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of June 18, 2017.
Identification of Factor H−like Protein 1 as
the Predominant Complement Regulator in
Bruch's Membrane: Implications for
Age-Related Macular Degeneration
Simon J. Clark, Christoph Q. Schmidt, Anne M. White,
Svetlana Hakobyan, B. Paul Morgan and Paul N. Bishop
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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 © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 193:4962-4970; Prepublished online 10
October 2014;
doi: 10.4049/jimmunol.1401613
http://www.jimmunol.org/content/193/10/4962
The Journal of Immunology
Identification of Factor H–like Protein 1 as the Predominant
Complement Regulator in Bruch’s Membrane: Implications
for Age-Related Macular Degeneration
Simon J. Clark,*,† Christoph Q. Schmidt,‡ Anne M. White,*,† Svetlana Hakobyan,x
B. Paul Morgan,x and Paul N. Bishop*,†,{
A
ge-related macular degeneration (AMD) is the leading
cause of blindness in the developed world, affecting ∼50
million people worldwide. The prevalence of this condition is predicted to rise as the elderly population expands: in the
*Centre for Hearing and Vision Research, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; †Centre for Advanced
Discovery and Experimental Therapeutics, University of Manchester and Central
Manchester University Hospitals NHS Foundation Trust, Manchester Academic
Health Science Centre, Manchester M13 9WL, United Kingdom; ‡Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, 89081
Ulm, Germany; xComplement Biology Group, Institute of Infection and Immunity,
School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom; and
{
Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS
Foundation Trust, Manchester M13 9WL, United Kingdom
ORCID: 0000-0001-8394-8355 (S.J.C.).
Received for publication June 26, 2014. Accepted for publication September 11,
2014.
S.J.C. is a recipient of Medical Research Council Career Development Fellowship
MR/K024418/1 and had previously been supported by a Stepping Stones Fellowship
from the Faculty of Medicine and Human Sciences, University of Manchester. The
eye tissue holdings were initiated and supported by Medical Research Council Grants
G0900592 and MR/K004441/1. The Bioimaging Facility microscopes used in this
study were purchased with support from the Biotechnology and Biological Sciences
Research Council, the Wellcome Trust, and the University of Manchester Strategic
Fund.
S.J.C. designed the study, performed experiments, and wrote the manuscript; C.Q.S.
expressed the recombinant FHL-1 proteins; A.M.W. helped with the design and
execution of PCR experiments; S.H. and B.P.M. purified FH from human donors;
and P.N.B. advised on experimental design and writing the manuscript.
Address correspondence and reprint requests to Dr. Simon J. Clark, Centre for Hearing and Vision Research, University of Manchester, AV Hill Building, Oxford Road,
Manchester M13 9PT, U.K. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AMD, age-related macular degeneration; CCP,
complement control protein; ECM, extracellular matrix; FB, complement factor B;
FH, complement factor H; FHL-1, factor H–like protein 1; FHR, factor H–related; FI,
complement factor I; GAG, glycosaminoglycan; HS, heparan sulfate; RPE, retinal
pigment epithelium.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401613
United States it has been estimated that there will be a 50% increase in the number of affected individuals between 2004 and
2020 (1). This debilitating disease can be subdivided into neovascular (“wet”) and atrophic (“dry”) AMD (2), both of which are
usually preceded by the formation of drusen. These aggregates of
lipids, proteins, and cellular debris accumulate within Bruch’s
membrane, a sheet of extracellular matrix (ECM) that separates
the retinal pigment epithelium (RPE) from the blood vessels of the
choroid. The presence of complement proteins and downstream
inflammatory markers in drusen has led to the hypothesis that
chronic local inflammation in Bruch’s membrane and surrounding
structures, resulting from inappropriate complement activation,
has a major influence on the pathogenesis of AMD (3–5).
Genetic alterations are a major risk factor for AMD. Two major
loci have been identified: one is on chromosome 10 near the ARMS2/
HTRA1 genes and the other is on chromosome 1 involving complement factor H (FH) and the FH-related (FHR) proteins (6).
Additionally, genes encoding members of the alternative complement pathway have been implicated, including C3, complement
factor I (FI), and complement factor B (FB), thereby providing
strong evidence that this pathway is involved in AMD pathogenesis
(6–8). The locus on chromosome 1 is complex with multiple haplotypes having been identified that modify AMD risk (9).
Within the chromosome 1 locus the Y402H polymorphism in FH
represents a major risk factor for AMD (10–14). The frequency of
this risk allele is ∼35% in individuals of European descent and
results in a tyrosine being replaced by a histidine residue at position 402 (using the preprotein sequence numbering) (15). The
effects of the Y402H polymorphism appear to be mediated locally,
as the polymorphism does not promote complement activation in
the blood (14), unlike some mutations found in the C-terminal
complement control protein (CCP) domains of FH.
