Download Distinct and Separable Roles of the Complement System in Factor H

Distinct and Separable Roles of the Complement System in
Factor H–Deficient Bone Marrow Chimeric Mice with
Immune Complex Disease
Jessy J. Alexander,* O.G.B. Aneziokoro,* Anthony Chang,† Bradley K. Hack,*
Adam Markaryan,* Alexander Jacob,* Roger Luo,‡ Michael Thirman,‡ Mark Haas,§ and
Richard J. Quigg*
*Sections of Nephrology and ‡Hematology-Oncology, Department of Medicine, and †Department of Pathology, The
University of Chicago, Chicago, Illinois; and the §Department of Pathology, Johns Hopkins University School of
Medicine, Baltimore, Maryland
Plasma complement factor H (Cfh) is a potent complement regulator, whereas Cfh on the surface of rodent platelets is
responsible for immune complex processing. For dissection between the two, bone marrow chimeras between Cfh-deficient
(Cfh⫺/⫺) and wild-type C57BL/6 mice were created. Platelet Cfh protein was tracked with the Cfh status of the bone marrow
donor, indicating that platelet Cfh is of intrinsic origin. In an active model of immune complex disease, Cfh⫺/⫺ mice that were
reconstituted with wild-type bone marrow had levels of platelet-associated immune complexes comparable to those of
wild-type mice and were protected against the excessive glomerular deposition of immune complexes seen in Cfh⫺/⫺ mice, yet
these mice still developed glomerular inflammation. In contrast, wild-type mice with Cfh⫺/⫺ bone marrow had reduced
platelet-associated immune complexes and extensive glomerular deposition of complement-activating immune complexes, but
they did not develop glomerular pathology. The large quantities of glomerular C3 in wild-type mice with Cfh⫺/⫺ bone marrow
were in the form of iC3b and C3dg, whereas active C3b remained in Cfh⫺/⫺ recipients of wild-type bone marrow. These data
show that plasma Cfh limits complement activation in the circulation and other accessible sites such as the glomerulus,
whereas platelet Cfh is responsible for immune complex processing.
J Am Soc Nephrol 17: 1354 –1361, 2006. doi: 10.1681/ASN.2006020138
T
he complement system of proteins identifies pathogens
and apoptotic material on which a directed activation
can ensue to result in their efficient disposal (1). Besides
this important contribution to innate immunity, the complement system is involved in adaptive immunity, such as by
providing cues for a directed immune response by B and T cells
(2). In addition, complement is crucial for the proper disposal of
immune complexes. This multistep process requires complement activation by immune complex Ig that leads to immune
complex incorporation of C3b and binding to CR1 on primate
erythrocytes, which shuttles and transfers them to CR3 and Fc
receptor– bearing cells of the mononuclear phagocyte system
(3). Although it is clear that primates use the erythrocyte and its
CR1 protein for this function, other species, such as the mouse,
rely on platelets to process immune complexes (4). We previously provided evidence that the platelet protein that is responsible for binding C3b in these complexes is complement factor
H (Cfh) (5).
Received February 12, 2006. Accepted March 4, 2006.
Published online ahead of print. Publication date available at www.jasn.org.
Address correspondence to: Dr. Richard Quigg, Section of Nephrology, The
University of Chicago, AMB S-508, MC 5100, 5841 S. Maryland Avenue, Chicago,
IL 60637. Phone: 773-702-0757; Fax: 773-702-4816; E-mail: [email protected].
uchicago.edu
Copyright © 2006 by the American Society of Nephrology
Like CR1, Cfh is a member of the regulators of complement
activation gene family (6,7). All family members have short
consensus repeats (SCR) that are arranged in tandem and that
each contain approximately 60 amino acids with a conserved
core structure, including four cysteines that form two intra-SCR
disulfide bonds. Both CR1 and Cfh have affinity for C3b and
can serve as co-factor for the complement factor I (Cfi)-mediated cleavage of C3b to iC3b, which allows transfer of immune
complexes to CR3-bearing cells. However, unlike CR1, which is
a type I membrane protein with a specific cellular distribution,
Cfh exists in the plasma as a soluble protein. Cfh does have the
capacity to bind to cells (and other noncellular sites) with
specificity, a phenomenon that can lead to protection from
complement activation on normal cells, as well as malignant
cells and adapted microorganisms (8,9). The inability of Cfh to
bind to host tissue sites, particularly because of mutations that
affect the final SCR, also may underlie the pathogenesis of
atypical hemolytic uremic syndrome (10).
