Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo.

Abstract

New therapies that target chronic inflammation with fibrosis are urgently required. Increasing evidence points to innate activation of inflammatory cells in driving chronic organ fibrosis. Serum amyloid P is a naturally circulating soluble pattern recognition receptor, a member of the family of pentraxin proteins. It links danger-associated molecular pattern recognition to Fc gamma receptor-mediated phagocytosis. Here we show that fibrosis progression in the mouse kidney is significantly inhibited by therapeutic administration of human serum amyloid P, regulated by activating Fc gamma receptors, and dependent on inflammatory monocytes and macrophages, but not fibrocytes. Human serum amyloid P-mediated inhibition of mouse kidney fibrosis correlated with specific binding of human serum amyloid P to cell debris and with subsequent suppression of inflammatory monocytes and kidney macrophages in vitro and in vivo, and was dependent on regulated binding to activating Fc gamma receptors and interleukin-10 expression. These studies uncover previously unidentified roles for Fc gamma receptors in sterile inflammation and highlight serum amyloid P as a potential antifibrotic therapy through local generation of interleukin-10.

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FIBROSIS
Serum Amyloid P Inhibits Fibrosis Through
FcgR-Dependent Monocyte-Macrophage
Regulation in Vivo
Ana P. Castaño,1,2* Shuei-Liong Lin,1,2* Teresa Surowy,3Brian T. Nowlin,1Swathi A. Turlapati,1
Tejas Patel,2Ajay Singh,2Shawn Li,3Mark L. Lupher Jr.,3Jeremy S. Duffield1,2†
(Published 4 November 2009; Volume 1 Issue 5 5ra13)
New therapies that target chronic inflammation with fibrosis are urgently required. Increasing evidence points
to innate activation of inflammatory cells in driving chronic organ fibrosis. Serum amyloid P is a naturally circulat-
ing soluble pattern recognition receptor, a member of the family of pentraxin proteins. It links danger-associated
molecular pattern recognition to Fcg receptor–mediated phagocytosis. Here we show that fibrosis progression in
the mouse kidney is significantly inhibited by therapeutic administration of human serum amyloid P, regulated by
activating Fcg receptors, and dependent on inflammatory monocytes and macrophages, but not fibrocytes.
Human serum amyloid P–mediated inhibition of mouse kidney fibrosis correlated with specific binding of human
serum amyloid P to cell debris and with subsequent suppression of inflammatory monocytes and kidney macro-
phages in vitro and in vivo, and was dependent on regulated binding to activating Fcg receptors and interleukin-10
expression. These studies uncover previously unidentified roles for Fcg receptors in sterile inflammation and
highlight serum amyloid P as a potential antifibrotic therapy through local generation of interleukin-10.
INTRODUCTION
Many modern human diseases, including those of heart, lung, liver,
gut,kidney,brain,andlargebloodvessels,arecharacterizedbychronic
inflammation with fibrosis, loss of microvasculature, loss of organ
parenchyma, and loss of function. Increasing evidence points to ac-
tivation of the innate immune system, recruited in response to tissue
injury in these disease processes (1). Currently, few effective therapies
targetthesefibroticinflammatorydiseases.Fibrosisitselfcausesparen-
chymal cell ischemia, distortion, and contraction of normal organ
architecture and contributes directly to functional demise (2). Despite
theprevalenceoforganfibrosis,notherapiesdirectlytargetthefibrotic
process. There is a pandemic of such fibrotic diseases of the kidney in
Western societies, ultimately leading to organ failure and the need for
lifesaving dialysis or organ transplantation.
Both chronic and acute tissue injuries stimulate a primary innate
injury response that is broadly similar across all tissues, including the
kidney. This response involves the sequential, regulated recruitment
and activation of multiple cell populations of hematopoietic and mes-
enchymal origin (3). The process proceeds through several phases in-
cluding an initial classical inflammatory influx of neutrophils and
monocytes, generation of excessive apoptotic and/or necrotic tissue,
recruitment and activation of myofibroblasts, significant extracellular
matrix deposition, and dynamic extracellular matrix remodeling.
Whether the outcome of this innate injury response is resolution of
injury and restoration of normal tissue homeostasis (wound healing)
or progressive fibrotic disease is controlled by the type of cell popula-
tions that are recruited to and activated at the site of injury. There is
accumulating evidence that monocyte-derived cell populations can
dynamically control this process through both direct effects on matrix
remodelingandindirecteffectsonregulationofactivatedmyofibroblasts
and their precursor populations (4–12).
Serum amyloid P (SAP), also known as pentraxin-2, is a highly
conserved, naturally circulating serum protein and one of two short
pentraxin protein family members, the other being C-reactive protein
(CRP) (13–17). SAP is produced in the liver and circulates as a highly
stable 135-kD pentamer (18) composed of five noncovalently linked
27-kD protomers associated into a ring-like structure (19). Each proto-
merofSAPcontainstwouniquebindingsites:aCa2+-dependentligand-
bindingsiteononefaceoftheprotomerandareceptor-bindingsiteonthe
opposite face for recognition of specific Fcg receptors (FcgRs) (20). The
calcium-dependent ligands recognized by SAP include both pathogen-
associated molecular patterns [PAMPs; for example, lipopolysaccharide
(LPS)andzymosan]anddanger-ordamage-associatedmolecularpatterns
(DAMPs; for example, DNA, chromatin, and phosphorylethanolamine)
presented on the membranes of apoptotic cells. SAP binding to Ca2+-
dependent ligands promotes subsequent FcgR-dependent phagocytosis
(21–26).
Although SAP was initially identified as a minor component of
amyloid plaque, which led to its nomenclature (27), it is structurally
unrelated to b-amyloid (Ab) or amyloid precursor protein (28). SAP
associationwithamyloidplaquelikelyreflectsahumoralresponseto
amyloid deposition, because amyloid fibrils are also recognized by
SAP as Ca2+-dependent ligands. These unique binding activities of
SAP and in vitro biology studies suggest that SAP may localize specif-
ically to sites of injury and aid in the removal of damaged tissue and
pathogenic organisms.
Because FcgR expression is restricted predominantly to cells of the
innate immune system, and many of the ligands for SAP are concen-
1Laboratory of Inflammation Research, Harvard Institutes of Medicine, 5th Floor, 4
Blackfan Circle, Boston, MA 02115, USA.2Renal Division, Department of Medicine,
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA.
3Promedior Inc., Malvern, PA 19355, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: jduffield@rics.bwh.harvard.edu
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tratedatsitesoftissueinjury,wepredictedthatSAPbindingtoligands
mightaffectinnateimmunecellactivationeventsinalocalizedfashion
and thereby potentially modulate the innate injury response. Despite
extensivecharacterizationofSAPinvitro(13,15–17),itspotentialpar-
ticipation in natural regulation of the innate injury response has only
recently been appreciated (10, 29–32). Pilling et al. first demonstrated
that SAP could suppress the differentiation of monocytes into fibro-
cytes, a monocyte cell lineage implicated in fibrotic disease of the lung
andotherorgans(29).TheysubsequentlyshowedthatpurifiedratSAP
could suppress development of lung fibrosis in the bleomycin model
(30), whichcorrelatedwith reducedfibrocytenumbers withinthelung
tissue. However, fibrocytesplay no obvious role in the development of
fibrosis of the kidney (33); therefore, we wished to determine whether
SAP would have an antifibrotic effect in this tissue setting and, if so,
what mechanisms mediated its biologic effect.