The central activating mechanism of the alternative pathway is
the covalent deposition of the protein C3b (an opsonin) on all local
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The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human
body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against
damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein
1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch’s membrane. Bruch’s membrane is
a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can
passively diffuse through Bruch’s membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch’s
membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene,
associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is
retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel
mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies. The
Journal of Immunology, 2014, 193: 4962–4970.
The Journal of Immunology
Materials and Methods
Primary Abs and protein reagents
Both recombinant 402H and 402Y forms of FHL-1 were generated according
to the methodology described previously (30). The full-length FH protein was
purified from human plasma, as described by Hakobyan et al. (31). Commercial Abs used in this study were OX23 (AbD Serotec, Kidlington, U.K.)
(32) and L20/3 (Hycult Biotech, Uden, the Netherlands), which has been
published previously with the clone name C02 (33). Both of these Abs
recognize different epitopes on FH (see Fig. 1A). Anti–FHL-1 was generated
against the peptide sequence CIRVSFTL (Mimotopes, Clayton, Australia).
Anti–FHL-1 IgG was purified using an affinity column: recombinant FHL-1
was coupled to cyanogen bromide–activated Sepharose according to the
manufacturer’s instructions (GE Healthcare, Buckinghamshire, U.K.).
Briefly, rabbit antiserum was centrifuged at 12,000 3 g for 5 min at room
temperature to remove particulate matter. The antiserum was then run down
the FHL-1 affinity column in PBS, 1 mM EDTA. The column was washed
thoroughly with 5 column volumes 0.4 M NaCl to remove weakly bound
material. Two column volumes 3 M MgCl2 was used to elute bound protein.
The column was then regenerated by washing with 5 column volumes 0.4 M
NaCl and re-equilibrating with 5 column volumes PBS, 1 mM EDTA. Eluted
protein was dialyzed into 2 l Milli-Q H2O for 16 h at 4˚C using 10 kDa cutoff
dialysis tubing before being further dialyzed into 20 mM NaH2PO4, 150mM
NaCl (pH 8.0) for a further 16 h at 4˚C. Rabbit IgG was purified from the
dialysate using protein A–Sepharose, following the manufacturer’s instructions (Sigma-Aldrich, Poole, U.K.). Isolated IgG was run on 4–12%
NuPAGE reducing gels to check for purity and tested for specificity to
FHL-1 in solid phase assays (see Supplemental Fig. 1).
Eye tissue preparation
Details of donor eye tissue used in this study are listed in Table I. Human
eyes were obtained from the Manchester Royal Eye Hospital Eye Bank
after removal of the corneas for transplantation. Our research adhered to
the tenets of the Declaration of Helsinki. In all cases, there was prior
consent for the eye tissue to be used for research, and guidelines established in the Human Tissue Act of 2004 (U.K.) were followed. Except in
the case of donor tissue used for the staining of drusen, none of the other
donors had a history of visual impairment or eye disease.
For the donor eye pairs used for immunohistochemistry, PCR analysis,
and Western blotting, one globe was designated for RPE cell isolation and
Bruch’s membrane enrichment. These eyes were opened by making three
incisions into the eyecup and flattening out the tissue. The vitreous and
neurosensory retina were removed, RPE cells were harvested with gentle
scraping, and RNA was isolated (see below). The Bruch’s membrane was
enriched by removal of the sclera and choroid. The Bruch’s membrane was
washed multiple times with PBS and either frozen for analysis by Western
blotting or used in Ussing chamber diffusion experiments (both methodologies are described below).
The second globe from each donor was used to prepare frozen tissue
sections and was fixed in 4% (v/v) formaldehyde for 2 h. Eyes were then
processed and serial sections of 5 mm were prepared using a cryostat, as
described previously (23, 34).
Fluorescent immunohistochemistry
Tissue sections were stained for the presence of endogenous FH or FHL-1
using methods described previously (23). Briefly, tissue sections were incubated with chilled (220˚C) histological grade acetone (Sigma-Aldrich)
for 20 s before thorough washing with PBS. Tissue sections were blocked
with 0.1% (w/v) BSA, 1% (v/v) goat serum, and 0.1% (v/v) Triton X-100
in PBS for 1 h at room temperature. After washing with PBS, tissue
sections were incubated with Ab combinations of either 10 mg/ml mix of
L20/3 and anti–FHL-1 or of OX23 and anti–FHL-1 for 16 h at 4˚C.
Sections were washed and Ab binding was detected using fluorescently
labeled secondary Abs (Life Technologies, Paisley, U.K.). An equal 1:5000
dilution mix of Alexa Fluor 488–conjugated goat anti-rabbit (to detect
anti–FHL-1) and Alexa Fluor 594–conjugated goat anti-mouse (against
OX23 or L20/3) was added to sections for 2 h at room temperature. Finally,
DAPI was applied as a nuclear counterstain (at 0.3 mM for 5 min) prior to
mounting with medium (Vectashield; Vector Laboratories, Peterborough,
UK) and application of a coverslip.
In some experiments the enzymatic pretreatment of tissue was performed as described previously (23). Briefly, 20 U/ml each of heparinase I, II,
and III (all from Flavobacterium heparinum, Sigma-Aldrich) in PBS was
applied to tissue sections for 1.5 h at 37˚C; this was performed after fixation
in acetone and followed by washing with PBS prior to the blocking step.