Furthermore, age-related macular degeneration was associated recently with the Tyr402His polymorphism in the seventh
SCR, a site for binding C-reactive protein and heparin (11).
There also is evidence that cells can produce Cfh and have it
remain plasma membrane associated (12,13).
Given these various functions of Cfh, along with our uncertainties of its origin on platelets, here we studied cultured
ISSN: 1046-6673/1705-1354
J Am Soc Nephrol 17: 1354 –1361, 2006
Distinct Roles of Platelet and Plasma Factor H
platelets as well as those that originated in animals with adoptive bone marrow transfers. In the latter, we also investigated
an active model of immune complex disease in which immune
complex processing and complement activation are key.
Materials and Methods
Animals
Normal wild-type C57BL/6 mice were purchased from Jackson Laboratories. Cfh⫺/⫺ mice (14) were backcrossed ⬎10 generations onto the
C57BL/6 strain.
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each was reactive with the 110-kD C3b ␣’ chain, attributable to nonoverlapping reactivities of anti-C3 with ␣’67 and anti-C3d with ␣’43.
Platelet Culture
Published protocols were used with minor modifications (20,21).
CD-117–positive progenitor cells were isolated and cultured in Iscove’s
Modified Dulbecco’s Medium supplemented with 1% Nutridoma
(Roche Applied Science) and 3 ng/ml murine thrombopoietin (Cell
Sciences, Canton, MA) for 3 d. Megakaryocytes were isolated on a
discontinuous BSA density gradient and cultured again in the same
medium.
Bone Marrow Chimeric Mice
Wild-type or Cfh⫺/⫺ C57BL/6 mice at 4 to 6 wk of age received 1050
cGy (irradiation). Bone marrow cells were isolated by standard techniques from femurs of wild-type or Cfh⫺/⫺ C57BL/6 mice, and CD-117
(c-kit)-positive progenitor cells were isolated from bone marrow cells
using a mAb magnetic positive selection technique (Miltenyi CD117
MicroBeads, Auburn, CA). One day after irradiation, mice received 1 ⫻
106 CD-117–positive progenitor cells intravenously. In pilot studies,
this protocol rescued animals from lethality and led to full hematopoietic reconstitution within 3 wk.
Serum Sickness
Immune complex disease was induced by actively immunizing chimeric mice 6 wk after bone marrow transfer with a daily intraperitoneal
dose of 4 mg of horse spleen apoferritin (Calzyme Laboratories, San
Luis Obispo, CA) (15–18). Controls included wild-type mice into which
wild-type bone marrow was transferred and wild-type and Cfh⫺/⫺ mice
that were not subjected to bone marrow transfer yet were immunized
with the same schedule of apoferritin or saline vehicle alone. After 5
wk, mice were killed and renal disease was characterized.
Flow Cytometry
Platelets were isolated ex vivo (5,19) or from culture supernatants and
stained either directly with FITC-goat anti-mouse IgG (Cappel) or
indirectly with sheep anti-rat factor H or goat anti-mouse thrombocyte
antibodies (Accurate Scientific, Westbury, NY) followed by FITC-conjugated antibodies to sheep or goat IgG (Cappel) (5). Samples were
analyzed using the Becton Dickinson FACSCalibur and CellQuest software (BD Biosciences, Bedford, MA).
Immunofluorescence Microscopy
Four-micrometer sections from frozen mouse kidneys were fixed in
ethanol:ether (1:1) for 10 min followed by 95% ethanol for 20 min and
washed with PBS and stained with FITC-conjugated goat antibodies to
mouse IgG or C3 (Cappel). Slides were viewed with an Olympus BX-60
IF microscope (Melville, NY) and scored in a masked manner from 0 to
4. Representative photomicrographs were taken at identical settings.
Renal Pathology
Western Blots
Glomeruli were isolated from 4-␮m frozen sections by laser capture
microdissection using a Leica AS LMD microdissection microscope. A
total of 450 glomerular sections in 150 mM sodium chloride, 1% NP-40,
50 mM Tris (pH 8.0), and protease inhibitor cocktail (Roche Applied
Science, Indianapolis, IN) were separated by SDS-PAGE (10%), transferred onto polyvinylidene difluoride membranes (Millipore, Bedford,
MA), and incubated with horseradish peroxidase–anti-mouse C3 (Cappel, MP Biomedicals, Irvine, CA) or rabbit anti-human C3d (Dako,
Carpinteria, CA) followed by horseradish peroxidase–anti-rabbit IgG
(Sigma-Aldrich, St. Louis, MO). Bound antibodies were detected with
enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL).