RESULTS
Systemic administration of human SAP
inhibits fibrosis in the kidney
Miceunderwentunilateraluretericobstruction(UUO)surgerytogen-
erate mechanical injury to the kidney, which results in a rapid, highly
reproducible interstitial fibrosis independent of T and B lymphocytes
(34).HumanSAP(hSAP)orhumanserumalbumin(HSA)asacontrol
was administered every 48 hours by intraperitoneal injection. hSAP
specifically inhibited the development of fibrosis at both day 7 and
day14(Fig.1,AtoC).hSAPwasnextadministeredtomicecontaining
the Coll1a1-GFP transgene (Coll-GFP) that had undergone UUO sur-
gery. Green fluorescent protein (GFP) expression by the kidney, indic-
ative of myofibroblast activation, was markedly reduced by hSAP
relativetoHSAcontrol(Fig.1,A,D,and E)(33).Cohortsofmicewith
UUO kidney disease were also treated with low-dose and high-dose
hSAP at different time intervals (Fig. 1F), showing that the effect of
hSAP was dose-dependent. hSAP injections had no effect on the total
numberofasmoothmuscleactin–positive(aSMA+)fibroblasts(Fig.1,
G and H); rather, the proportion of aSMA+fibroblasts that expressed
Coll-GFP was decreased by hSAP treatment (Fig. 1G). In addition, the
extent of macrophage infiltration measured in tissue sections was un-
affected by hSAP (Fig. 1H).
To confirm the broad antifibrotic effects of hSAP in kidney injury,
we induced a secondmodel of kidney fibrosis, the unilateral ischemia-
reperfusion injury (IRI) model. In this model, acute injury to the kid-
ney is followed by repair, but nevertheless, significant fibrosis ensues
(35).hSAPwasadministeredafterkidneyIRIandtheextentoffibrosis
wasdeterminedatdays7and15.SimilartodataintheUUOmodel,the
extentoffibrosiswasmarkedlydecreasedatbothtimepoints(Fig.2,A
toC),butthenumberofinterstitialmacrophageswasunaffected(HSA,
8.9 ± 2.0% versus hSAP, 7.4 ± 1.6%; area of F4/80 stain on day 15).
Together these data indicate that fibroblasts are present in similar
numbers after hSAP treatment, but that they show down-regulated
fibrotic collagen gene transcription and collagen protein deposition,
therebyidentifyinghSAPasanaturalinhibitoroffibrosisduringinjury
ofthekidney.OtherstudieshaveidentifiedSAPadministrationasanti-
fibrotic in injury to lung and heart, supporting a generalized role for
SAP in inhibition of fibrosis progression (30, 32); however, in lung
and heart, the antifibrotic effects of SAP correlated with decreased re-
cruitment and/or activation of fibrocytes, myeloid lineage cells that
generate collagen matrix directly. In recent comprehensive studies of
fibrocytes in kidney fibrosis models using Coll-GFP reporter mice, fi-
brocytes made no significant contribution to fibrosis (33). Although
specific culture of mouse monocytes in vitro readily induced fibrocyte
morphologythatwasinhibitedequivalentlybyhumanandmouseSAP
(fig. S1), fibrocytes were rarely identified in diseased kidneys treated
with HSA despite significant fibrosis progression (Fig. 1), confirming
Fig. 1. SAP inhibits kidney fibrosis after unilateral ureteral obstruction.
(A) Photomicrograph of Sirius red stain or GFP immunofluorescence of
day 14 UUO kidneys in Coll1a1-GFP mice treated with HSA or hSAP.
Scale bar, 50 mm. (B and C) Morphometric quantification of Sirius red–
stained area on (B) day 7 (d7) after UUO or (C) d14 after UUO in kidneys
of mice treated with HSA or hSAP [20 mg/kg every 2 days (q2d), n = 6
per group]. CON, control. (D and E) Morphometric quantification of GFP
area on (D) d7 after UUO or (E) d14 after UUO in kidneys of Coll-GFP
mice treated with HSA or hSAP (n = 6 per group). (F) Graph showing
effect of different doses and frequencies of hSAP on Trichrome-stained
fibrosis. qd, every day. (G) Immunofluorescence of aSMA-positive (a myo-
fibroblast marker) area in kidneys of Coll-GFP mice on d7 after UUO treated
with HSA or hSAP (left panel), and percent of aSMA-positive cells that were
Coll1a1-GFP–positive in kidneys treated with SAP or HSA (right panel). Note:
hSAP inhibits Coll1a1 gene transcription in kidney myofibroblasts. Marker,
50 mm. (H) Morphometric quantification of F4/80 positive (a macrophage
marker) area on d7 after UUO in kidneys of mice treated with HSA or hSAP
(n = 6 per group). *P < 0.05, **P < 0.01.
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that fibrocytes do not contribute to kidney fibrosis. Therefore, we
wished to determine what mechanisms mediated the biologic effect
of hSAP in the kidney.
ManyofthereportedligandsforSAP,includingcomponentsofcell
debris, are concentrated at sites of tissue injury, and SAP staining is
increased in injured skin aftersystemic administration (36). We tested
whether systemically administered hSAP selectively localized to
injured kidneys. We detected a marked increase of hSAP specifically
in the injured kidney in both models of injury (Fig. 2, D to F). This
retained hSAP was predominantly associated with apoptotic and ne-
crotic cells (Fig. 2E), and many of the hSAP-stained cell debris were
inside recruited macrophages (Fig. 2E).
Kidney disease in humans correlates with
reduced SAP concentrations
The studies in mice revealed that not only was hSAP antifibrotic, but
thathSAPwasdepositedselectivelyintheinjuredkidney.Inthissense,
hSAP maysharesimilaritieswiththecomplementproteinsthatcanbe
“consumed” at sites of inflammation, resulting in depressed plasma
concentrations.ThatdosedhSAPamelioratesfibrosisandsystemicad-
ministration increases hSAP deposition in the kidney also suggest that
injurystatesmightrepresentSAPdeficiency.Totestthis,plasmahSAP
concentrationswerestudiedinacohortofpatientswithchronickidney
diseases(fig.S2A).Patientswithmoreseverekidneydiseasehadsignif-
icantly lower concentrations of hSAP in plasma relative to that of pa-
tients with mild kidney diseases and normal function, and there was a
correlation between plasma hSAP concentration and loss of kidney
function (fig. S2, B and C). Patients with more active kidney disease,
indicatedbyanalbuminleakof>300mgadayfromthekidneyintothe
urine, also had lower hSAP concentrations (fig. S2D). Because lower
plasma hSAP concentrations in patients with more severe kidney dis-
easecould be interpreted aseitherconsumption of hSAP inthekidney
orleakofhSAPintotheurine,kidneysfrompatientswithkidneyinjury
werelabeledfordepositionofhSAP.Thesekidneysectionsalsoshowed
increaseddepositionofhSAP(fig.S2E),consistentwithourfindingsin
mice and in keeping with the hypothesis of SAP consumption by
injured tissues.
Human SAP binds directly to monocytes and macrophages,
but not to fibroblasts
We next studied the effect of hSAP on fibroblasts, monocytes, and
macrophages, using fluorescently labeled and functionally specific
hSAP (hSAP conjugates with Alexa 594 or Alexa 488: SAP-594 or
SAP-488)andsimilarlylabeledHSAascontrol(fig.S3A).Primarycell
cultures of kidney fibroblasts were established from kidneys of Coll-
GFP mice on 7 days after UUO but did not bind to SAP-594 (Fig. 3A).
Furthermore,whenthesefibroblastswereculturedinmediumcontain-
ing hSAP but no other serum proteins or in medium containing 0.1%
albumin, hSAP had no effect on collagen 1a1 promoter activity (Fig.