Images were collected on a snapshot widefield microscope (Olympus
BX51) using a 340/0.30 Plan Fln objective. Microscopy images were
captured using a CoolSNAP ES camera (Photometrics) via MetaVue
software (Molecular Devices). To prevent bleed-through of color from one
channel to the next, specific band pass filter sets were used for DAPI,
FITC, and Texas Red. All images were handled using ImageJ64 (version
1.40g; http://rsb.info.nih.gov/ij).
Western blotting
Samples were run on 4–12% NuPAGE Bis-Tris gels (Life Technologies) at
200 V for 60 min and transferred onto nitrocellulose membranes at 80 mA
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surfaces. Surface-linked C3b can react with other complement
proteins to form an active enzyme, the C3 convertase that is able to
produce further (surface-attachable) C3b molecules. This is achieved
as the C3 convertase proteolytically processes C3 in blood into
fresh C3b molecules, producing at the same time the anaphylatoxin
C3a. Insufficient control of the C3 convertase results in massive
production of C3b and C3a molecules and a shift of the complement cascade to its terminal lytic pathway. This produces the
most potent anaphylatoxin, C5a, and the cell lytic protein complex
termed the membrane attack complex; both C5a and the membrane
attack complex provide strong inflammatory signals.
However, the presence of FH on host tissues, and its cofactor
activity for FI, results in C3b breakdown (resulting in the formation of inactive iC3b), thereby preventing inappropriate complement activation and inflammation (16). FH also exerts decayaccelerating activity, which can assist in the deconstruction of
already formed C3 convertases. Whereas cell surface–expressed
complement regulators also prevent complement activation on
cells, the blood-borne FH is currently the only known complement
regulator to bind and confer protection to ECM such as Bruch’s
membrane (17). The protective FH is recruited to self-surfaces, at
least in part, by binding specific polyanions such as glycosaminoglycan (GAG) chains or sialic acid groups, which are not normally present on potential pathogens. GAGs are long, unbranched
polysaccharides made up of repeating disaccharide units that can
be variably sulfated: the sulfation pattern can drive specific protein
recruitment (18). One ubiquitously expressed GAG, and a major
ligand for FH, is heparan sulfate (HS), which comprises both lowand high-sulfated regions (18). These sulfated ligands are essential
ECM components responsible for a range of biological processes,
including immune homeostasis (19, 20).
FH comprises 20 CCP domains and contains two main GAGbinding regions, in CCP7 (with some contribution from CCPs 6
and 8) and CCP20 (21) (see Fig. 1A): the Y402H polymorphism
resides in CCP7 and alters FH binding to sulfated GAGs (22). The
402H disease-associated variant binds significantly less well to
human Bruch’s membrane (an important site in AMD pathogenesis)
than does the 402Y form when applied exogenously to tissue sections (23). We have demonstrated that the GAG binding region in
CCP6–8 is responsible for surface anchoring and hence host recognition in eye structures, including Bruch’s membrane, whereas
the CCP19–20 region anchors FH to ECM in the kidney (24).
In this study we investigated the distribution of a naturally
occurring truncated form of FH called FH-like protein 1 (FHL-1),
which arises from alternative splicing of the CFH gene (25). FHL-1
is identical to FH for the first seven CCP domains before
terminating with a unique 4-aa C terminus (see Fig. 1A). Importantly, FHL-1 retains all the necessary domains for function and is
also subject to the Y402H polymorphism. Previous studies have
demonstrated FHL-1 expression by RPE cells (13, 26) and it has
been identified in the vitreous of the eye where FH and FHL-1
were reported to be in equimolar concentrations (27). In human
blood the concentration of FH is ∼300 mg/ml (28) and FHL-1
∼50 mg/ml (29), resulting in a molar ratio of ∼2:1.
4963
4964
for 2.5 h using semidry transfer apparatus in transfer buffer (25 mM Tris,
192 mM glycine, 10% [v/v] methanol). The membranes were blocked in
PBS, 10% (w/v) milk, and 0.02% (w/v) BSA for 16 h at 4˚C before the
addition of an OX23 and anti–FHL-1 Ab mix, each at 0.5 mg/ml, in PBS
with 0.2% (v/v) Tween 20 (PBST) for 2 h at room temperature. Membranes were washed twice for 30 min in PBST before the addition of
a 1:2000 dilution of IRDye 680RD–conjugated goat anti-mouse and IRDye
800CW–conjugated goat anti-rabbit (LI-COR Biosciences U.K., Cambridge, U.K.) for 2 h at room temperature, protected from light. Membranes were washed and protein bands were visualized using a LI-COR
Odyssey infrared imaging system and Image Studio software.
RPE cell complement gene expression
Ussing chamber diffusion experiments
The macular region of enriched Bruch’s membrane isolated from donor
eyes (described above) was mounted in an Ussing chamber (Harvard
Apparatus, Hamden, CT). Once mounted, the 5-mm-diameter macular area
was the only barrier between two identical compartments (see Fig. 3A).