As controls for mouse C3 cleavage products, C3 was purified from
normal mouse plasma using previously described techniques (19) and
incubated in trypsin (1% wt/wt) at 37°C for various times, followed by
neutralization with soybean trypsin inhibitor. The resultant C3b and
further cleavage fragments were subjected to Western blotting with the
anti-C3 and anti-C3d antibodies that were used for glomerular immunoblotting. As in our previous studies with these same antibodies (19),
Tissues were fixed in 10% buffered formalin and embedded in paraffin, from which 4-␮m-thick sections were cut and stained with periodic acid-Schiff. Each slide was scored in a blinded manner by a renal
pathologist (M.H.) for the extent of glomerulonephritis (GN) on a scale
of 0 to 4 (in increments of 0.5) according to the schema of Passwell et al.
(22) as described previously (23). In addition, the fraction of glomeruli
with segmental sclerosis and/or hyalinosis was determined; no crescents were seen in any of the specimens.
Statistical Analyses
Numerical data are presented in the text or Table 1 as medians with
ranges in parentheses. Graphical data are presented from individual
mice with different experiments depicted with different symbols or as
means ⫾ SEM. Statistical significance between groups was determined
by Mann-Whitney testing (Minitab v.12) or by ANOVA followed by
Fisher pairwise tests. Potential correlation among variables was determined by calculating the Pearson product moment correlation coefficient.
Table 1. Immunologic features in blood of bone marrow chimeric mice with serum sicknessa
a
BM 3 Host
Anti-Apoferritin IgG (U)
Circulating Immune Complexes (U)
Platelet Cfh (MCF)
Cfh⫺/⫺ 3 wt
wt 3 Cfh⫺/⫺
0.80 (0.72 to 0.99)
1.19 (0.67 to 1.41)
0.35 (0.32 to 0.67)
0.50 (0.31 to 1.20)
204 (16 to 794)
3934 (220 to 4829)b
Data are medians (ranges); n ⫽ 5 per group. Cfh, complement factor H; MCF, mean channel fluorescence.
P ⫽ 0.02 versus Cfh⫺/⫺ 3 wt.
b
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Journal of the American Society of Nephrology
J Am Soc Nephrol 17: 1354 –1361, 2006
Results
Platelet Cfh Is of Intrinsic Origin
Although Cfh is an abundant plasma protein, our previous
studies demonstrated that it was specifically expressed on the
platelet and not other blood cells, such as the erythrocyte,
despite their continuous exposure to plasma proteins (5). By
analogy to erythrocyte CR1, we considered it possible that
platelets are endowed with Cfh upon exit from the bone marrow. To investigate this, we cultured megakaryocytes from
normal C57BL/6 mice. These had the expected arborizing processes that released into the culture supernatant a continuous
supply of platelets with features that were comparable to those
of blood platelets (20,21). Megakaryocytes and platelets had
mRNA for Cfh (Figure 1A), and cultured platelets bore Cfh
protein on their surface (Figure 1B). These results provide proof
that platelets and their precursors have the intrinsic capacity to
produce Cfh as a plasma membrane protein.
To investigate this in vivo, we performed adoptive bone
marrow transfers between mice with targeted deficiency of Cfh
(14) (ⱖN10 C57BL/6) and wild-type C57BL/6 mice. For elimination of any contribution from native bone marrow cells, mice
were lethally irradiated 1 d before transfer of bone marrow
cells. Platelets from Cfh⫺/⫺ recipients of wild-type bone marrow
had quantities of Cfh that were comparable to those from
unmanipulated mice, whereas platelets in wild-type recipients
of Cfh⫺/⫺ bone marrow had a marked reduction in Cfh (Figure
2). These results are consistent with data from cultured platelets
and indicate that platelet-bound Cfh is largely of intrinsic (i.e.,
megakaryocyte) origin. Because in these studies Cfh-deficient
platelets were bathed in a Cfh-rich plasma, it is likely that what
platelet-associated Cfh was identified in these studies could be
attributable to its absorption from plasma onto the platelet
surface.