3B), suggesting that hSAP does not exert its in vivo antifibrotic effects
on kidney fibroblasts directly.
Human SAP was reported to bind to FcgRs in indirect binding
studies (21–23). Although mouse kidney fibroblasts do not express
FcgRs (fig. S3C), monocytes and macrophages do (figs. S3D and S4, A
and B). Human monocytes (CD11bhigh) selectively bound SAP-488,
whereas lymphocytes (CD11blow) did not (Fig. 3, C and D). Mature
purified mouse bone marrow monocytes (BMMs) avidly bound SAP-
488 (Fig. 3E). Similarly, peripheral blood monocytes (PBMos) from
mice taken 7 days after UUO surgery bound SAP-488 avidly (Fig.
3G) and with binding similar to that observed for human peripheral
blood mononuclear cells (PBMCs) (Fig. 3C). By contrast, the majority
of mouse PBMos from healthy mice did not bind hSAP (Fig. 3F). Be-
cause SAP-488 also bound to primary cultures of macrophages (fig.
S3D), we purified inflammatory kidney macrophages from day 3, day
7, and day 10 UUO kidneys. At each time point, SAP-488 selectively
and specifically bound to CD11b+ kidney macrophages (Fig. 3H). Col-
lectively these studies indicated that in mice, inflammatory monocytes
held in bone marrow, those released into the circulation during kidney
injury, and kidney inflammatory macrophages, all bind hSAP.
Fig. 2. SAP inhibits fibrosis after IRI and is selectively deposited in the
injured kidney. (A) Sirius red stain of d15 post-IRI kidneys in C57BL6
mice treated with HSA or hSAP (20 mg/kg q2d) (marker, 100 mm). (B
and C) Morphometric quantification of Sirius red area in (B) d7 post-IRI
kidneys or (C) d15 post-IRI of mice treated with HSA or hSAP. (D) Morpho-
metric quantification of SAP deposition detected by immunofluorescence
in d7 post-IRI kidney treated with hSAP or HSA compared with a control,
nondiseased kidney. (E) Immunofluorescence images (left panel) of SAP
(green) detected in post-IRI kidney after alternate-day intraperitoneal
injections of hSAP or HSA. Note that SAP is detected in intratubular cellular
debris (left) where nuclear debris can be seen (arrows), but is also seen in
interstitial macrophages (F4/80, green) (center) predominantly in endo-
somes and phagosomes (arrowheads) (F) Western blot of whole-kidney
proteins detecting SAP in kidneys d10 after UUO and normal kidney. n = 6
per group. Marker, 50 mm. *P < 0.05, **P < 0.01.
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FcgRs are up-regulated by monocytes and macrophages
in the injured kidney
Our studies indicated that hSAP bound to inflammatory monocytes and
macrophages, possibly through FcgRs (20, 21). We therefore assessed
monocytes and macrophages for murine FcgR (mFcgR) expression. In-
flammatory monocytes residing in bone marrow or circulating as
PBMos expressed high concentrations of mFcgRI and mFcgRIII, mod-
erate concentrations of mFcgRIV, and low concentrations of mFcgRII
(fig. S4A). By contrast, only a minority of normal monocytes expressed
mFcgRI and none expressed mFcgRII. Inflammatory macrophages
isolated from day 7 UUO kidney expressed increased concentrations
of mFcgRI, II, III, and IV, compared with inflammatory monocytes
(fig. S4B), and distinct populations of kidney macrophages were identi-
fied based on levels of mFcgR expression.
Human SAP binds to FcgRs
Previously published studies suggested hSAP binds to human FcgRs
(hFcgRs) (20–23). However, no study has compared all four mFcgRs
and the effect of the common FcRg-chain on binding activity. We
generated mouse fibroblast (3T3) cell lines exclusively expressing
mFcgRI ± FcRg, mFcgRII, mFcgRIII ± FcRg, or mFcgRIV ± FcRg
(Fig. 3I). Control and receptor-expressing cells were incubated with
SAP-488atphysiologicalconcentrationsandwereassessedforspecific
binding (Fig. 3I). 3T3-mFcgRI cells alone did not bind SAP-488, but
when coexpressed with FcRg, binding activity was detected. 3T3-
mFcgRII also bound SAP-488, albeit with substantially lower binding
activity than the other receptors. 3T3-mFcgRIII cells and 3T3-
mFcgRIV cells exhibited binding activity to SAP-488 that was also
enhanced by coexpression of FcRg (Fig. 3I and fig. S4C), and 3T3-
mFcgRIVcellsexhibitedthegreatestshiftinSAPbinding. Thus,hSAP
has the capacity to bind all mouse FcgRs with a relative rank order of
mFcgRIV > mFcgRIII ≥ mFcgRI > mFcgRII.
To characterize the direct association of hSAP protein with the
hFcgR, using label-free surface plasmon resonance (Biacore), we
studied the binding affinity of hSAP for all forms of hFcgRs (Fig.
3, J to L, fig. S4, and Table 1). In contrast to a recent publication (20),
Fig. 3. SAP binds to the surface of monocyte lineage cells and FcgRs.
(A) Histogram plot of binding of SAP-594 compared with that of HSA-
594 to primary cultured mouse kidney interstitial fibroblasts purified
from Coll-GFP mice. (B) Histogram plot of Coll-GFP fluorescence intensity
in primary kidney fibroblasts from Coll-GFP mice cultured for 24 hours
with HSA (50 mg/ml; black), hSAP (25 mg/ml; gray), or hSAP (50 mg/ml; light
gray). (C) Plot of SAP-488 binding to human PBMCs separated by CD11b.
SAP-488 binds selectively to CD11b high leukocytes (monocytes) (high
FSC, low SSC) and does not bind to other leukocytes. (D) Histogram
showing binding of HSA-488 (dark gray) and SAP-488 (light gray) to hu-
man monocytes. (E to H) Histograms showing HSA-488 (black) or SAP-488
(white) binding to (E) purified, mature mouse BMMs, (F) mouse PBM from
healthy mice, (G) PBM from mice d7 after UUO, (H) purified kidney macro-
phages from mice d7 after UUO compared. (I) Histogram plots of 3T3
mouse fibroblast cell lines expressing individual FcgRs with or without
the co-receptor FcRg-chain. Cell surface FcgR expression (white) is shown
for individual cell lines (left columns) compared with isotype control
(black). Binding of SAP-488 (white) compared with HSA-488 (black) is
shown for individual cells lines (right columns). 7-AAD-positive cells are
excluded from these binding studies. (J to L) Biochemical characterization
of hSAP binding to hFcgRs with Biacore surface plasmon resonance tech-
nology. (J) Sensorgrams of human IgG1(upper panel) binding to hFcgRI
(recombinant ectodomain) when the receptor is immobilized on a Biacore
CM5 dextran chip, but hSAP (lower panel) was unable to bind to hFcgRI in
this orientation. (K) Sensorgram of Ca2+-dependent binding of hSAP to
CM5 dextran followed by dissociation in the presence of Ca2+chelation
(10 mM EDTA). (L) Sensorgram of single-cycle kinetic analysis of hFcgRIIIB
binding to chip-bound hSAP oriented through Ca2+-dependent binding to
the CM5 dextran. Five different receptor concentrations injected in order
of increasing concentration were used to obtain data for affinity calcula-
tion. Association time was 180 s and the final long dissociation was 7200 s
for hFcgRIIIB (attenuated for presentation). Both raw data (black) and data
fitted for 1:1 binding (red) are shown on the same sensorgram and repre-
sent data after background subtraction. Off-rate is very slow and drives the
high affinity of interaction we observed. Results are representative of three
independent experiments for each FcgR.
