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The Inhibiting Fc Receptor for IgG, Fc RIIB, Is a Modifier of Autoimmune Susceptibility
BOROSS, P., et al.
BOROSS, P., et al . The Inhibiting Fc Receptor for IgG, Fc RIIB, Is a Modifier of Autoimmune Susceptibility. Journal of Immunology , 2011, vol. 187, no. 3, p. 1304-1313
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Is a Modifier of Autoimmune Susceptibility RIIB, γ
The Inhibiting Fc Receptor for IgG, Fc
Verbeek Terence Cook, Shozo Izui, Marina Botto and J. Sjef
Maria Pia Rastaldi, Ferry Ossendorp, Mohamed R. Daha, H.
Claassens, Marcel Camps, Erik Lubberts, Daniela Salvatori, Flierman, Jos van der Kaa, Cor Breukel, Jill W. C.
Marie-Laure Santiago-Raber, Francesco Carlucci, Roelof Peter Boross, Victoria L. Arandhara, Javier Martin-Ramirez,
http://www.jimmunol.org/content/187/3/1304 doi: 10.4049/jimmunol.1101194
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2011; 187:1304-1313; Prepublished online 1 July J Immunol
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The Journal of Immunology
The Inhibiting Fc Receptor for IgG, FcgRIIB, Is a Modifier of Autoimmune Susceptibility
Peter Boross,*
,1,2Victoria L. Arandhara,*
,1,3Javier Martin-Ramirez,*
,1,4Marie-Laure Santiago-Raber,
†Francesco Carlucci,
‡Roelof Flierman,
xJos van der Kaa,*
Cor Breukel,* Jill W. C. Claassens,* Marcel Camps,
{Erik Lubberts,
‖,#Daniela Salvatori,**
Maria Pia Rastaldi,
††Ferry Ossendorp,
{Mohamed R. Daha,
xH. Terence Cook,
‡Shozo Izui,
†Marina Botto,
‡and J. Sjef Verbeek*
FcgRIIB-deficient mice generated in 129 background (FcgRIIB
1292/2
) if back-crossed into C57BL/6 background exhibit a hyper- active phenotype and develop lethal lupus. Both in mice and humans, the
Fcgr2bgene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named
Sle16,causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of FcgRIIB deficiency to the development of lupus in FcgRIIB
1292/2mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of “loss of function” mutations in the
Fcgr2bgene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that FcgRIIB
2/2mice generated by gene targeting in B6-derived ES cells (FcgRIIB
B62/2
), lacking the 129-derived flanking
Sle16region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in FcgRIIB
1292/2
mice, not FcgRIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived
Fcgr2bflanking region cause loss of toler- ance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of FcgRIIB, culminating in lethal lupus. In the presence of the
Yaalupus-susceptibility locus, FcgRIIB
B62/2mice do develop lethal lupus, confirming that FcgRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci.
The Journal of Immunology, 2011, 187: 1304–1313.T he hyperactive phenotype of FcgRIIB KO mice confirms the important role of this inhibiting receptor for IgG in the negative regulation of activating Fc receptors on mye- loid effector cells and in a negative feedback mechanism in B cells, controlling Ab production. However, the lupus phenotype of FcgRIIB2/2mice generated by gene targeting in 129-derived ES cells (FcgRIIB
1292/2) and back-crossed into C57BL/6 (B6) background (1) is surprising because genetic studies revealed that lupus susceptibility is a multigenic phenotype (2).
Genetic and functional studies have revealed contradictory data, making the role of FcgRIIB in autoimmunity controversial.
The Fcgr2b gene is located in a locus within the distal region of
mouse chromosome (Chr) 1, which is orthologous with an SLE- associated locus on human Chr 1. All lupus-prone mouse strains share the lupus-associated Sle1 haplotype of that locus, but B6 mice do not. In B6 congenic mice, the Sle1 locus causes loss of tolerance leading to anti-nuclear Ab (ANA) production, mild splenomegaly, but no fatal glomerulonephritis. The following ob- servations in autoimmune-prone mice and systemic lupus erythe- matosis (SLE) patients suggest that the association with lupus of the Sle1 locus and its human ortholog can be attributed, at least partially, to polymorphisms in the Fcgr2b gene. Autoimmune- prone strains share a promoter haplotype and/or polymorphisms in the third intron of the Fcgr2b gene, which are associated with
*Department of Human Genetics, Leiden University Medical Center, 2333 ZA Lei- den, The Netherlands;†Department of Pathology and Immunology, University of Geneva, 1211 Geneva, Switzerland;‡Division of Immunology and Inflammation, Centre for Complement and Inflammation Research, Imperial College, London W12 ONN, United Kingdom;xDepartment of Nephrology, Leiden University Med- ical Center, 2333 ZA Leiden, The Netherlands;{Department of Immunohematology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands;‖Department of Rheumatology, Erasmus MC University Medical Center, 3000 CA Rotterdam, The Netherlands;#Department of Immunology, Erasmus MC University Medical Center, 3000 CA Rotterdam, The Netherlands; **Department of Anatomy, Leiden Univer- sity Medical Center, 2333 ZA Leiden, The Netherlands; and††Laboratorio di Ricerca Nefrologica, Fondazione Instituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20122 Milan, Italy
1P.B., V.L.A., and J.M.-R. contributed equally to this work.