Platelet Cfh Binds Immune Complexes In Vivo
To investigate the role of platelet Cfh in immune complex
metabolism, we studied chronic serum sickness in bone mar-
Figure 1. Complement factor H (Cfh) is present in cultured
platelets. (A) Reverse transcription–PCR for Cfh and ␤-actin
mRNA was performed on blood platelets isolated ex vivo and
on cultured megakaryocytes and platelets using primers and
conditions as described previously (5). (B) Flow cytometry was
performed on cultured platelets using antibodies to Cfh and
mouse platelets or nonimmune IgG as control.
Figure 2. Platelet-associated Cfh in Cfh⫺/⫺ bone marrow chimeric mice as determined by flow cytometry. Platelet-associated Cfh was restored in Cfh⫺/⫺ mice by transfer of wild-type
bone marrow, whereas platelets from wild-type mice with
Cfh⫺/⫺ bone marrow had markedly reduced Cfh. Median mean
channel fluorescence (MCF) values were 1182 and 35, respectively.
row chimeric mice. Mice immunized with apoferritin generated
an appropriate anti-apoferritin IgG immune response. After 5
wk of daily immunization with apoferritin, there were no differences in antibody titers between wild-type recipients of
Cfh⫺/⫺ bone marrow and Cfh⫺/⫺ recipients of wild-type bone
marrow (Table 1). Similar to animals without serum sickness,
platelets from wild-type recipients of Cfh⫺/⫺ bone marrow had
markedly decreased platelet-associated Cfh, whereas the transfer of wild-type bone marrow to Cfh⫺/⫺ hosts was capable of
reconstituting platelet Cfh (Table 1).
As a measure of immune complexes bound to platelets, the
quantities of IgG that were associated with platelets were determined. To explore this in more detail, we also examined
platelets from wild-type and Cfh⫺/⫺ mice (i.e., without bone
marrow transplant) that were immunized with apoferritin or
saline as controls. As shown in Figure 3, low levels of plateletassociated IgG were found in both wild-type and Cfh⫺/⫺ mice
that were immunized with saline. In wild-type mice that were
immunized with apoferritin, there was a significant increase in
platelet-associated IgG, which was not seen in Cfh⫺/⫺ mice. As
with platelet-associated Cfh, IgG on platelets was reduced in
wild-type recipients of Cfh⫺/⫺ bone marrow, whereas Cfh⫺/⫺
recipients of wild-type bone marrow had amounts of plateletassociated IgG that were comparable to apoferritin-immunized
wild-type animals. These data are consistent with the hypothesis that intrinsically produced platelet Cfh binds immune complexes. Further support for this is the significant correlation
between platelet-associated Cfh and IgG levels (r ⫽ 0.68, P ⫽
0.03). There was not total elimination of IgG from Cfh-deficient
platelets could reflect that acquired from plasma was some
platelet Cfh that was capable of binding immune complexes,
J Am Soc Nephrol 17: 1354 –1361, 2006
Figure 3. Platelet-associated IgG in mice with serum sickness.
Platelets from wild-type and Cfh⫺/⫺ mice and bone marrow
chimeric mice that had been immunized with apoferritin to
induce chronic serum sickness were studied, as were control
mice that were immunized with saline. Platelet-associated IgG
was increased significantly in wild-type mice and Cfh⫺/⫺ mice
with wild-type bone marrow in which serum sickness was
induced (*P ⬍ 0.01 versus all other groups). The number of mice
studied in each group is given in the bars.