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we did not observe specific binding of hFcgRs to hSAP when hSAP was
directly coupled to the dextran surface, using the standard amine cou-
pling procedure (fig. S4D). When the orientation was reversed and
hFcgRs were coupled to the chip surface via amine coupling, binding
to immunoglobulin G (IgG) was readily detected (Fig. 3J, upper panel),
but there was no specific binding of hSAP to hFcgRs in this orientation
either (Fig. 3J, lower panel).
SAPwasreportedtopromotephagocytosisthroughcouplingCa2+-
dependent binding and opsonization of ligands to FcgR-mediated cell
recognition (21–23). Therefore, we investigated whether hSAP would
specifically bind hFcgRs if the hSAP was first oriented on a Ca2+-
dependent ligand. SAP recognizes several glycosaminoglycans as
Ca2+-dependent ligands, and dextran sulfate can competitively inhibit
Ca2+-dependent self-association of SAP (37). We tested whether
hSAP would specifically bind to the dextran surface of the CM5 chip
inaCa2+-dependentmannerintheabsenceofamine-couplingreagents.
We found that hSAP readily bound to the CM5 chip dextran surface
and could be removed by 10 mM EDTA (Fig. 3K). To stabilize the
oriented hSAP for subsequent binding to FcgRs, we followed hSAP-
dextran association with a brief amine coupling pulse, yielding an
oriented and highly-stable hSAP surface. Subsequent investigation of
eachhFcgRshowedspecificandhigh-affinityassociationofhSAPwith
most forms of hFcgRs, in the following order of affinity: hFcgRIIA ~
hFcgRIII>hFcgRI≫hFcgRIIB(Fig.3L,fig.S4E,andTable1).There-
fore, although there are some differences between mouse and human
specificities, in both species SAP preferentially binds to the immuno-
receptortyrosine–basedactivationmotif(ITAM)–containingactivating
FcgRs, rather than the immunotyrosine-inhibitory motif (ITIM)–
containinginhibitorymFcgRII(mouse)orhFcgRIIB(human).Thefact
that both human and mouse lymphocytes and NK (natural killer) cells
express FcgRIII isoforms, but the predominant binding leukocyte in
bothspeciesisthemonocyte,indicatesthatcellsurfaceexpressionalone
is not sufficient for binding and that FcgRIII expression and binding to
hSAP are not linearly related. Further, our biochemical studies suggest
that hSAP only binds to hFcgRs with high affinity once hSAP is bound
toaligand(Fig.3,JtoL,andfig.S4E),suchaswouldoccurwhenSAPis
bound to apoptotic and necrotic tissue at an injury site or when such
debris is released into circulation. hSAP did not bind to 3T3-FcgR cell
linesintheabsenceofbovineserumalbumin(BSA)(alow-affinitySAP
ligand). This regulated receptor preference could be important in SAP
function by localizing SAP activity to sites of damaged tissue exposure,
and suggests that SAP is a soluble danger-associated molecular pattern
receptor (DAMP).
Development of fibrosis in the kidney is
dependent on monocytes/macrophages
Because hSAP bound selectively to monocyte lineage cells and FcgRs,
yet was antifibrotic in the absence of fibrocytes, we explored a possi-
ble role for monocytes/macrophages in the progression of kidney fi-
brosis by paracrine mechanisms. Using diphtheria toxin (DT) to
induce conditional ablation of monocytes/macrophages in the
Cd11b-DTR mouse (12, 38) from day 4 to day 7 resulted in a signif-
icant decrease in both fibrosis and monocyte/macrophage accumula-
tion on day 7 (Fig. 4, C and D). In another cohort, kidneys were
assessed at day 10 (Fig. 4, A and E) after DT administration on days
7 to 10. As expected there was a high level of ablation of monocytes
in blood and of kidney macrophages (Fig. 4, A to C). Collagen accu-
mulation was measured morphometrically by staining with Sirius red
(Fig. 4, A and E), and fibrosis was markedly reduced at both day 7
and day 10 compared with vehicle-treated mice. In previous studies
we have confirmed that DT has no direct effect on fibroblasts from
CD11b-DTR mice and that these mice retain the normal complement
of neutrophils (38). These findings are consistent with a paracrine
role for macrophages in fibrosis progression.
Fig. 4. Conditionalmacrophageablationinhibitskidneyfibrosisinureteral
obstruction. (A) Photomicrographs of F4/80 immunostaining for macro-
phagesorSiriusredstainingforfibrosisinCD11b-DTRkidneystreatedwith
DT or vehicle to ablate macrophages on d10 after UUO. (B) FACS plots of
PBMCsfrom thesame mice labeled withantibodiesagainst CD11band7/4
tolabelmonocytes,showingthatDTalsoablatedcirculatingmonocytes.(C
to E) Morphometric quantification of F4/80 immunostain (C), collagen III
immunostain(D)orSiriusredstaininkidneys10daysafterUUOinvehicle
(VEH)-treated or DT-treated CD11b-DTR mice. *P < 0.05, **P < 0.01.
Table 1. Affinities of FcgRs for human SAP. Single-cycle kinetics meth-
ods and analysis on the Biacore X100 were used to obtain binding affi-
nities for interaction of different hFcgRs with hSAP. The SAP was
orientedviaCa2+-dependentdextranbindingtothechip.Foreachanalysis,
an ascending concentration series of five different hFcgR concentrations
was used. The results shown represent the average of atleast two separate
analyses for each receptor.
FcgR
KD(M)
hFcgRI 4.3 × 10–9
2.9 × 10–10
hFcgRIIA (H131)
hFcgRIIBNo binding
3.7 × 10–10
hFcgRIIIB
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Human SAP triggers an anti-inflammatory signature
in infiltrating macrophages
Because hSAP was predominantly detected bound to dead cells and
debris in vivo (Fig. 2, D to G, and fig. S2), we hypothesized that hSAP
opsonizes apoptotic cells, permitting FcgR-dependent clearance by
macrophages as opposed toclearanceviaotherreceptors,and thatthis
method of clearance may affect activation. First, we confirmed the
binding of hSAP to cells undergoing UV-mediated apoptosis (Fig.
5A). Whereas there was little binding to healthy cultured human lym-
phocytes, there was progressive and striking binding of hSAP to lym-
phocytesearlyintheprocessofapoptosis.Thesestudiessuggestedthat
hSAP-opsonized apoptotic cells would preferentially bind FcgRs. To
test this, we incubated 3T3 cell lines expressing single mFcgRs with
hSAP-opsonized or unopsonized apoptotic mouse lymphocytes and
assessed the cell lines by flow cytometry for phagocytosis using
established phagocytosis protocols (39). There was little difference
in phagocytosis of hSAP-opsonized versus unopsonized apoptotic
cells by cell lines expressing mFcgRII or mFcgRI (Fig. 5B). However,
hSAP-opsonized apoptotic cells were more avidly phagocytosed by
mFcgRIII- and mFcgRIV-expressing cell lines, implicating these two
receptors in hSAP-mediated inhibition of fibrosis.
