FcR-Bearing Myeloid Cells Are Responsible for Triggering
Murine Lupus Nephritis1
Amy Bergtold,* Anamika Gavhane,†Vivette D’Agati,‡Michael Madaio,§and Raphael Clynes2†
Lupus glomerulonephritis is initiated by deposition of IgG-containing immune complexes in renal glomeruli. FcR engagement by
immune complexes (IC) is crucial to disease development as uncoupling this pathway in FcR??/?abrogates inflammatory re-
sponses in (NZB ? NZW)F1mice. To define the roles of FcR-bearing hemopoietic cells and of kidney resident mesangial cells in
pathogenesis, (NZB ? NZW)F1bone marrow chimeras were generated. Nephritis developed in (NZB ? NZW)F1mice expressing
activating FcRs in hemopoietic cells. Conversely, recipients of FcR??/?bone marrow were protected from disease development
despite persistent expression of FcR? in mesangial cell populations. Thus, activating FcRs on circulating hemopoietic cells, rather
than on mesangial cells, are required for IC-mediated pathogenesis in (NZB ? NZW)F1. Transgenic FcR??/?mice expressing
FcR? limited to the CD11b?monocyte/macrophage compartment developed glomerulonephritis in the anti-glomerular basement
disease model, whereas nontransgenic FcR??/?mice were completely protected. Thus, direct activation of circulating FcR-bearing
myeloid cells, including monocytes/macrophages, by glomerular IC deposits is sufficient to initiate inflammatory responses. The
Journal of Immunology, 2006, 177: 7287–7295.
tis. Studies of acute murine models of Ab-mediated inflammation
in the skin (1–4), joints (5–12), lungs (13), kidneys (14–19), and
peritoneum (20, 21) in gene-deficient mice permit the general con-
clusion that the coordinate expression of activating and inhibitory
FcRs on effector cells regulates inflammatory responses. Comple-
ment components including C5a contribute directly as chemoat-
tractants and as inducers of preferential up-regulation of activating
FcRs on effector cells (20, 22–24).
The initial events following IC deposition in the tissues include
the local activation of complement and the triggering of tissue-
resident cells though their Fc and complement receptors. The re-
sultant collective action of locally produced chemokines, cyto-
kines, and small molecule mediators of inflammation activates
endothelial cells and promotes the adhesion and diapedesis of ac-
tivated bloodborne effectors, including monocytes and neutrophils,
into the tissue. In this scenario, the recruitment of circulating cel-
lular effectors is expected to occur as a consequence of local ac-
tivation of resident tissue cells. The importance of resident cells
including tissue macrophages and mast cells in the initiation of the
mmune complex (IC)3deposition in tissue contributes to
many autoimmune disease states including systemic vascu-
litis, arthritis, blistering skin diseases, and glomerulonephri-
inflammatory cascade and subsequent recruitment of circulating
neutrophils has been demonstrated in the joints (25–27) and in
Arthus reactions in the lungs (13, 22, 28), peritoneum (21, 29, 30),
and skin (3).
In the kidney, the relevant resident cell that would be expected
to initiate the inflammatory response to ICs deposited in glomeruli
is the mesangial cell (MC). MC activation contributes directly to
glomerular pathogenesis through proliferation and collagen depo-
sition and indirectly through the production of the inflammatory
mediators (31, 32) cytokines and chemokines (31, 33). Indeed,
FcRs are expressed on cultured rodent and human MC (34–36),
and Fc?R cross-linking on cultured MC induces matrix deposition
(34) and the production of inflammatory mediators including che-
mokines (37, 38) and cytokines (39). Numerous studies have im-
plicated Fc?R cross-linking on MC as a proximal and key step in
IC-mediated nephritis, yet few studies have directly demonstrated
mesangial expression of Fc?R in vivo. Low-level expression of the
inhibitory Fc?RIIB on glomerular cells was detectable by immu-
nohistochemistry (17), but other studies have failed to detect Fc?R
at the RNA level (40). FcR??/?mice are protected from the de-
velopment of nephritis despite IC mesangial deposition (18, 19).