Both sides of Bruch’s membrane were washed with 2 ml PBS for 5 min at
room temperature. Human serum (Sigma-Aldrich) was diluted 1:1 with
PBS and 2 ml was added to the Ussing compartment representing the
choroidal side of Bruch’s membrane. After 1 min when no leaks were
detected into the second compartment (which would indicate a compromise in membrane integrity), 2 ml PBS alone was added to the second
compartment and the Ussing chamber was left at room temperature for
24 h with gentle stirring in each compartment to avoid generating gradients
of diffusing proteins. Samples from each chamber were analyzed by gel
electrophoresis. Gels were either stained with Instant Blue stain (Expedeon,
Harston, U.K.) for 60 min at room temperature or subject to Western
blotting (see above).
In some experiments the above protocol was repeated using 100 mg/ml
FH purified from human plasma (31) on three donor Bruch’s membranes
(see Table I). After 24 h the entire PBS compartment, where FH would
diffuse into, was collected and StrataClean beads (Agilent Technologies,
Cheadle, U.K.) used to pull all proteins out of solution. The entire content
of the PBS compartment was analyzed by gel electrophoresis as described
above. Similarly, to ascertain whether the AMD-associated polymorphism
altered the diffusion properties of FHL-1, 100 mg/ml recombinant 402H or
402Y forms of FHL-1 were separately tested on three donor Bruch’s
membranes (see Table I). The protein content of each Ussing chamber
compartment was analyzed by gel electrophoresis and band densities were
normalized and compared with a 0 h, 100 mg/ml FHL-1 sample.
FHL-1 fluid-phase cofactor activity
The fluid-phase cofactor activity of the FHL-1 402H and 402Y variants
were measured by incubating FHL-1 (either form), C3b, and FI together in
a total volume of 20 ml PBS for 15 min at 37˚C. For each reaction, 2 mg
C3b and 0.04 mg of FI were used with varying concentrations of FHL-1
ranging from 0.0125 to 0.8 mg per reaction. The assay was stopped with
the addition of 5 ml 53 SDS reducing sample buffer and boiling for 10 min
at 100˚C. Samples were run on a 4–12% NuPAGE Bis-Tris gel at 200 V for
60 min to maximize the separation of the C3b breakdown product bands.
The density of the 68-kDa iC3b product band was measured using
ImageJ64 (version 1.40g; http://rsb.info.nih.gov/ij) and used to track C3b
breakdown efficiency of the FHL-1 proteins. For both forms of FHL-1,
averaged data from three separate experiments were used.
Solid-phase plate assays
The heparin-binding characteristics of the FHL-1 (402H and 402Y variants)
proteins were analyzed using microtiter plate-based assays, where either
heparin or one of its selectively desulfated derivatives (all from Iduron) was
immobilized noncovalently on allylamine-coated heparin-binding plates
(BD Biosciences, Oxford, U.K.) as described previously (22, 35). The
selectively desulfated heparin samples used in this study were 2-O–
desulfated, 6-O–desulfated, and N-desulfated heparin. HS from either
porcine mucosa (Iduron) or bovine kidney (Sigma-Aldrich) was also tested
under the same conditions.
All GAGs were diluted in PBS and immobilized at 1 mg/well in a volume
of 100 ml/well overnight at room temperature. Plates were blocked for
90 min at 37˚C with 300 ml/well 1% (w/v) BSA in assay buffer (20 mM
HEPES, 130 mM NaCl, 0.05% [v/v] Tween 20 [pH 7.3]). This standard
assay buffer was used for all subsequent incubations, dilutions, and
washes, and all steps were performed at room temperature. FHL-1 protein
was incubated with the immobilized GAGs for 4 h. After washing, bound
protein was detected by the addition of 100 ml/well 0.5 mg/ml OX23 Ab
and incubated for 30 min followed by washing and a 30-min incubation in
100 ml of a 1:1000 dilution of alkaline phosphatase–conjugated anti-mouse
IgG (Sigma-Aldrich). Plates were developed using 100 ml/well 1 mg/ml
disodium p-nitrophenylphosphate solution (Sigma-Aldrich) in 0.05 M TrisHCl, 0.1 M NaCl (pH 9.3). The absorbance values at 405 nm were determined after 10 min of development at room temperature and corrected
against blank wells (i.e., those with no immobilized GAGs).
Heparin affinity chromatography
The heparin-binding properties of the 402H and 402Y FHL-1 variants were
compared by affinity chromatography on a heparin affinity column in which
30 mg heparin (Iduron) was coupled to 1.5 ml cyanogen bromide–activated
Sepharose (GE Healthcare) in 0.1 M NaHCO3, 0.5 M NaCl (pH 8.3) using
the manufacturer’s protocol. Before sample loading, the column was
equilibrated in 5 ml PBS (Sigma-Aldrich). Purified recombinant protein
(100 mg) was loaded onto the column in a total volume of 5 ml PBS. The
column was washed with 4 column volumes PBS before bound protein was
eluted with a linear salt gradient of 130 mM–1 M NaCl over 20 ml by
mixing elution buffer (PBS, 1 M NaCl) with equilibration buffer at a flow
rate of 1 ml/min; fractions (1 ml) were collected throughout the protocol.
