid/polyanion binding sites (1–11). Moreover, age- related macular degeneration suggested a similar role might exist for the polyanion binding site in CCP 7 (49–52). Our results ex- tend the existing understanding of the mechanism by which factor H discriminates between activator and nonactivator surfaces. Fig. 1 shows that rH 19–20 and full-length factor H each bind 7-fold better to cells bearing C3b and polyanions than to cells bearing C3b but lacking polyanions. A maximum difference of 10-fold for this effect has been reported for full-length factor H (37, 48). This observation suggests that the two C-terminal domains may account

for most of the discriminating ability of factor H, at least for cells Downloaded from

where sialic acid is thought to play the primary protective role (ES and EH). It is important to note that this interaction is C3b as well as sialic acid dependent. It is difﬁcult to show any binding of factor H to most cells lacking C3b, although recently endothelial cells were shown to bind factor H directly via the C-terminal domains

at reduced ionic strength (18). The nature of the receptor on en- http://www.jimmunol.org/ dothelial cells has not been identiﬁed but has been proposed to be glycosaminoglycans (18). In our model the presence of C3b on the cell surface is essential for detectable factor H binding under phys-

iological conditions, as shown by the absence of binding to ES alone (Fig. 1B). Studies analyzing the functions of deletion mutants of factor H support the conclusions drawn from the present studies. These studies (62) showed that deletion of the C-terminal 10 domains by guest on September 25, 2021 caused a 40-fold reduction in binding to ESC3b. Similarly, a protein lacking the last 5 domains resulted in a 50-fold reduction in binding. Both results suggested that the C-terminal domains play a dominant role in binding to cells bearing C3b and polyanions. When the effect on decay acceleration of the complement C3/C5 convertase was measured (62) recombinant proteins lacking the C-terminal 5 domains exhibited an 11-fold reduction in functional activity. Mutants lacking the entire C-terminal half of the protein exhibited a 30-fold reduction in decay accelerating activity on

sheep ESC3b,Bb. Signiﬁcantly, systematic removal of clusters of domains spanning the entire factor H molecule showed that no other region, aside from the N terminus, affected factor H function

on ESC3b,Bb as much as removal of the C-terminal domains. FIGURE 7. rH 19–20 inhibits the factor H-mediated control of alter- In this study, the use of rH 19–20 as a site-specific competitor native pathway complement lysis of host-like polyanion-bearing cells (ES allowed the dissection of the protective functions of factor H. rH and EH). A, A total of 40% NHS was incubated with ES or EH in the 19–20 was able to enhance complement-mediated lysis of nor- presence of 0–24.1 ␮M (0–350 ␮g/ml) rH 19–20, for 20 min at 37°C. The mally complement-resistant cells (Fig. 7) and to act against control assays contained 5 mM MgEGTA to inhibit classical and lectin pathway of the surface-bound C3 convertase (Figs. 4 and 5), without af- activation and restrict activation to the calcium-independent alternative fecting fluid-phase functions of factor H (Fig. 6). This facilitated pathway of complement. Lysis was subsequently measured by hemoglobin the study of the cell surface-specific regulatory functions of factor release (A412) after centrifugation to remove unlysed cells. B, Same as H, within the context of a fully functional fluid phase complement above, except that the E were preincubated for 20 min on ice with satu- H system. rH 19–20 was able to compete with full length factor H for rating concentrations of neutralizing anti-CD59 or -CD55 Abs, before the binding to cell-bound C3b more efficiently than the other C3b/ addition of NHS and rH 19–20. C,EH were preincubated for 20 min on ice with or without saturating concentrations of neutralizing anti-CD55 Abs. polyanion binding sites of factor H (Fig. 2B). Although rH 1–6 C8 depleted sera, in the presence or absence of 12 ␮M rH 19–20, was showed significant inhibition of factor H binding, it is the active site and thus cannot be used as a functional inhibitor. The use of added and incubated for 20 min at 37°C. C3b deposition on EH was detected with FITC-conjugated goat anti-human C3 Ab, by FACS analysis. domain-specific inhibitory Abs for this purpose has the disadvan-

Deposition of C3b on EH is expressed as mean ﬂuorescence intensity tage that the Abs bind directly to factor H and have variable dose- (MFI). A minimum of 10,000 events per sample were counted. Results are dependent effects on function, probably due to dimerization or representative of three separate experiments, and the data show the mean steric hindrance of the 150-kDa factor H by the 150-kDa Abs (41, of duplicate observations. 46, 65, 66). 6314 SURFACE-SPECIFIC REGULATION OF COMPLEMENT ACTIVATION