The leukocyte cell surface marker Ly6C labels subpopulations of
circulating monocytes (40). Inflammatory monocytes that are selec-
tively recruited to inflamed sites express high concentrations of Ly6C
(Ly6Chigh) and hSAP selectively bound to Ly6Chighmonocytes (Fig. 3,
E toG, and fig.S5).To explore further the role ofFcgR-mediated phago-
cytosis of apoptotic cells, mature BMMs that have high concentra-
tions of Ly6C and express only the activating FcgRs (figs. S4, A
and B, and fig. S5), were purified, incubated with unopsonized or
hSAP-opsonized apoptotic cells, and coactivated with interferon-g
(IFN-g) or immobilized IgG (iIgG) (Fig. 5C). Twenty-four hours later,
hSAP-opsonized apoptotic cells had potently inhibited the activation
of monocytes compared with unopsonized apoptotic cells. In these
experiments, monocytes generated no detectable Il-6, Il-12, or Pdgfb,
andhSAPopsonizationhadnoeffectonTgfb transcriptconcentrations.
To study the functional consequences of hSAP opsonization and
macrophage phagocytosis in vivo in the progression of kidney fibrosis,
we purified kidney macrophages from day 7 UUO diseased kidney of
mice treated with either HSA or hSAP by fluorescence-activated cell
sorting (FACS) and measured messenger RNA (mRNA) transcript
levels by branched-chain DNA (bDNA) amplification or quantitative
polymerase chain reaction (Q-PCR). The leukocyte cell surface marker
Fig. 5. hSAP inhibits activation of mouse monocytes and kidney macrophages
in vivo and ex vivo by triggering FcgR-dependent uptake of hSAP-opsonized
apoptotic cells. (A) Histogram plots showing a time course (hours) of SAP-488
binding to Jurkat T lymphocytes after UV irradiation to induce apoptosis. (B)
Representative plot of percentage phagocytosis of hSAP-opsonized apoptotic
thymocytes by 3T3-FcgR/FcRg-chain cell lines after a 4-hour co-incubation. (C)
Relative transcript expression, assessed by bDNA amplification (normalized to
housekeeping gene Hprt1), by purified mouse BMMs incubated with apoptotic
cells (AC), unopsonized (black) or opsonized with hSAP (white), and coactivated with either no stimulus, immobilized IgG, or IFN-g. (D) Relative
transcript expression, assessed by bDNA amplification (normalized to housekeeping gene HPRT1) by three subpopulations of purified kidney
macrophages 7 days after UUO, distinguished by the markers Ly6Chi, Ly6Cint, and Ly6Clo, from mice treated with HSA (black) or hSAP (white) by
intraperitoneal injections for 7 days. In addition, relative Il-10 transcript expression assessed by Q-PCR (normalized to housekeeping gene GAPDH)
is markedly increased in all kidney macrophage populations by hSAP treatment. (E) Western blot of whole-kidney lysates showing IL-10 (18 kD) in d10
UUO kidneys of mice treated with hSAP but only weakly detected in mice treated with HSA. (F) Human monocytes incubated with SAP (5 mg/ml)
triggers release of IL-10 protein in supernatants (n = ≥ 3 per group, *P < 0.05).
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Ly6C also labels three discrete subpopulations of kidney macrophages
designated Ly6Chigh, Ly6Cint, and Ly6Clow(fig. S5), which separates
macrophagesintoM1type(Ly6Chigh)andM2type(Ly6Clow)functions.
Ly6Cintcells representboth Ly6ChighcellstransitioningtoLy6Clowcells
andtheresidentmacrophagepopulation.Asexpected,thethreepopu-
lations of kidney macrophages differed greatly in transcript profiles.
The Ly6Chighmacrophages were activated and transcribed high con-
centrationsof M1 typetranscriptsincluding Mip2a andIl-1b, andthe
Ly6Clowmacrophages transcribed high concentrations of M2-type
transcripts including Ccl17 and Ccl22. Systemic administration of
hSAP inhibited transcription of both M1-type and M2-type cytokines
(Fig.5D),indicatingthathSAPfunctionstobufferactivationofinflam-
matorymonocytesandmacrophagesinvivo.Inaddition,transcriptsof
the anti-inflammatory and antifibrotic cytokine Il-10 were markedly
up-regulated in all three populations of kidney macrophages (Fig.
5D). In whole kidney 10 days after UUO, IL-10 protein was increased
selectively in mice treated with hSAP (Fig. 5E), confirming the tran-
scriptional studies. Furthermore, human monocytes cultured with
hSAProbustlygeneratedIL-10protein(Fig.5F),collectivelysuggesting
local IL-10 production by inflammatory macrophages may be central
to the efficacy of hSAP.
FibrosisprogressionisdependentonactivatingFcgRsandIL-10
Our data implicate macrophage FcgRs in the progression of kidney
fibrosis. To examine the role of these receptors further, we induced
UUO in kidneys from mice lacking FcRg (FcRg–/–). Leukocytes from
these mice have no cell surface expression of activating receptors
FcgRI, III, and IV. After 10 days of UUO, the extent of fibrosis in
FcRg–/–and strain-matchedwild-type control mice was compared. Fi-
brosis was significantly reduced in mice without FcRg (Fig. 6A). To
understand whether there was any role for the inhibitory receptor
FcgRIIinthisprocess,FcgRII–/–micehadUUOsurgeryandwerecom-
pared with strain-matched controls. The absence of FcgRII resulted in
enhanced accumulation of collagen matrix relative to strain-matched
controls(Fig.6B).However,micelackingonlyFcgRIIIthatunderwent
UUO surgery developed fibrosis comparable with that of wild-type
controls(Fig.6C),indicatinganegligibleorredundantroleforFcgRIII
function in kidney fibrosis. These studies suggested that ITAM-
bearing FcgRs played a positive role in leukocyte activation. Never-
theless, when hSAP was given to FcRg–/–mice or wild-type controls
withUUO-inducedkidneyfibrosis,theantifibroticeffectofhSAPwas
markedly attenuated in the absence of activating FcgRs (Fig. 6D),
confirming the in vivo role of activating FcgRs in hSAP-dependent
signaling. To explore the role of activating FcgRs further, monocytes
from FcRg–/–or wild-type mice were incubated with hSAP and coac-
tivated with immobilized IgG, IFN-g, the TLR4 (Toll-like receptor 4)
ligand LPS, or combinations of these activating stimuli. Strikingly,
FcRg–/–monocyteswereresistanttoactivationbyallpro-inflammatory
cytokines (Fig. 6E). Furthermore, hSAP had no effect on inhibition of
cytokine production by FcRg–/–monocytes, in distinction from wild-
type monocytes (Figs. 5 and 6E), indicating that hSAP signals through
activating FcgRs in monocytes and that activating FcgRs play broad
roles in regulating monocyte activation.
Soluble immunoglobulins are readily detected in injured but not in
healthy tissues. Whereas monocytes are susceptible to activation by
complexed or immobilized IgG, but not soluble IgG, mature primary
macrophages are susceptible to activation by soluble IgG (fig. S6A),
particularlyafterIFN-gincubation.IncubationwithIFN-ghadnoeffect
on FcgR expression. Strikingly, hSAP (in BSA containing serum-free
buffer) inhibited TNFa (tumor necrosis factor–a) release triggered
by soluble IgG (fig. S5B).