However, a requisite inflammatory role of FcRs on MC in vivo
Recent work in the anti-glomerular basement membrane (anti-
GBM) model suggests instead that circulating hemopoietic cells
directly engage immune deposits in the mesangium, initiating the
inflammatory response without prior recruitment by FcR-engaged
MC. Transferred wild-type (WT) neutrophils become activated in
FcR??/?hosts bearing IC mesangial deposits, arguing that acute
injury can be initiated by FcR cross-linking-circulating neutrophils
(41). In bone marrow (BM) chimeras (42) using FcR??/?and
FcR??/?donors and recipients, anti-GBM nephritis required FcR-
bearing cells in the hemopoietic compartment, suggesting that MC
FcR engagement is not necessary for the induction of the IC-me-
diated inflammatory responses (42). Although these short-term
acute models provide important mechanistic insights, the sponta-
neous nephritis model in (NZB ? NZW)F1mice most closely
approximates pathogenetic mechanisms mediating human lupus
nephritis. We have addressed the role of FcR? in intrinsic renal
*Integrated Program in Cellular, Molecular, and Biophysical Studies,†Department of
Microbiology and Medicine,‡Department of Pathology, Columbia University, Col-
lege of Physicians and Surgeons, New York, NY 10032; and§Department of Med-
icine, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104
Received for publication December 20, 2005. Accepted for publication September
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Immunology Training Program (T32 AI 07525 (to
A.B.), the National Institutes of Health/National Institute of Allergy and Infectious
Diseases RO3AR45764, and by an Investigator Award of the Arthritis Foundation
2Address correspondence and reprint requests to Dr. Raphael Clynes, Columbia Uni-
versity, College of Physicians and Surgeons, P & S Building, Room 8-510, 630 West
168th Street, New York, NY 10032. E-mail address: email@example.com
3Abbreviations used in this paper: IC, immune complex; MC, mesangial cell; GBM,
glomerular basement membrane; WT, wild type; BM, bone marrow; PAS, periodic
acid-Schiff; SLE, systemic lupus erythematosus.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
cells or hemopoietic cells in the spontaneous NZB/NZW lupus
nephritis model and find that mesangial FcR expression is not re-
quired for disease development. Furthermore, we have partially
reconstituted anti-GBM nephritis in FcR??/?by transgenic ex-
pression of FcR? in the monocyte/macrophage compartment, im-
plicating direct activation of this FcR-bearing cellular subset in the
initiation of the inflammatory phase of IC-mediated nephritis.
Thus, direct activation of FcR-bearing monocyte/macrophages is
sufficient to induce inflammatory responses in response to glomer-
ular IC deposition.
Materials and Methods
(NZB ? NZW)F1FcR??/?mice were generated from an intercross of
NZB FcR??/?male and NZW FcR??/?female mice (18). To generate
BM chimeras, 10 ? 106BM cells obtained from 3-wk-old (NZB ?
NZW)F1FcR??/?and FcR??/?mice (The Jackson Laboratory) were in-
jected i.v. into the tail vein of lethally irradiated recipients (1000 rad ? 1
dose). Chimeric mice were given oral ciprofloxacin in the water ad libitum
for 14 days after reconstitution and followed for the development of pro-
teinuria weekly for 9 mo. Proteinuria was read using Urostix for the NZB/
NZW mice and scored positive if 2? measurements (?250 mg/dl) were
recorded for two successive readings. A subset of mice was sacrificed at 6
mo for histopathological analysis of the kidney. These studies were re-
viewed and approved by the Institution Animal Care and Use Committee
of Columbia University.
CD11b-? Tg?mice were generated after injection of oocytes obtained
from FcR??/?mice. The transgenic construct was generated by insertion
of the murine FcR? cDNA (550-bp fragment) as an EcoRI fragment (43)
into pB203 (a gift from Dr. D. G. Tenen, Harvard Medical School, Boston,
MA; see Ref 44) containing the 1.7-kb 5?-flanking sequences of the mouse
CD11b promoter and the 3?-flanking region from the human growth hor-
mone gene. A NotI/HindIII fragment (containing 5?-CD11bpromoter-FcR?
cDNA-hGH-3?) was injected into the oocytes and three founder lines har-
boring the transgene were further analyzed for expression. Of these three
founders, only one (line 14) expressed the FcR? chain in peritoneal
Accelerated anti-GBM nephritis
Mice were immunized with 100 ?g of sheep IgG in CFA 3 days before i.v.
injection of 150 ?l of specific sheep anti-mouse GBM sera. Urine was
obtained daily and blood obtained on the day before injection with anti-
GBM sera and then at the time of sacrifice 7 days later. Urine samples were
diluted in PBS and protein quantified by the Bradford method (Bio-Rad)
using an ELISA plate reader at OD570.