Results
FHL-1 is present in the human Bruch’s membrane
To determine whether FHL-1 was present in human Bruch’s membrane we employed the Abs OX23 (which recognizes both FH and
FHL-1) (32), L20/3 (previously published as clone C02 and recognizes only FH) (33), and a specific anti–FHL-1 Ab that we generated
(Fig. 1A). The anti–FHL-1 and L20/3 Abs were shown to be specific
for their intended targets and exert no cross-reactivity when tested
against recombinant FHL-1 or FH purified from human serum
(Supplemental Fig. 1A–C). Fluorescent staining of six separate human
maculae (Table I) identified FHL-1 throughout Bruch’s membrane
(Fig. 1B). In contrast, full-length FH protein was identified on the
choroidal side of Bruch’s membrane (with particular accumulation in
the choriocapillaris), and small amounts were present in patches on
the RPE-facing side, but staining was not seen within Bruch’s membrane (Fig. 1C, 1D). The choroidal stroma stained for (full-length) FH,
and there was weak staining for FHL-1, especially near Bruch’s
membrane. FHL-1 staining could be ablated by preabsorbing the Ab
with recombinant protein (Supplemental Fig. 1D, 1E), thereby providing further evidence that the staining identified endogenous protein.
Additionally, as expected, we demonstrated colocalization of anti–
FHL-1 and OX23 Abs throughout Bruch’s membrane (Supplemental
Fig. 1F). Analyses of drusen from four donors (Table I) demonstrate
staining for endogenous FH in the periphery of the lesions whereas
staining with FHL-1 appeared throughout the lesions (Fig. 1E).
We performed Western blots on extracts from isolated human
Bruch’s membrane and probed them with the anti–FHL-1 and OX23
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Total RNA was isolated from human RPE cells isolated from the donors listed
in Table I, using standard extraction and purification methods. Briefly, RPE
cells were homogenized in 1 ml cold TRI Reagent (Life Technologies) per
30 mg tissue using a TissueLyser II bead mill (Qiagen). After homogenization, RNA was isolated following TRI Reagent/BCP disruption and phase
separation. RNA was further purified by absorption to an RNeasy mini spin
column (Qiagen) with on-column DNAse I treatment. RNA purity and
concentration, as measured by a NanoDrop spectrophotometer, were determined by absorbance at 230, 260, and 280 nm. cDNA was synthesized using
a Transcriptor High Fidelity cDNA synthesis kit (Roche Diagnostics,
Burgess Hill, U.K.) with 1 mg RNA and primed with oligo(dT)18 primer
according to the manufacturer’s instructions.
Target gene sequences were obtained from GenBank and PCR primers to
specific targets were designed with Primer3 software (http://www.ncbi.nlm.
nih.gov/tools/primer-blast/) and are shown in Supplemental Table I. Primers for the RPE cell–specific genes (Bestrophin-1 and RPE65) were also
included to ensure purified cDNA was indeed obtained from RPE cells.
cDNA was amplified using the following PCR protocol: 95˚C for 5 min
followed by 40 cycles of 95˚C for 10 s, 60˚C for 15 s, and 72˚C for 20 s,
followed by a melting program. The integrity of the PCR reactions was
verified by detection of a single band of the correct size by agarose gel
electrophoresis. The same experiments were also performed on pooled
liver cDNA (Sigma-Aldrich).
FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE
The Journal of Immunology
4965
Abs. We identified an ∼49-kDa band that migrated to the same
position as recombinant FHL-1 protein control (Fig. 1F). This was
identified by both OX23 and anti–FHL-1, thereby confirming that
this band was FHL-1 and not one of a number of known tryptic
fragments of FH (25, 36). The OX23 Ab was, however, unable to
detect a 155k-Da species corresponding to FH in the extracts.
Human RPE transcription of FHL-1, FH, and genes associated
with the alternative complement pathway
We investigated mRNA expression of a number of complement
genes by RPE cells isolated from donor tissue (for a list, see
Supplemental Table I). We found that most genes involved in an
alternative pathway complement response were transcribed, including C3, FB, FI, FHL-1, and FH (Fig. 2A).
FHL-1 can diffuse across the Bruch’s membrane
Next we investigated whether FH and FHL-1 could contribute to
the protection of the Bruch’s membrane/RPE interface by diffusing across Bruch’s membrane from the blood supply. Isolated
Bruch’s membrane was sandwiched between two compartments
of an Ussing chamber (see Fig. 3A) as previously described (37),
and the diffusion of FH and FHL-1 from human serum across the
membrane was investigated. After 24 h only an ∼49-kDa band
was identified in the diffusate by Western blot using OX23
(Fig. 3B). Dual staining of this band with OX23 and anti–FHL-1
confirmed that this was indeed FHL-1 traversing the Bruch’s
membrane (Fig. 3B). In contrast, FH (155 kDa) was not detected.