Although little IL-10 was detected in fibrotic kidney after UUO in
the absence of SAP injections, it was synthesized by macrophages af-
ter FcgR ligation by SAP (Fig. 5, D to F). As expected, mice deficient
in IL-10 had similar levels of fibrosis after UUO-induced injury of the
kidney (Fig. 6F). However, hSAP no longer inhibited the progression
of fibrosis in IL-10–deficient mice. These genetic studies implicate IL-10
release by macrophages in the kidney in the mechanism of action of
hSAP. To test whether IL-10 alone could inhibit fibrosis, using adeno-
viruses, we overexpressed IL-10 systemically in mice. Systemic IL-10
expression in plasma at the time of ureteral ligation surgery was con-
firmed (38.0 ± 6.7 ng/ml [Ad-IL-10] versus 4.9 ± 2.3 ng/ml [AdMock]
versus 0 ± 0 ng/ml [no virus]) and fibrosis progression was markedly
Fig. 6. hSAP-inhibited kidney fibrosis is dependent on FcgR and IL-10
expression. (A to C) Morphometric quantification of Sirius red–stained
kidney fibrosis 10 days after UUO in age-and strain-matched wild-type
(WT) mice compared with (A) FcRg-chain–/–mice (lacking FcgRI, III, and
IV), (B) FcgRII–/–mice, and (C) FcgRIII–/–mice. (D) Morphometric quanti-
fication of Sirius red fibrosis. hSAP treatment of FcRg-chain–/–mice fibrosis
results in a significantly smaller reduction in fibrosis on d10 after UUO
compared with strain-matched wild-type mice treated with hSAP. (E)
CXCL2 relative gene transcription 24 hours after activation with inflamma-
tory cytokines (IFN-g, iIgG, LPS, or combinations) by wild-type monocytes,
FcRg-chain–/–monocytes or FcRg-chain–/–monocyte coactivated with
hSAP (25 mg/ml). (F) Morphometric quantification of Sirius red–stained kid-
ney fibrosis d10 after UUO in strain-matched wild-type and IL-10–/–mice,
treated with either HSA or hSAP. (G) Morphometric quantification of Sirius
red–stained kidney fibrosis 10 days after UUO in C57BL6 mice treated sys-
temically with Ad-IL-10, Ad-Mock, or no virus. (H) Histogram plots of
cultured Coll-GFP kidney fibroblasts incubated with IL-10 for 8 hours as-
sessed by flow cytometry show decreased Coll1a1 gene transcription.
(n = 6 per group; *P < 0.05, **P < 0.01).
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inhibited(Fig.6G).WhereasligationoftheIL-10receptoronmacrophages
inhibits activation, IL-10 can also directly inhibit collagen production
by myofibroblasts, which also express the IL-10 receptor (Fig. 6H).
DISCUSSION
These studies identify SAP as a natural inhibitor of fibrosis during
inflammatory injury of the kidney. Moreover they show a potential
role for hSAP as a therapeutic compound in the treatment of inflam-
mation associated with fibrosis. We identified previously unknown
mechanisms of action of hSAP in regulating anti-inflammatory clear-
ance of apoptotic and necrotic cell debris in injured tissues and down-
regulating activation of monocytes and macrophages that direct
fibrosis, and identified hSAP as a soluble DAMP receptor, coupling
injury to recognition by activating FcgRs (Fig. 7).
ThemostobviouscelltypestobindhSAPandmediateanantifibrotic
effect are the fibroblast and fibrocyte. However, our in vivo and in vitro
studiesprovidenosupportivedataforadirectinteractionbetweenfibro-
blasts and hSAP in the kidney. Further, although we did confirm that
mouse monocyte culture can induce differentiation into fibrocytes,
and that both hSAP and mSAP inhibit this differentiation with similar
dosetitrations(fig.S1A),wedidnotfindanyevidencetosupportarolefor
fibrocytes in contributing to the kidney myofibroblast pool (fig. S1, B
andC). Instead, our results highlight the importance ofthe monocyte/
macrophage in mediating SAP’s antifibrotic effect within the kidney.
PreviousstudieshaveidentifiedbindingligandsforhSAP,including
apoptotic and necrotic cells and binding receptors for hSAP including
FcgRs (21–24, 26). Our studies confirm that hSAP rapidly recognizes
bothearlyand late apoptoticcellsandshow thatinvivoadministration
ofhSAPresultsindepositionofhSAPintheinjuredorganwithbinding
to both dead cells and interstitial macrophages in vivo. hSAP behaves
similarlytocomplementcomponentsinthatitopsonizesdeadcellsand
binds specifically to receptors on macrophages. Moreover, the recruit-
ment of hSAP to the site of injury may explain why plasma concentra-
tions of hSAP fall in patients with inflammatory diseases of the kidney;
the fall is most likely due to consumption.
Our studies independently confirm with the use of previously un-
described methods that hSAP is a ligand for FcgRs. Although in the
mouseitbindstoallFcgRs,hSAPshowedpreferencefortheactivating
receptors (RIV, RI, and RIII) over the inhibitory receptor (RII), and a
similar preference was shown in our studies with human receptors
wherelittlebindingactivitywasobserved for hFcgRIIB,thehumanin-
hibitory receptor, but hSAP has the greatest affinity for the activating
receptors hFcgRIIA and hFcgRIII. We also demonstrated that high-
affinitybindingofhSAPtohFcgRoccursuponCa2+-dependentligand
binding.Thisligand-dependentreceptorpreferencemaybeimportant
in SAP function by localizing SAP activity selectively to sites of dam-
agedtissueexposureandidentifyingSAPasasolubleDAMPreceptor.
On murine monocytes and inflammatory kidney macrophages,
mFcgRII represents only a small proportion of the target FcgRs for
hSAP. In addition, hSAP-opsonized apoptotic cells are preferentially
recognized by mFcgRIII and mFcgRIV, not mFcgRII, which suggests
that hSAP in rodents also likely functions by binding to the activating
FcgRs.
The prevailing model of FcgR function is that activating receptors,
mFcgRI, III, and IV (hFcgRI, IIA, and III) activate leukocytes and
that mFcgRII (hFcgRIIB) inhibits activation. Our data, however, in-
dicatethatligationofmFcgRIVand/ormFcgRIIIbyhSAPorhFcgRIIA
and/or hFcgRIII results in potent inhibition of leukocyte activation,
pointingtomorecomplexsignalinginmonocytes/macrophagesinduced
by ITAM signaling through activating FcgRs than has previously been
appreciated. One possible mechanism is that in vivo, SAP effectively
competes for IgG binding to mFcgRIV and mFcgRI without inducing
a productive activating signal, although at the same time allowing IgG
to ligate mFcgRII, thereby indirectly amplifying the negative regulatory
activity of mFcgRII. Alternatively, ligation of activating FcgR by SAP
may induce an anergic signaling pathway in monocytes/macrophages
similartotheeffectofpartialagonistpeptidesonTCRsignaling.Presen-
tation of such altered peptide ligands results in recruitment but not ac-
tivation of the Syk-family kinase ZAP-70 (41, 42). SAP-mediated
inhibition of monocyte to fibrocyte differentiation is dependent on Src
familykinasesignaling,butnotSykfamilykinasesignaling(31).Further
studieswillberequiredtodeterminewhetherhSAPligationofactivating
FcgRs results in a distinct intracellular signaling signature compared
with ligation by IgGs.
IndissectingthemechanismofactionofhSAP,wehavedetermined
that FcgRs participate in fibrosis progression. Because FcRg–/–mono-
cytes are hypoactivated by a range of stimuli, FcgRI, III, and IV may
help to regulate leukocyte activation. Thus, activating FcgRs may be
generallyimportantinmacrophageactivationduringsterileinflamma-
tion and suggest that additional therapies targeting FcgRs could also
regulate the progression of fibrosis.