The data shown in Figs. 4–7 indicate that there is a fundamental not support the presence of a cryptic functional site at the N ter- difference between the control of soluble C3b and that of cell- minus, which is revealed upon engagement of the C terminus with bound C3b. Nilsson et al. (67) describe antigenic differences be- ligands. Furthermore, this model is not supported by the ability of tween bound and soluble C3b. Conformational neo-epitopes were aHUS patients, with mutations affecting the C-terminal domains, suggested to be present on surface-bound C3b, which are not to control fluid phase complement activation (7, 68). present on soluble C3b, although these epitopes have not been HUS (shiga-toxin-related, familial, and sporadic) and various characterized. This difference may also be due to multisite attach- other diseases, presents with thrombotic microangiopathy which ment of factor H to C3b clusters and to bifunctional binding by defines a lesion of vessel wall thickening (mainly arterioles and domains 19–20 to polyanions and C3b. Notably, one interpretation capillaries), intraluminal platelet thrombosis, and partial or com- of the recently determined structure of rH 19–20 suggested bind- plete obstruction of the vessel lumina, secondary to endothelial cell ing sites for C3b and polyanions on opposite faces of CCP 20 (16). damage (69). The enhanced shear stress in the narrowed microcir- According to such a model, the C-terminal site of factor H would culation causes erythrocytes to be traumatized, which can result in bind poorly to C3b on cells lacking the second cell surface ligand, hemolysis. Our data shows that factor H is important in maintain- i.e., a polyanion. However, domain 20 would bind bifunctionally ing erythrocyte homeostasis, presenting an additional factor that between C3b and the polyanion, providing that the surface bears a may contribute to the damage of erythrocytes and other cell types sufficient density of polyanions. This would raise the apparent af- in factor H- and factor I-related aHUS. Deposition of C3b (Fig. finity of factor H for the C3b on these cells and would increase 7C) as well as up to 20% lysis of human erythrocytes (Fig. 7, A and regulation of complement activation on these surfaces. The degree B) was observed when factor H-mediated cell surface control was of enhancement of factor H binding by polyanions appears to be inhibited by rH 19–20, even when the function of the membrane- Downloaded from limited to 5- to 10-fold (37, 48) raising the question of how this bound regulatory proteins was intact and when shear stress was modest difference could be sufficient to account for the difference absent. Interestingly, eryptosis, a pathophysiological mechanism between activation or no activation of this system. One rationale of RBC death, has recently been shown in vitro to be linked to for the adequacy of this mechanism is the following: because the HUS, to be induced by complement activation, and may be factor alternative pathway is dependent on a feedback loop and because H dependent (70). Studies defining the relative contribution of

all three reactions in that loop are inhibited by factor H, a 10-fold each of these mechanisms to erythrocyte destruction in aHUS are http://www.jimmunol.org/ increase in the affinity of H for the activation site could be as warranted. Likewise, erythrocytes from individuals with aHUS effective as a 1000-fold increase in factor H function. caused by mutations in CD46, which is not present on erythro- There is a growing body of evidence that each polyanion-bind- cytes, may be more resistant to complement-mediated lysis, but ing site on factor H has unique specificities. Recently, CCP domain will still be affected by enhanced shear stress encountered in the 7 was shown to exhibit a polyanion recognition profile different microvasculature due to endothelial cell damage. from that of CCP 19–20 (38). It is thus probable that factor H Host cells are normally protected from autologous complement- exhibits cell type-specific recognition depending on the array of mediated lysis due to the complex, often redundant array of CRP polyanions that the particular cell expresses on its surface. Thus, in that control activation on the surface of the cell. These CRP can be the case of endothelial cells (18), the presence of sulfated glycos- membrane-bound (CD59, CD55, CD46, CD35), as well as fluid by guest on September 25, 2021 aminoglycans would appear to be sufficient for weak initial bind- phase (C4BP and factors H and I) (12, 64). Human erythrocytes as ing of factor H, in the possible absence of C3b. Whichever the well as nucleated human cells are known to be protected by CD59 case, the conclusion is the same in the sense that the C terminus is and CD55 and the present data suggest a significant role for factor crucial for the ability of factor H to bind to cell surfaces, be it H. Use of rH 19–20 as a cell-specific inhibitor overcomes the through interaction with the C3b-polyanion interface, or through primary difficulty in analyzing the role of factor H. As shown in direct interaction with polyanions. Further studies are needed to Fig. 7 human cells activate the alternative pathway and are lysed comprehend how mutations in other regions affect the function of (20% in 20 min) in the presence of the factor H inhibitor rH 19–20. factor H and predispose to diseases, such as macular degeneration Completely blocking CD55 with polyclonal Abs produced no lysis (49–52). and minimal C3b deposition unless rH 19–20 was present. Inhib- All of the regulatory activities of factor H are expressed by the iting CD59 with excess mAbs resulted in levels of lysis similar to N-terminal four domains. In this study, all the activities of full- those produced by inhibition of factor H by rH 19–20. These re- length factor H for cell-bound C3b were inhibited by rH 19–20 sults suggest that control of complement activation on human (Figs. 2–5). If rH 19–20 competes for the binding site on C3b erythrocytes by fluid phase factor H is at least as important to red where domains 1–4 bind, then the nearly complete inhibition cell homeostasis as CD55 or CD59. The results also show (Fig. shown in Fig. 2A is easily explained. However, direct competition 7B) that on EH, inhibition of any two of these three regulators with the N-terminal site is unlikely to explain this because rH results in aggressive activation of the alternative pathway and lysis 19–20 does not compete with factor H (Fig. 6) or with rH 1–7 of the cells indicating that each of the three regulators performs a (data not shown) for control of fluid-phase C3b. In addition, rH critical function in protecting host cells from the cytotoxic effects 1–7 does not compete with rH 19–20 for the binding to membrane of autologous activation of complement. bound C3b (Fig. 3). This is in agreement with previous findings that these sites are distinct (42). However, these results do not Acknowledgments agree with the model proposed recently (68) in which the N-ter- We express our appreciation to Kerry L. Wadey-Pangburn, Connie Elliot, minal site is blocked by intramolecular folding in solution and is and Kristi Mckee for their excellent technical work. freed upon binding of the C-terminal site to C3b, C3d, or polya- Disclosures nions. According to such a model, rH 19–20 would prevent ex- M. K. Pangburn is an officer of and has a financial interest in posure of the N-terminal site of full-length factor H by competing Complement Technology, a supplier of complement reagents. for binding of the C terminus to ligands, which should block fluid- phase cofactor activity of factor H, but no evidence of inhibition References was found in our study (Fig. 6). Therefore, although both studies 1. Bettinaglio, C. J., P. F. Zipfel, B. Amadei, E. Daina, S. Gamba, C. Skerka, support an important role of the C-terminal region, our results do N. Marziliano, G. Remuzzi, and M. Noris. 2001. The molecular basis of familial The Journal of Immunology 6315

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