Distinct Roles of Platelet and Plasma Factor H
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apoferritin immunization and examining glomeruli for immune reactants. Cfh⫺/⫺ mice with wild-type bone marrow had
moderate glomerular deposition of IgG (Figure 4B) and C3
(Figure 4F), primarily in the mesangium, findings that are
comparable to that seen in wild-type mice (but not Cfh⫺/⫺ mice)
with serum sickness (18). Similar findings were seen in control
studies in which serum sickness was induced in wild-type mice
that had received wild-type bone marrow (Figure 4, C and G),
thereby excluding an effect of the bone marrow transfer procedure itself on immune complex processing and complement
activation in glomeruli of C57BL/6 mice. In contrast, wild-type
mice with Cfh⫺/⫺ bone marrow had significantly greater deposition of IgG (Figure 4, A and D) and C3 (Figure 4, E and H)
in glomeruli, with these being distributed throughout the glomerular capillary wall, a pattern that otherwise was observed
only in Cfh⫺/⫺ mice with serum sickness and also in the case of
IgG and C3 in unmanipulated Cfh⫺/⫺ mice as a spontaneous
manifestation (14,18). These data indicate that the platelet Cfh
limits glomerular accumulation of immune complexes in situations in which they are formed in excess, such as in serum
sickness. Furthermore, it seems that the presence of immune
complexes in glomeruli and not the deficiency of fluid-phase
Cfh is responsible for the activation of complement in glomeruli in these settings; presumably, this is initiated through the
classical pathway that is not appreciably affected by Cfh.
and/or binding occurred in a Cfh-independent manner, such as
to Fc receptor on the platelet surface (24).
Platelet Cfh Limits Deposition of Immune Complexes, whereas
Plasma Cfh Restricts Complement Activation in Glomeruli
Glomerular manifestations of serum sickness in the chimeric
mice were investigated next by killing of mice after 5 wk of
Glomerular Inflammation Occurs When C3 Is Not
Inactivated by Cfh
Despite the presence of abundant IgG-containing immune
complexes and complement activation products, glomeruli of
wild-type mice with Cfh⫺/⫺ bone marrow were completely
Figure 4. Immunohistologic manifestations in glomeruli from Cfh⫺/⫺ bone marrow chimeric mice with serum sickness. Shown are
representative photomicrographs for glomerular IgG (A through C) and C3 (E through G) staining in wild-type recipients of
Cfh⫺/⫺ bone marrow (A and E), Cfh⫺/⫺ recipients of wild-type bone marrow (B and F), and wild-type recipients of wild-type bone
marrow (C and G). The staining intensity scores for IgG (D) and C3 (H) from individual mice are shown.
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Journal of the American Society of Nephrology
normal when examined histopathologically (Figure 5A). In contrast and despite having markedly less IgG and C3 evident in
glomeruli, every Cfh⫺/⫺ mouse with wild-type bone marrow
developed significant GN, characterized by increased cellularity as well as the accumulation of matrix material (Figure 5B
and quantified in all animals in Figure 5, D and E). This is a
pattern with similarities to the membranoproliferative GN
(MPGN) that can occur spontaneously in Cfh⫺/⫺ mice on
129/Sv ⫻ C57BL/6 mixed backgrounds as well as that actively
produced by serum sickness in C57BL/6 Cfh⫺/⫺ mice (14,18).
As in native wild-type mice, serum sickness in wild-type mice
that had received wild-type bone marrow failed to induce
histologic glomerular disease (Figure 5C), thereby excluding an
effect of the bone marrow transfer procedure itself on this
disease in C57BL/6 mice. Therefore, deficiency of fluid-phase
Cfh seems to be the key aspect accounting for the susceptibility
of Cfh⫺/⫺ mice to develop glomerular histopathology. In contrast, deficiency of platelet Cfh, with the attendant marked
deposition of IgG and complement activation products in glomeruli, is not sufficient for GN to develop.
As shown above, the presence of C3 tracked closely with the
extent and distribution of IgG in glomeruli. Because it was
surprising that the Cfh⫺/⫺ mice with wild-type bone marrow
had much less C3 deposition in glomeruli but developed GN,
we examined the form of C3 in glomeruli by Western blotting.
J Am Soc Nephrol 17: 1354 –1361, 2006
To examine specifically glomerular C3 proteins (rather than
renal cortex with its significant plasma contribution), we isolated glomeruli by laser capture microdissection and subjected
them to immunoblotting with anti-C3 antibodies. Consistent
with the immunohistological data, wild-type recipients of
Cfh⫺/⫺ bone marrow had significantly greater amounts of C3
activation products in the kidney compared with Cfh⫺/⫺ recipients of wild-type bone marrow (Figure 6A). However, despite
the marked increase in C3 quantity in the former group, there
also was evidence of efficient cleavage of the C3b 110-kD ␣’chain to iC3b (containing ␣’67, ␣’43, and ␣’40 chains) and C3dg
(Figure 6, B and C), which presumably occurred because the Cfi
co-factor function of plasma Cfh was intact in these animals. In
contrast, Cfh⫺/⫺ recipients of wild-type bone marrow had intact
␣’110-chain, as shown with antibodies to C3 (Figure 6A) and
C3d (Figure 6B), and therefore seemed to have impaired inactivation of C3b to iC3b as a result of the deficiency of plasma
Cfh.