Inbothhumanandmousemacrophages,inhibitionofactivationby
hSAP is at least in part due to release of IL-10 (Fig. 7). This immuno-
modulatory cytokine inhibits macrophage activation, and the studies
presented here implicate local IL-10 release in both indirect (de-
activation of macrophages, which results in down-regulation of para-
crine signals thatactivatemyofibroblasts) and direct(down-regulation
of collagen transcription in myofibroblasts) actions on collagen-
producing cells. IL-10 has a short circulating half-life, limiting its sys-
temictherapeuticapplication.Inaddition,itstimulatesBcellproliferation
Fig. 7. The antifibrotic mechanism of action of SAP. Apoptotic cells or
debris are opsonized by SAP, which in turn renders SAP a high-affinity
ligand for hFcgRIIA or hFcgRIII. Both Ly6Chiand Ly6Cloinflammatory
macrophages bind SAP-opsonized debris, which triggers IL-10 release,
inhibiting macrophage profibrotic function (through recruitment and
activation of pericytes/myofibroblasts) and directly inhibiting myofibro-
blast production of collagen 1a1. Although SAP inhibits fibrocyte ap-
pearance, fibrocytes play no role in kidney fibrosis.
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andmaybedeleteriousifadministeredsystemically.BecausehSAPad-
ministration results in local release of IL-10 at the site of injury,
potentialsystemicsideeffectsofIL-10maybeavoided,andhSAPmod-
ulation of monocyte/macrophage production of IL-10 may result in
longer exposure of the tissue to the regulatory capacity of this cytokine
than its systemic application could accomplish.
ThatadministrationofhSAPpromotesanti-inflammatoryresponses
inourstudiesindicatesthatinflammationinthekidneymayrepresent
a local state of functional deficiency in SAP. Alternatively, consump-
tionofhSAPthroughrecruitmenttothesiteofinjurymayexplainwhy
plasma concentrations fall in patients with inflammatory kidney dis-
eases. SAP may be tonically produced by the liver in health as a mech-
anismtolimitleukocyteactivationexceptinoverwhelmingcircumstances.
Our data identify hSAP as a DAMP receptor that then serves as a ligand
for activating FcgRs, triggering anti-inflammatory clearance of apoptotic
andnecroticcelldebrisintheinjured tissuesanddown-regulatingactiva-
tion of monocytes and macrophages. Moreover, they demonstrate a
strong potential for hSAP as a therapeutic compound in the treatment
of inflammation and fibrosis. A recombinant form of hSAP, PRM-151,
has been produced by Promedior, Inc., and is currently being tested in
a phase 1 single ascending dose clinical trial to assess safety and pharma-
cokineticsinhealthyvolunteers(Promediorpressrelease).Wearehopeful
that subsequent testing in patients with active fibrotic disease will de-
terminewhetherthestrongtherapeuticpotentialindicatedherecanbe
translated into appropriate clinical practice.
MATERIALS AND METHODS
Mouse breeding and genotyping
Wild-type C57BL/6 mice were from Charles River Laboratories.
Cd11b-DTR mice (FVB/N) were generated and maintained as pre-
viously described (12). Coll-GFP transgenic mice were generated on
the C57BL6 background and validated as previously described (33).
The presence of CD11b-DTR was confirmed by genomic DNA PCR
with the following primers: 5′-TTCCACTGGATCTACGGACC-3′,
5′-TGTCGGCCATGATATAGACG-3′. FcRg-chain–/–mice and strain-
matchedcontrols(B6;129P2-Fcer1gtm1Rav/J-B6;129P2-Fcer1gtm1Rav/J)
were from Taconic Laboratories (43); FcgRII–/–mice and strain-matched
controls(B6;129S4-Fcgr2btm1Ttk/J-B6129SF2/J)(44)andFcgRIII–/–mice
were from Jackson Laboratories (B6.129P2-Fcgr3tm1Sjv/J- C57BL/6J)
(45); and IL-10–/–(B6.129P2-Il-10tm1Cgn/J) mice were from Jackson
Laboratories Genotypingofthemousestrainswascarried outaccord-
ing to protocols available at the Jackson Laboratory Web site.
Mouse models of fibrosis
Adult (12 to 20 weeks) male mice [C57BL6, Coll-GFP, CD11b-DTR,
FcgRII–/–, FcgRIII–/–, FcRg-chain–/–, IL-10–/–and age-matched con-
trols (n ≥ 5 per group)] were anesthetized with ketamine-xylazine
[100/10 mg/kg intraperitoneally (ip)] before surgery. UUO surgery
wascarriedoutaspreviouslydescribed(46),andkidneys,blood,spleen,
andbonemarrowwerecollectedondays0,7,10,or14.Renalunilateral
IRI was performed as previously described (35), and tissues were har-
vestedonday7orday15afterIRI.Insomeexperiments,hSAP(20mg/g)
was administered intraperitoneally or intravenously on alternate days
ordailyfromday0ofdiseaseinductionuntilharvestingoforgans.HSA
(20 mg/g) was administered to separate cohorts of mice as a control
protein. In experiments with macrophage ablation, 25 ng/g DT (List
Biological Laboratories Inc.) in 100 ml or vehicle was given on day 4
and day 6 or day 7 and day 9 by tail vein injection (n ≥ 5 per group);
separate cohorts of mice received equal volumes of vehicle. Kidneys
and blood were collected on day 7 or day 10 (12, 33). In experiments
usingmutantmice,age,weight,sex,andstrain-matchedwildtypemice
wereusedascontrols,treatedidentically,andblindedtothesurgeon.In
some experiments adenoviruses[5 × 108plaque-forming units (PFU)]
weregiventomice3daysbeforesurgerybytailveininjectionin200ml
ofvehicle. Plasma concentrations of IL-10 were quantified by ELISA
(enzyme-linked immunosorbent assay) on day 0 and day 10 of kidney
injury. All surgical procedures were carried out in accordance with
ProtocolsapprovedbytheHarvardAnimalResourcesandComparative
Medicine Committee.
Tissue preparation and histology
Mousetissueswereprepared,fixed,stored,andsectionedaspreviously
described (47). PeriodicacidSchiffstainingwasperformedonparaffin
sections using standard methods. Primary antibodies against the fol-
lowing proteins were used for immunolabeling: aSMA-Cy3 (1:200,
clone 1A4, Sigma), CD11b (1:200, eBioscience), F4/80 (1:200, Caltag),
CD45[fluoresceinisothiocyanate(FITC)-conjugated1:400,EBioscience]
CD34 (1:200, Pharmingen), and collagen III (1:400, Southern Biotech).
Fluorescence-conjugated affinity-purified secondary antibody labeling
(1:400-1:800,Jackson),mountingwithVectashield/DAPI(4′,6-diamidino-
2-phenylindole), image capture, and processing were carried out as
previously described (39, 47). For morphometric analysis of collagen
fibrilstaining,deparaffinizedsectionswerestainedwith0.1%picrosirius
red or trichrome stain (12). For morphometric analyses of collagen III
deposition and macrophage infiltration (F4/80 staining), deparaf-
finized sections or air-dried PLP (periodate-lysine-paraformaldehyde)-
fixed cryosectionswereincubated withprimary antibody,followedby
biotinylated secondary antibodies (DAKO) amplified with Vectastain
Elite ABC peroxidase staining kit, and the final stain was generated
with DAB (diaminobenzidine) as previously described (12). For mor-
phometric analysis of Coll1a1-GFP, cryosections of kidneys from
Coll-GFPmiceweremountedwithVectashield,andthedigitalimages
were captured at 200× magnification with a Nikon TE2000 micro-
scope, a Coolsnapdigital camera, andIPLabsoftware(46).For quan-
tificationofaSMAcells,CD11bcells,orCD45+,CD34+cells,counting
of random sections was carried out as previously described (29). In
brief, sections were co-labeled with DAPI, and Coll1a1-GFP–positive
cells were identified by blue and green nuclear colocalization; aSMA-
positive cells were identified by greater than 75% of the cell area im-
mediatelysurroundingthenuclei(detectedbyDAPI)stainingpositive
with Cy3 fluorescence indicative of the antigen expression. Low-
power field images were captured at 100× magnification, and high-
power field images at 400×.