The experiments were repeated using purified FH, and after 24 h
the entire protein content of the diffusate chamber was concentrated and analyzed, and we were unable to detect any FH protein
having crossed Bruch’s membrane (Fig. 3C). To ascertain whether
the Y402H AMD-associated polymorphism affected FHL-1
diffusion, we repeated the experiments using purified 402H
and 402Y forms of FHL-1 (Fig. 3D, 3E). In the case of both
variants, equilibrium was reached, although a reduction in the
calculated protein recovery for the 402Y form was observed (an
average of 73% over three separate experiments opposed to 92%
for the 402H form).
The AMD-associated 402H polymorphism does not affect C3b
cofactor activity of FHL-1
We assessed whether the AMD-associated Y402H polymorphism
would alter the ability of FHL-1 to regulate complement activation
on host surfaces by catalyzing the FI-mediated breakdown of C3b
to iC3b. When run on reducing gradient gels, the a- and b-chains
of C3b can be clearly separated (Supplemental Fig. 3A). When
C3b is incubated with FI and either the 402Y or 402H form of
FHL-1, the proteolytic cleavage of the C3b a-chain by FI yeilds
bands at 68 and 43 kDa (38), the appearance of which can be used
as a measure of FHL-1 cofactor activity. In the present study, we
noted no difference in the ability of the two FHL-1 forms to break
down C3b (see Supplemental Fig. 3B). This result was predictable
because the C3b/FI binding regions and the Y402H polymorphism
site are at opposite ends of the FHL-1 protein (Fig. 1A) (39, 40).
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FIGURE 1. FHL-1 rather than FH is the predominant complement regulator in human Bruch’s membrane. (A) Schematic indicating the CCP regions of
FH and FHL-1 recognized by the OX23, anti–FHL-1, and L20/3 Abs. The AMD-associated Y402H polymorphism is located in CCP7 of both FH and
FHL-1. (B) Gray-scale fluorescent staining of a human macula with anti–FHL-1. (C) Gray-scale staining of the same donor as shown in (B) but with the Ab
L20/3. (D) Fluorescent staining of human macula with an equal mix of both anti–FHL-1 (green) and L20/3 (red). (E) Labeling of a druse with FHL-1
(green) and FH (red) Abs. (F) Western blot of solubilized Bruch’s membrane from four donors stained with anti–FHL-1 (green) and OX23 (red): yellow
staining is indicative of colocalization of both Abs. Blue color represents DAPI staining of cell nuclei. Images in (B)–(D) are representative of six individual
donors. Scale bars, 10 mm. Image in (E) is representative of four donors. Scale bar, 5 mm.
4966
FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE
Table I. Details of donor eyes used in this study
Donor
Sex
Age (y)
a
72
73
89
83
92
78
82
84
70
86
67
69
78
58
75
63
65
78
84
a
Donors without known eye disease used for immunohistochemistry, Western
blot analysis, and RPE gene transcription.
402H and 402Y forms of FHL-1 differentially bind sulfated
self-surface markers
Although the Y402H polymorphism does not affect the ability of
FHL-1 to inactivate C3b, it does reside in the protein’s only surface
recognition domain in CCP7 (Fig. 1A). Previously using FH and
recombinant proteins representing the CCP6–8 region of FH, the
Y402H polymorphism has been shown to affect GAG binding (22,
23). In this study, we investigated the binding of the FHL-1 402H
and 402Y proteins to selectively desulfated heparin. The heparin
was desulfated at the 6-O, 2-O, and N positions (see Fig. 4A), and
although this did not affect the binding of the 402Y form of
FHL-1, desulfation did significantly affect the binding of the
AMD-associated 402H form (Fig. 4B, 4C).
FIGURE 2. Detection of complement gene transcription by human RPE
cells. Pooled RNA from five donors’ RPE cells was used to detect complement gene transcription. (A) RNA for a number of genes central to the
alternative pathway of complement were detected in both liver and RPE
cells, including FHL-1, FH, C3, FB, and FI. (B) Two RPE-specific genes,
Best-1 and RPE65, were used as positive controls for RPE cell RNA, and
b-actin and TATA-binding protein (TBP) were selected as housekeeping
gene controls.
Discussion
Since 2005, a large body of work has focused on alterations in FH
as major genetic contributors to AMD pathogenesis (6, 41–44) and
the importance of the alternative complement pathway. This has
been supported by evidence of complement proteins being present
in AMD macular tissue, including markers of dysregulation (3, 22,
23, 45). Previous studies have concluded that FH, as a blood-borne
protein, is the only complement regulator that would confer protection to ECM such as Bruch’s membrane. Genetic studies have
not distinguished between FH and FHL-1 (given that they share
the same gene), and although FHL-1–specific Abs have been made
in the past (46) they have not been used to probe eye tissue (27).