Discussion
Besides its physiologic roles, activation of the complement
system can contribute to pathology. In the case of a number of
immunologic diseases that affect the renal glomerulus, immune
complexes and complement activation products are found in
affected glomeruli, supporting a pathogenic role for immune
Figure 5. Glomerular pathologic features in Cfh⫺/⫺ bone marrow chimeric mice with serum sickness. Representative photomicrographs are shown from a wild-type recipient of Cfh⫺/⫺ bone marrow (A), a Cfh⫺/⫺ recipient of wild-type bone marrow (B), and
a wild-type recipient of wild-type bone marrow (C). Scores for the extent of glomerulonephritis (D) and glomerular sclerosis/
hyalinosis (E) from individual animals are shown graphically. The arrow in B points to an area of segmental hyalinosis in a
glomerulus from a Cfh⫺/⫺ mouse with wild-type bone marrow.
J Am Soc Nephrol 17: 1354 –1361, 2006
Figure 6. Glomerular C3 in Cfh⫺/⫺ bone marrow chimeric mice
with serum sickness. Isolated glomeruli were subjected to
Western blotting with anti-C3 (A) and anti-C3d (B) antibodies,
which have nonoverlapping reactivities (C). Consistent with
immunohistologic data (cf., Figure 4E), there was a marked
increase in glomerular C3 in wild-type mice with Cfh⫺/⫺ bone
marrow, which was in the form of iC3b and C3dg. In contrast,
Cfh⫺/⫺ mice with wild-type bone marrow had markedly less C3
but a relatively high quantity of the 110-kD ␣’ chain, indicating
the persistence of intact C3b. All samples were run on the same
blot. That Cfh-independent cleavage of C3b into iC3b1, iC3b2,
and C3dg could occur also is shown by Western blotting of
glomeruli from globally Cfh-deficient mice with serum sickness
(A, right).
complex– directed complement activation (25). Further evidence for the role of complement activation in glomerular
diseases also has been provided by years of study in rodent
models of disease (26,27). For example, in glomeruli of Cfh⫺/⫺
mice on a mixed 129/Sv ⫻ C57BL/6 background, there is
unrestricted alternative complement activation and accumulation of immune complexes (14). At a relatively advanced age,
these animals can spontaneously develop glomerular disease
that has pathologic similarities to human MPGN.
Serum sickness and the resultant GN have been studied in
various animal species, using a variety of immunization protocols. Rats of several strains have been used to generate serum
sickness nephropathy (28 –34). The resultant glomerular lesions
seem to depend in large part on the amount of free antigen
administered and range from deposition of immune deposits in
the mesangium without cellular proliferation to a severe exudative GN (28,29,31). Depletion of complement at the end of the
disease protocol with cobra venom factor or selective inhibition
of C5 reduces proteinuria in this model of GN (33,34). The latter
finding, combined with the observation by electron microscopic
immunohistochemistry that podocyte membranes stain intensely for C5b-9 (35), implicate C5b-9 –mediated podocyte injury as a cause of proteinuria in this model (36).