To identify hSAP in kidney sections, tissue sections were deparaf-
finized, rehydrated, and boiled in citric buffer for 15 min for epitope
retrieval.Afterrinsinginphosphate-bufferedsaline(PBS),thesections
were incubatedwith 1%normal rabbitserum (30 min)then incubated
overnight at 4°C with rabbit antibody against hSAP (1:300 dilution,
Abcam, cat. no. ab 45151). After washing with PBS, the sections were
then incubated for 20 min at room temperature with Alexa Fluor
488–labeled goat secondary antibody against rabbit (1:500 dilution;
Invitrogen,cat.no.A11008).AfterwashingwithPBS,thesectionswere
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mounted with antifade mounting medium containing DAPI. Mor-
phometry was performed as previously described. To co-label hSAP
and macrophages, PLP-fixed cryosections were prepared and rabbit
antibodies against hSAP were applied in 10% goat serum (as above
but without antigen retrieval), followed by goat Cy3-conjugated
secondary antibodies against rabbit, followed by antibodies against
CD11b-FITC, with methods previously described (33), and confocal
imageswerecapturedwithaNikonC1D-Eclipseconfocalmicroscope.
Single-cell preparation from blood, bone marrow, and kidney
Mouse blood (500 ml) was collected from the inferior vena cava into
sodiumcitrate(0.38%).Micewereflushedwithice-coldPBStoremove
remainingblood,andkidneysandbonemarrowwereharvestedaspre-
viouslydescribed(33).PBMCswereisolatedfromcitratedwholeblood
with Ficoll-Paque PLUS (GE Healthcare). Single cells were resus-
pended in FACS buffer (33, 47) after centrifugation. The kidney was
decapsulated, diced, incubated (37°C, 30 min) with liberase (0.5 mg/ml,
Roche) and deoxyribonuclease (100 U/ml, Roche) in HBSS (Hanks’
balanced salt solution), then resuspended in 10 ml of FACS buffer or
PBS/1% BSA, and cells were filtered (40 mm). In some cases, leukocyte
enrichmentwascarriedoutbyresuspendingthesingle-cellsuspension
in PBS, then overlaying it on a discontinuous Percoll gradient (33%,
66% in PBS), followed by centrifugation (20 min, 620g). Mouse bone
marrow was flushed out of the femur, filtered through a 70-mm cell
strainer, and resuspended in FACS buffer or PBS/1% BSA.
MousePBMoswerepurifiedfromPBMCsbyFACSsortingCD11b+,
forward scatter (FSC) high, side scatter (SSC) low cells (FACSAria).
Human monocytes were purified from 20 ml of citrated whole blood
fromhealthynormalvolunteers.PBMCswereseparatedbyFicoll-Paque
PLUS (GE Healthcare) density centrifugation. Mouse BMMs were puri-
fied from erythrocyte-depleted bone marrow by negative selection
with MACS beads (Miltenyi Biotech). In brief, bone marrow in buffer
was incubatedwith phycoerythrin(PE)-conjugated antibodiesagainst
Ly6G, B220, and CD90. Anti-PE magnabeads were then incubated
with the leukocytes and the whole incubation mixture was passed
through a magnetic column. Nonadherent cells were washed through
the column and tested for purity by flow cytometry. There was >90%
depletion of CD90, B220, and LyG+ cells (not shown).
Flow cytometric analysis
Single cells (2 × 105) from kidney, PBMCs, or bone marrow were re-
suspendedinFACSbuffer(39)andincubatedfor30minat4°Cwithanti-
bodies against CD11b, CD16/32, F4/80 (APC-conjugated, eBioscience),
CD64 (Alexa Fluor 647–conjugated, 1:200, BD Biosciences), and
Ly6C (FITC-conjugated,1:200,BD Biosciences)at4°Cinthepresence
of 2% mouse serum. Cells were washed three times with FACS wash
buffer, then fixed in 200 ml of FACS buffer/1% PFA (paraformaldehyde),
and analyzed with a BD FACSCalibur flow cytometer. Note that in
C57Bl/6 mice, antibodies against CD16/32 recognize only CD16 (48).
For detection of mFcgRII, specific antibody against Ly-17.2 (clone
K9.361) (1:500, Sloan Kettering) (48) in 2% mouse serum was used,
followedbyFITC-conjugated,affinitypurified,goatsecondaryantibodies
againstmouseIgG(1:500,JacksonImmunoresearch).TodetectmFcgRIV,
clone 9G8.1 (1:800, Sloan Kettering)–specific antibody in 2% mouse
serum was used (49), followed by PE-conjugated goat secondary anti-
bodies against hamster IgG (BD Biosciences). In preliminary experi-
ments, isotype controls followed by secondary antibodies showed no
specific binding, and primary dilution was optimized. Specificity of
antibodies was confirmed with 3T3 cell lines expressing single FcgRs
(not shown). All studies were repeated at least three times and gave
results comparable to those presented.
Statistical analysis
Error bars represent SEM. One-way analysis of variance or t test was
carried out with GraphPad Prizm (GraphPad Software).
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50. Acknowledgments: We thank D. Brenner, B.-L. Chiang and the Common Laboratory at Na-
tional Taiwan University Hospital, D. Pilling, R. Gomer, D. Hesson, M. Kramer, L. Murray,
M. Sayegh, D. Kozoriz, and S. Jogani for assistance or advice; L.-L. Hsiao, R. Jensen, and
M. Lombardi for initial analysis of macrophage transcriptomes; and M. L. Hawes, B. A. Watkins,
and E. G. Fey (Biomodels LLC) for technical assistance in some of the UUO experiments.
Funding: Supported byNIH (grantsDK73299, DK84077,and DK87389 to J.S.D.),PromediorInc.
(J.S.D.), American Society of Nephrology (Gottschalk Award), and an award from the National
Taiwan Science Council (S.L.L.).
Author contributions: J.S.D. and M.L.L. designed and performed experiments, interpreted and
presented data, and wrote the manuscript.A.P.C, S.L.L., T.S, B.T.N, S.A.T., and S.L performed
experiments and interpreted data. T.V.P. and A.S. maintained and analyzed patient cohort.
Competing interests: T.S., M.L.L., S.L., and J.S.D. own stock options in Promedior Inc., a com-
pany that develops therapeutic agents for the treatment of fibrotic disorders and diseases.
M.L.L.isanofficeratPromedior,andJ.S.D.isapaidmemberoftheScientificAdvisoryBoard.
Promedior has assignment of or license to severalpatent applications relating to the use of
hSAP for treatment of various fibrotic disorders, including kidney fibrosis.
Submitted 22 April 2009
Accepted 16 October 2009
Published 4 November 2009
10.1126/scitranslmed.3000111
Citation: A. P. Castaño, S.-L. Lin, T. Surowy, B. T. Nowlin, S. A. Turlapati, T. Patel, A. Singh, S. Li,
M. L. Lupher Jr., J. S. Duffield, Serum amyloid P inhibits fibrosis through FcgR-dependent
monocyte-macrophage regulation in vivo. Sci. Transl. Med. 1, 5ra13 (2009).
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