In this study, we show that FHL-1 is likely to be conferring
greater protection to Bruch’s membrane than does FH, whereas the
latter is the predominant form protecting the ECM of the choroid
(Fig. 1C, 1D). Furthermore, we confirm that the RPE cells transcribe genes involved in complement activation and regulation on
Bruch’s membrane, and our data demonstrate that FHL-1 is locally transcribed (Fig. 2). As well as a local contribution to FHL-1
accumulation in the Bruch’s membrane, we have demonstrated
that the 49-kDa protein could passively diffuse across Bruch’s
membrane from the choroidal vasculature, whereas the 155-kDa
glycosylated FH protein cannot (Fig. 3B, 3C). This latter observation supports the previous finding that the Bruch’s membrane
confers a size limit on proteins able to diffuse passively across it,
the size of which decreases with age (37). The AMD-associated
Y402H polymorphism does not affect the diffusion of FHL-1
across Bruch’s membrane (Fig. 3D, 3E), but differences in the
amount of recovered protein between the 402H and 402Y forms
may be indicative of more 402Y FHL-1 binding to Bruch’s
membrane in the Ussing chamber as it passes through compared
with the 402H form.
Small patches of FH were observed on the RPE-facing side of
Bruch’s membrane (Fig. 1D), which are likely to originate from
RPE cells. Local synthesis would also explain the distinctive FH
labeling on the surface of drusen, which may be unable to penetrate into them, whereas the smaller FHL-1 can penetrate into the
drusen (Fig. 1E). Interestingly, the FH staining around drusen is
removed by heparinase treatment (Fig. 5), suggesting that the
protein is retained on the surface of drusen by binding HS chains.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Donors without known eye disease
M17000
M
M17055
F
M17119
F
M17126
M
M17130
M
M17143
M
Dual FHL-1/FH staining of drusen
M17142
F
M12106
M
M12109
F
M14557
M
Ussing chamber experiments
Whole serum
M20053
M
M14479
F
M14507
F
Recombinant FHL-1 402H and 402Y variants
M20854
F
M20860
M
M20866
M
FH
M20879
M
M20882
F
M20884
M
Heparin is, however, a convenient highly sulfated model of the
HS that is found in ECM. HS contains large regions with low
sulfated disaccharides and overall has much less sulfation than
heparin (Fig. 4D) (18). The experiments with selectively desulfated heparin demonstrated that the disease-associated 402H variant of FHL-1 relies heavily on highly sulfated GAG sequences
and therefore is likely to bind less well to HS than heparin. To
confirm this, solid-phase binding experiments with two sources of
HS (porcine mucosa and bovine kidney) were performed and these
confirmed that the 402H form of FHL-1 binds significantly less
well than does the 402Y form to HS (Fig. 4E, 4F). Conversely, the
402H form of FHL-1 bound better than did the 402Y form to
highly sulfated heparin (Supplemental Fig. 2), which agrees with
the influence of sulfation availability on 402H binding, but also
highlights the unsuitability of heparin as a physiological model for
FHL-1/HS interactions.
The removal of HS from macula tissue sections by pretreatment
with a heparinase I/II/III mix pretreatment reduced the signal seen for
endogenous FHL-1 (Fig. 5B), indicating that HS is indeed one of the
main ligands anchoring FHL-1 to the Bruch’s membrane. Interestingly, enzymatic treatment of drusen did not appear to alter detectable levels of FHL-1 within the druse, but did remove the distinctive
staining of FH around the edges of the lesions (Fig. 5C, 5D).
The Journal of Immunology
4967
The lack of heparinase effect on FHL-1 staining within drusen
suggests that the protein is either interacting with a currently
unidentified ligand, or it is trapped among the plethora of components that make up these hallmark lesions of AMD. Similarly, it
may be possible that the heparinase itself is not able to penetrate
the tight matrix of the druse. Whether FHL-1 retains activity in
this environment is unclear.
We show that the FHL-1 within Bruch’s membrane is immobilized there largely through interactions with HS. In other recent
work we have shown that there is a marked decrease in the levels
of HS GAGs in Bruch’s membrane with age (47). This decrease in
HS levels in Bruch’s membrane coupled with the poorer binding
of the 402H form of FHL-1 could explain why AMD is a disease
of aging. Individuals with the 402H polymorphism may, as they
age, be unable to localize sufficient FHL-1 to Bruch’s membrane
so that its protective effects are lost and the complement cascade is
activated with damaging consequences that ultimately lead to
AMD. Furthermore, the dominant role of FHL-1 in complement
regulation at this site provides, to our knowledge for the first time,
an explanation of why the GAG-binding region in CCP7 was
found to be vital for protein localization in the eye (23), whereas
CCPs 19–20 of FH are more important in the kidney (24, 33, 48).
The absence of full-length FH in Bruch’s membrane (and thus
the CCPs 19–20) may have resulted in evolutionary pressure to
express HS species capable of recruiting FHL-1 via its CCP7
domain.