Anti-dsDNA and soluble immune complex ELISAs
Diluted serum (1/100) from 6- to 7-mo-old NZB/NZW-??/?and NZB/
NZW-??/?mice were added to ELISA plates coated with C1q (Sigma-
Aldrich) for detection of ICs (45, 46) and to dsDNA-coated plates (United
Biotech) for detection of Abs to chromatin. After washing away unbound
serum, rat anti-mouse IgG (BD Pharmingen) was added. Alkaline phos-
phatase-conjugated AKP polyclonal anti-rat IgG (BD Pharmingen) was
used as secondary Ab. After incubation with p-nitrophenyl phosphate sub-
strate, the samples were read spectrophotometrically at 405 nm with an
ELISA reader (Molecular Devices).
Immunofluorescence and immunohistochemistry
For histological analysis, formalin-fixed sections were stained with H&E or
periodic acid-Schiff (PAS). To detect IC deposition, paraformaldehyde- or
acetone-fixed cryosections were stained with (1/1000 diluted) FITC goat
anti-mouse C3 and IgG (Valeant Pharmaceuticals). To detect FcR?, a poly-
clonal anti-FcR? rabbit IgG (gift from Dr. J. Ravetch, The Rockefeller
University) or rat anti-Mac-1 (clone C71/16; BD Pharmingen) followed by
rabbit anti-rat IgG Alexa594 (Molecular Probes). Biotinylated goat anti-
rabbit IgG, followed by either streptavidin-FITC or streptavidin-HRP was
used for detection. A Nikon Eclipse 600 microscope equipped with a RT
Spot digital camera was used for imaging.
Renal pathological assessment
PAS sections were prepared from WT, FcR??/?, and FcR??/?CD11b-?
Tg?kidneys on day 7 after induction of accelerated glomerulonephritis
(five per group). Slides were examined in a blinded fashion by one of us
(V. d’A.). Severity of the following seven categories of histological activity
were semiquantitatively graded as follows: glomerular fibrinoid necrosis
0–4, endocapillary hypercellularity 0–4, glomerular leukocyte infiltration
0–4, crescents 0–4, tubular degeneration 0–4, casts 0–4, and interstitial
inflammation 0–4.The cumulative pathological score is the sum of all
seven categories and has a possible range of 0–28.
MC and NK culture
Glomeruli were isolated with successive sieving (47). Kidneys were
minced with scissors and tissue fragments were passed through a no. 60
mesh sieve (Fisher Scientific) and then sequentially passed through no. 100
and no. 200 sieves. Glomeruli were digested with 0.1% collagenase type IV
(Sigma-Aldrich) and 0.1% trypsin (Invitrogen Life Technologies) for 30
min at 37°C before plating in 24 wells in DMEM/10% FCS. Cells were
passaged in D-valine-substituted medium to eliminate fibroblasts. After 2
wk in culture, cells exhibited a stellate morphology and were replated.
Immunostains were smooth muscle actin-positive, weakly 2.4G2?and
Mac-1?, confirming their MC origin. RNA was prepared from MCs using
TRIzol and cDNA was generated using the cloned avian myeloblastosis
virus first-strand synthesis kit according to the manufacturer’s protocol
(Invitrogen Life Technologies). Primer sequences for RT-PCR amplifica-
tion (30 cycles) of FcR? were as follows: 5?-CCAGGATGATCTC
AGCCG-3? and 5?-ACAGTAGAGTAGGGTAAG-3?. These primers am-
plify a 137-bp band corresponding to exons 1 and 2 of the ? subunit. The
band is not amplified in genomic DNA due to intervening intronic se-
quences. The housekeeping gene, HPRT, was amplified from cDNA using
the following primer sequences: 5?-AGCTACTGTAATGATCAGTCA
ACG-3? and 5?-AGAGGTCCTTTTCACCAGCA-3?. For assessing MC
chimerism, genomic DNA was subjected to PCR analysis using the fol-
lowing primer sequences: neo, CTCGTGCTTTACGGTATCGCC; ?-1,
TATAGCTGCCTT. Annealing temperature was 62°C. Knockout and WT-
amplified products were 260 and 224 bp, respectively.