2Current address: Immunotherapy Laboratory, Department of Immunology, Univer- sity Medical Center Utrecht, Utrecht, The Netherlands.
3Current address: School of Sport, Exercise and Health Science, Loughborough University, Loughborough, United Kingdom.
4Current address: Sanquin, Research Department, Plasma Proteins U-019, Amster- dam, The Netherlands.
Received for publication April 29, 2011. Accepted for publication May 25, 2011.
This work was supported by the FP6 Marie Curie Research Training Network Im- mune Deficient Mice (MRTN-CT-2004-005632 to V.L.A. and J.M.-R.), the Dutch Arthritis Association (to J.M.-R.), and the Swiss National Foundation for Scientific Research (to S.I.).
P.B., F.O., M.R.D., H.T.C., S.I., M.B., and J.S.V. designed research; P.B., V.L.A., J.M.-R., M.-L.S.-R., F.C., R.F., J.v.d.K., C.B., J.W.C.C., M.C., E.L., D.S., and M.P.R. performed research; P.B., J.v.d.K., C.B., J.W.C.C., S.I., and M.B. contributed analytical tools; P.B., V.L.A., J.M.-R., R.F., E.L., D.S., M.P.R., F.O., M.R.D., H.T.C., S.I., M.B., and J.S.V.
analyzed data; and P.B., V.L.A., J.M.-R., S.I., M.B., and J.S.V. wrote the article.
Address correspondence and reprint requests to Dr. J. Sjef Verbeek, Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Lei- den, The Netherlands. E-mail address: j.s.verbeek@lumc.nl
The online version of this article contains supplemental material.
Abbreviations used in this article: ANA, anti-nuclear Ab; B6, C57BL/6; Chr, chro- mosome; CIA, collagen-induced arthritis; CII, collagen type II; HSA, human serum albumin; IC, immune complex; KO, knockout; NTN, nephrotoxic nephritis; PAS, periodic acid–Schiff; SLE, systemic lupus erythematosis; SNP, single nucleotide polymorphism; SRBC, sheep RBC; SSLP, simple sequence length polymorphism;
TNP-KLH, 2,4,6, trinitrophenyl hapten conjugated keyhole limpet hemocyanin.
CopyrightÓ2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
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reduced FcgRIIB expression in germinal center B cells and plasma cells (3, 4). In humans, the frequency of homozygosity for a single amino acid polymorphism (232T) in FcgRIIB, resulting in
“loss of function” (5), is at least doubled in individuals with SLE compared with that in healthy controls in three Asian and one Caucasian cohort (reviewed in Ref. 6).
Several other observations, however, argue against a role of FcgRIIB in lupus. In humans, the association between the 232T polymorphism and SLE could not be confirmed with an African–
American or Caucasian–American cohort (7). A recent genome- wide association study in women of European ancestry with SLE identified susceptibility variants in 15 different genes but not in the Fcgr2b gene (8). Moreover, a strong SLE association in Caucasians of a “gain of function” mutation in FcgRIIB, resulting in increased promoter activity, has been reported (9). In mice, within the Sle1 locus four subloci, Sle1a–d, have been identified.
In congenic B6 mice the presence of Sle1b is sufficient for the loss of tolerance resulting in ANA production. Surprisingly, genetic fine mapping placed the Fcgr2b gene not in, but just between, the Sle1a and Sle1b subloci (10). Moreover, a B6 strain congenic for the Fcgr2b allele derived from the NZW autoimmune-prone strain did not develop autoimmunity (11).
Importantly, the lupus-associated Sle1b haplotype is also present in the non-autoimmune strain 129. In agreement with this, a B6 strain congenic for the 129-derived distal part of Chr 1, encom- passing the Sle1b locus, named Sle16, develops an autoimmune phenotype (12). As a consequence, it may be the presence of the Sle16 locus that determines the autoimmune phenotype of FcgRIIB
1292/2mice. To analyze the biological function of FcgRIIB independently from the confounding effect of the Sle16 locus, we generated an FcgRIIB KO strain (FcgRIIB
B62/2) by gene targeting in B6-derived ES cells. FcgRIIB
B62/2mice displayed a hyperactive phenotype but did not develop spontaneous autoim- munity. However, when the FcgRIIB
B62/2strain was crossed with the autoimmune-prone Yaa B6 strain, FcgRIIB
B62/2.Yaa offspring did develop fatal lupus. These observations indicate that FcgRIIB deficiency does not cause autoimmunity but amplifies spontaneous autoimmunity determined by autoimmune susceptibility loci, such as Sle16 or Yaa.