Although our work suggests that FHL-1 is a major regulator of
complement in Bruch’s membrane, there is genetic evidence that
FH is also important. A highly penetrant mutation R1210C in the
C terminus of FH is a strong risk factor for AMD and is thought to
coincide with a 6-y earlier onset of the disease (9). The R1210C
form of FH is exclusively found covalently bound to albumin in
plasma (49), which would affect the proteins mobility, and although albumin binding has no effect on GAG binding, it does
alter FH binding to C3b (49, 50). As such, it is likely this mutation
hampers FH tissue penetration and surface protection. Furthermore, most AMD patients with the R1210C haplotype have the
FH/FHL-1 402H polymorphism on the other allele (9). It may be
the case that any contribution conferred by FH to immune regulation in the macula is perturbed by the R1210C mutation and
amplifies an already imbalanced immune homeostasis (conferred
by linked genetic factors such as the Y402H polymorphism).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. FHL-1 is able to diffuse through Bruch’s membrane from human serum. An Ussing chamber was used to compare the diffusion of FH and
FHL-1 from human serum across enriched Bruch’s membrane from human donor eyes. (A) Schematic of the Ussing chamber layout where (a) the Bruch’s
membrane, (b) sampling access points, and (c) magnetic stirrer bars are shown. (B) Fluorescent Western blot of one representative experiment from a total
of three showing 0 and 24 h samples from the PBS compartment: the left-hand lane shows a positive control sample containing 1 mg each of FH and FHL-1.
Red bands are recognized by OX23 alone, green by anti–FHL-1 alone, and yellow by both Abs. The Odyssey protein markers used are visualized in the red
channel. The Western blot shown is representative of three separate experiments. (C) Purified FH was placed in one compartment and after 24 h the entire
protein content of the other “PBS” compartment was concentrated, subjected to SDS-PAGE, and the resultant gel was stained with Coomassie blue. (D and
E) Diffusion experiments with purified recombinant FHL-1 proteins examined potential differences in the ability of the 402H and 402Y variants to cross
Bruch’s membrane. Both 402H and 402Y forms were tested separately using three donor Bruch’s membranes, and data are shown as percentage protein
detected in each chamber after 24 h at room temperature: the 50% mark is shown as a dashed line. The donor tissues used in (C)–(E) are listed in Table I.
4968
FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE
Our findings also have implications for understanding how the
five FHR proteins contribute to immune homeostasis in the eye.
Variations in the genes encoding the FHR proteins are associated
with alterations in AMD risk (41, 42), and the FHR proteins can
compete with FH binding to C3b and/or GAGs, and even to form
a novel C3 convertase (51). As such, they are fast being considered
FIGURE 5. Heparinase pretreatment alters
the pattern of FHL-1 and FH staining in the
macula. Both FHL-1 and FH localization
were visualized in eye tissue before and after
enzymatic pretreatment with a heparinase I/
II/III mix using the six normal donor eyes and
four drusen containing AMD (eyes as listed
in Table I). In each case green staining represents FHL-1 and red staining FH. (A) Distribution of FHL-1 and FH before removal of
HS, and (B) after enzymatic treatment in
macular tissue without AMD pathology.
FHL-1 and FH labeling of a druse without
(C) and with heparinase treatment (D).
Blue staining represents DAPI staining of
cell nuclei. Scale bars, 10 mm.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. The 402H form of FHL-1
shows greater dependency on GAG sulfation for binding. Heparin is a highly sulfated
model of HS. (A) Schematic showing the
basic iduronic acid–glucosamine backbone
disaccharide of heparin where all four possible sulfation positions are listed as follows: R1, 6-O sulfation; R2, N sulfation; R3,
2-O sulfation; and R4, 3-O sulfation. (B and
C) Plate assays demonstrating the binding
activities of FHL-1 402Y and 402H forms
for selectively desulfated heparin. (D) Schematic diagram demonstrating the different
disaccharide regions of an HS chain. GlcNAc,
N-acetylated glucosamine; GlcNS, N-sulfated glucosamine; GlcUA, glucuronic acid;
IdoUA, iduronic acid; 2S, sulfation in the
2-O position; 6S, sulfation in the 6-O position. (E and F) Plates assays demonstrating
the AMD-associated 402H form of FHL-1
binding relatively poorly to two forms of HS
compared with the 402Y form. Data in (B),
(C), (E), and (F) are n = 6, averaged from
two independent experiments 6 SEM.
The Journal of Immunology
4969
21.
Acknowledgments
22.
We thank Prof. Paul Barlow (University of Edinburgh, Edinburgh, U.K.) and
Prof. Anthony Day (University of Manchester, Manchester, U.K.) for advice, support, and direction during the early stages of this work. Also,
we thank Dr. Isaac Zambrano (Manchester Eye Bank, Manchester Royal
Eye Hospital, Manchester, U.K.) for assistance with obtaining the donor
eye tissue used in this study.
23.
12.
13.
14.
15.
16.
17.
18.
19.
20.
24.
Disclosures
The authors have no financial conflicts of interest.
25.
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FHL-1 REGULATES COMPLEMENT ON BRUCH’S MEMBRANE