Hemopoietic chimerism was assessed in cultured NK cells obtained af-
ter isolation of the adherent cell population from a 14-day culture of nylon
wool nonadherent splenocytes grown in IL-2 (10,000 U/ml). Flow cyto-
metric analysis used anti-NK1.1 PE and 2.4G2-FITC (BD Pharmingen).
Murine NK cells do not express FcRIIb (48) and thus the anti-FcRII/III
mAb (2.4G2) recognizes only FcRIII on these cells.
Western blot analysis of FcR? expression
Protein extracts were obtained from B cells, T cells, NK cells, and neu-
trophils were immunoblotted with polyclonal rabbit anti-mouse FcR?
chain IgG and anti-?-actin Abs. Neutrophils were obtained from thiogly-
colate- elicited peritoneal exudates (4 h after i.p. injection of thioglycolate)
after GR-1?bead selection (Miltenyi Biotec). Adherent peritoneal macro-
phages were obtained from thioglycolate-elicited exudates (72 h after i.p.
injection). B and T cells were obtained from CD43?and CD3?splenocyte
populations, respectively. All cell populations were lysed in TBS buffer
that contained 1% Triton X-100, 2 mM EDTA, and complete mini-protease
Rabbit IgG-opsonized SRBCs were prepared with subagglutinating quan-
tities of rabbit anti-sheep RBC IgG (MP Biomedicals). After washing away
free Ab, IgG-opsonized RBCs were added to adherent macrophages for 1 h
at 37°C. Unphagocytosed RBCs were removed by osmotic lysis, and
phagocytosis plates were fixed with PBS/0.25% glutaraldehyde before mi-
Blood albumin and urea nitrogen measurements
Blood samples were read by the Clinical Chemistry Laboratory of the
Irving Clinical Research Center at Columbia-Presbyterian Hospital.
BM chimeric NZB/NZW mice reveal a requirement for
FcR?-expressing hemopoietic cells for nephritis development
(NZB ? NZW)F1female mice develop a uniformly fatal rapidly
progressive IC nephritis heralded by the serological appearance of
anti-chromatin IgG autoantibodies at 4–6 mo of age. Disease pro-
gression is swift, with a median survival of 180 days. However, in
(NZB ? NZW)F1FcR ??/?female mice, IgG autoantibodies oc-
cur with equivalent titers and are deposited similarly in the kidney,
7288FcR-BEARING MYELOID CELLS TRIGGER LUPUS NEPHRITIS
46. Uwatoko, S., M. Mannik, I. R. Oppliger, M. Okawa-Takatsuji, S. Aotsuka,
R. Yokohari, G. Seki, S. Taniguchi, K. Suzuki, and K. Kurokawa. 1995. C1q-
binding immunoglobulin G in MRL/l mice consists of immune complexes con-
taining antibodies to DNA. Clin. Immunol. Immunopathol. 75: 140–146.
47. Luo, Y., C. Lloyd, J. C. Gutierrez-Ramos, and M. E. Dorf. 1999. Chemokine
amplification in mesangial cells. [Published erratum appears in 2000 J. Immunol.
164: 5332.] J. Immunol. 163: 3985–3992.
48. Takai, T., M. Li, D. Sylvestre, R. Clynes, and J. V. Ravetch. 1994. FcR ? chain
deletion results in pleiotrophic effector cell defects. Cell 76: 519–529.
49. Wakayama, H., Y. Hasegawa, T. Kawabe, T. Hara, S. Matsuo, M. Mizuno,
T. Takai, H. Kikutani, and K. Shimokata. 2000. Abolition of anti-glomerular
basement membrane antibody-mediated glomerulonephritis in FcR?-deficient
mice. Eur. J. Immunol. 30: 1182–1190.
50. Park, S. Y., S. Ueda, H. Ohno, Y. Hamano, M. Tanaka, T. Shiratori, T. Yamazaki,
H. Arase, N. Arase, A. Karasawa, et al. 1998. Resistance of Fc receptor- deficient
mice to fatal glomerulonephritis. J. Clin. Invest. 102: 1229–1238.