A mouse model of chronic serum sickness induced by repetitive immunization with horse spleen ferritin was developed by
Stilmant et al. (15) in 1975; Swiss-albino mice developed proliferative GN with intrinsic glomerular cell proliferation and neutrophil infiltration. Subsequent studies in this model using
iron-free apoferritin revealed similarities to the rat, including a
strain and dose dependence (16,37,38). The presence of C3 in
glomeruli suggested that complement was involved in the
pathogenesis of GN in this model (15,37). This later was proved
Distinct Roles of Platelet and Plasma Factor H
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by Falk and Jennette in their studies that compared the C5deficient B.10.D2.OSN strain with congenic C5-sufficient
B.10.D2.NSN mice (17). GN was reduced significantly in C5deficient mice; however, there still was detectable disease in
some of the C5-deficient B.10.D2.OSN mice in association with
glomerular deposition of immune complexes and C3. These
results indicated that GN is mediated in part by C5a recruitment and activation of inflammatory cells and/or by C5b-9 –
mediated glomerular cell injury with resultant proliferative
events (e.g., in the anti-thymocyte mesangial proliferative GN
model in rats [39]). Because disease was not eliminated completely in C5-deficient mice, proximal complement components, such as the C3a and C3b cleavage products of C3, or
noncomplement mediators of disease also must be playing a
role in this model of GN (17). The finding of C5-independent
yet C3-dependent GN has been observed in another murine
model of immune complex GN, in which mice were immunized
with cationized BSA (40).
It is important to note that active immunization of experimental animals with cationized antigens, as originally described by Border et al. (41) and also applied to the apoferritin
model by Iskandar et al. (42), is considered to result in their
being “planted” in the negatively charged glomerular capillary
wall, leading to in situ formation of immune complexes (43– 45).
In contrast, chronic immunization with the highly anionic unmodified apoferritin (46) results in the formation of circulating
immune complexes that subsequently deposit in glomeruli, as
we have shown in this and previous studies using this model
(18,47). In either circumstance, complement depletion or its
absence leads to decreased glomerular clearance of immune
complexes (34,44,47– 49), highlighting the relevance and complexities of the complement system in immune complex processing in the glomerulus (3,45).
Unlike some strains, C57BL/6 mice are resistant to developing glomerular inflammation in apoferritin-induced chronic
serum sickness (16 –18). This is despite the development of a
strong humoral immune response to the immunogen and the
deposition of immune complexes and complement activation
products in glomeruli. However, in our previous studies, all
C57BL/6 Cfh⫺/⫺ mice with serum sickness did develop GN
(18). Thus, Cfh deficiency converts the C57BL/6 strain from one
that is resistant to GN into one that is susceptible to GN.
Arguably, the most important function for Cfh is its ability to
limit complement activation in plasma and in sites that are not
served by other complement regulators, such as the glomerular
capillary wall and choroidal capillaries (10). However, it is a
versatile protein with a number of other functions, such as
serving as the platelet protein that is responsible for immune
complex processing in rodents (5). As such, it could not be
stated for certainty which of these predominated in the spontaneous glomerular disease that occurred in mixed background
Cfh⫺/⫺ mice or in the disease that was induced by serum
sickness in C57BL/6 Cfh⫺/⫺ mice.
The data presented in this study suggest that in a disease
such as serum sickness, immune complex deposition and complement activation occur in glomeruli but the C3b (generated
through the classical pathway but with the likely contribution
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of alternative pathway amplification) is efficiently inactivated
to iC3b when Cfh is present in plasma to serve as Cfi co-factor
(50). Cfh is an intrinsically derived platelet protein in mice that
is responsible for the processing of immune complexes in a
manner that is analogous to erythrocyte CR1 in primates.
Therefore, when absent, excessive immune complex accumulation occurs over time in glomeruli, which is accelerated in a
condition such as serum sickness. However, in these circumstances, the presence of plasma Cfh remains sufficient to limit
the proinflammatory effects of complement activation. In the
absence of Cfh in the plasma and glomerular capillaries, alternative pathway complement activation in glomeruli occurs
spontaneously over time in Cfh⫺/⫺ mice (14) and is accelerated
through the classical pathway in serum sickness (18), such as
also occurs in the prototypical immune complex disease systemic lupus erythematosus (51,52). This unrestricted alternative
complement pathway activation converts the normally resistant C57BL/6 strain into one that is susceptible to glomerular
disease, through the proinflammatory actions of C3b and other
downstream products of complement activation, such as C3a,
C5a, and C5b-9 (25). These data contribute to our growing
appreciation for the potency of alternative complement pathway activation (53) and the importance of Cfh as its physiologic
regulator.
Acknowledgments
This work was supported by National Institutes of Health grant
R01DK41873 and by a grant to J.J.A. from Kidneeds.
We thank Drs. Marina Botto and Matthew Pickering for supplying
the Cfh⫺/⫺ mice and for helpful advice with the studies and manuscript.
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