51. Ito, T., A. Suzuki, E. Imai, M. Okabe, and M. Hori. 2001. Bone marrow is a
reservoir of repopulating mesangial cells during glomerular remodeling. J. Am.
Soc. Nephrol. 12: 2625–2635.
52. Imasawa, T., Y. Utsunomiya, T. Kawamura, Y. Zhong, R. Nagasawa, M. Okabe,
N. Maruyama, T. Hosoya, and T. Ohno. 2001. The potential of bone marrow-
derived cells to differentiate to glomerular mesangial cells. J. Am. Soc. Nephrol.
53. Tang, T., A. Rosenkranz, K. J. Assmann, M. J. Goodman, J. C. Gutierrez-Ramos,
M. C. Carroll, R. S. Cotran, and T. N. Mayadas. 1997. A role for Mac-1 (CDIIb/
CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 defi-
ciency abrogates sustained Fc? receptor-dependent neutrophil adhesion and com-
plement-dependent proteinuria in acute glomerulonephritis. J. Exp. Med. 186:
54. Ikezumi, Y., L. A. Hurst, T. Masaki, R. C. Atkins, and D. J. Nikolic-Paterson.
2003. Adoptive transfer studies demonstrate that macrophages can induce pro-
teinuria and mesangial cell proliferation. Kidney Int. 63: 83–95.
55. Lea, P. J., M. Silverman, R. Hegele, and M. J. Hollenberg. 1989. Tridimensional
ultrastructure of glomerular capillary endothelium revealed by high-resolution
scanning electron microscopy. Microvasc. Res. 38: 296–308.
56. Drumond, M. C., and W. M. Deen. 1994. Structural determinants of glomerular
hydraulic permeability. Am. J. Physiol. 266: F1–F12.
57. Huang, X. R., P. G. Tipping, J. Apostolopoulos, C. Oettinger, M. D’Souza,
G. Milton, and S. R. Holdsworth. 1997. Mechanisms of T cell-induced glomer-
ular injury in anti-glomerular basement membrane (GBM) glomerulonephritis in
rats. Clin. Exp. Immunol. 109: 134–142.
58. Fujii, T., Y. Hamano, S. Ueda, B. Akikusa, S. Yamasaki, M. Ogawa, H. Saisho,
J. S. Verbeek, S. Taki, and T. Saito. 2003. Predominant role of Fc?RIII in the
induction of accelerated nephrotoxic glomerulonephritis. Kidney Int. 64:
59. Takai, T. 2005. Fc receptors and their role in immune regulation and autoimmu-
nity. J. Clin. Immunol. 25: 1–18.
60. Salmon, J. E., S. Millard, L. A. Schachter, F. C. Arnett, E. M. Ginzler,
M. F. Gourley, R. Ramsey-Goldman, M. G. Peterson, and R. P. Kimberly. 1996.
Fc?RIIA alleles are heritable risk factors for lupus nephritis in African Ameri-
cans. J. Clin. Invest. 97: 1348–1354.
61. Wu, J., J. C. Edberg, P. B. Redecha, V. Bansal, P. M. Guyre, K. Coleman,
J. E. Salmon, and R. P. Kimberly. 1997. A novel polymorphism of Fc?RIIIa
(CD16) alters receptor function and predisposes to autoimmune disease. J. Clin.
Invest. 100: 1059–1070.
62. Li, X., J. Wu, R. H. Carter, J. C. Edberg, K. Su, G. S. Cooper, and R. P. Kimberly.
2003. A novel polymorphism in the Fc? receptor IIB (CD32B) transmembrane
region alters receptor signaling. Arthritis Rheum. 48: 3242–3252.
63. Blank, M. C., R. N. Stefanescu, E. Masuda, F. Marti, P. D. King, P. B. Redecha,
R. J. Wurzburger, M. G. Peterson, S. Tanaka, and L. Pricop. 2005. Decreased
transcription of the human FCGR2B gene mediated by the ?343 G/C promoter
polymorphism and association with systemic Lupus Erythematosus. Hum. Genet.
7295 The Journal of Immunology