Materials and Methods
Generation of the conditional C57BL/6 FcgRIIB KO mouse model
For the generation of FcgRIIB2/2 mice on C57BL/6 background (FcgRIIBB62/2), a targeting vector was constructed based on a 12-kb fragment derived from the BAC clone RPCI23-87B18 of the RPCI 23 Female (C57BL/6J) mouse BAC genomic library (BACPAC Resources Center, Children’s Hospital Oakland Research Institute, Oakland, CA). By using conventional cloning techniques, a neomycin selection cassette, flanked by loxP sites, was inserted in intron 3, and a third loxP site was inserted in intron 5 (Supplemental Fig. 1A). Gene targeting was performed in C57BL/6-derived ES cells (Bruce4, kindly provided by Dr. Colin Stewart [National Cancer Institute, Frederick, MD]). Clones in which homologous recombination occurred were identified by Southern blotting (Supplemental Fig. 1B) and subsequently injected in albino C57BL/6 blastocysts. The obtained chimeras were crossed with albino C57BL/6 mice, and the F1 positive for the FcgRIIB-targeted allele was crossed with the EIIaCre deleter strain and the resulting F2 with C57BL/6. The EIIaCre negative F3 were intercrossed resulting in the following homo- zygous recombinant FcgRIIB strains: the FcgRIIBB6fl/fl with the floxed FcgRIIB gene without neo selection marker gene, and the FcgRIIBB62/2, the FcgRIIB null mutant, in which the exons 4 and 5, encoding the second extracellular ligand binding and transmembrane domains, are deleted (Supplemental Fig. 1A). Cre-mediated recombination was analyzed with PCR and Southern blot (Supplemental Fig. 1C, 1D). Primer sequences were as follows: P1, 59-CCTTCATCTTTCCTGCCAAG-39; P2, 59-AAC- ACCAGCTGAGGGGTCT-39; P3, 59-CACCACTGTGGAGAAGGATG-39;
P6, 59-TCACACCAGCAACACCTCTC-39.
The phenotype of FcgRIIBB62/2mouse was analyzed in a series of in vitro and in vivo assays and compared with the phenotype of the FcgRIIB1292/2 mouse (generated with 129-derived ES cells and sub- sequently back-crossed eight generations on C57BL/6 background).
FcgRIIBB6 fl/fl
and C57BL/6J mice, which responded similarly in all assays, were used as controls. FcgRIIBB62/2and FcgRIIBB6fl/flmice developed normally and showed normal breeding characteristics. No gross abnor- malities were observed in these mice. The expression of FcgRIIB on splenic B cells from FcgRIIBB6fl/fl
and C57BL/6 mice was similar, whereas no FcgRIIB expression was observed in FcgRIIBB62/2 mice (Supple- mental Fig. 1E). Similar results of FcgRIIB expression were obtained with B cells and myeloid cells isolated from peripheral blood, bone marrow, and peritoneal cavity (data not shown).
Mice
FcgRIIB1292/2 mice, generated on 129 background, were a gift of Dr.
T. Takai (Department of Experimental Immunology, Institute of Devel- opment, Aging and Cancer, Tohoku University, Sendai, Japan) (13) and back-crossed in our facility into C57BL/6J (Charles River, Maastricht, The Netherlands) background for eight generations. FcgRIII2/2 mice back- crossed into C57BL/6J background for 13 generations have been de- scribed previously (14). The C57BL6.Yaa congenic strain was generated as described (15). C57BL/6.Yaa FcgRIIBB62/2.Yaa mice were generated by intercrossing F1 progeny from FcgRIIBB62/2females and C57BL/6.Yaa males.
The EIIaCre deleter strain (n= 12 on C57BL/6J background), was a kind gift of Dr. Heiner Westphal (National Institute of Child Health and Human Development, Bethesda, MD). C57BL/6J control mice were purchased from Charles River. Mice were housed and all experiments were performed at the specified pathogen free animal facilities of the Leiden University Medical Center except for the aging experiment with cohorts of male mice of the C57BL/6.Yaa congenic strain and the FcgRIIBB62/2.Yaa strain, which was performed at the animal facilities of the University of Geneva. All experi- ments were approved by the local ethical committee and performed in ac- cordance with national guidelines and regulations. Six- to twelve-week-old mice were used for the experiments unless stated otherwise. Mice were routinely checked for their genotype by PCR and cytofluorimetry.
Simple sequence length polymorphism and single nucleotide polymorphism analysis
Genotyping of the mice was carried out using simple sequence length polymorphism (SSLP) markers (listed in Supplemental Table IA), standard PCR, and either 4% MetaPhor agarose (CambrexBioscience Rockland, Rockland, ME) or 16% polyacrylamide gels stained with ethidium bro- mide. The positions and sequences were determined from the Ensembl (http://www.ensembl.org/index.html) and Mouse Genome Informatics (http://www.informatics.jax.org) databases. Single nucleotide polymor- phisms (SNPs) in the FcgRIIB flanking regions reported to be different between the 129 and C57BL/6 mouse strains (Supplemental Table IA) were amplified by PCR. The PCR products were purified and their nu- cleotide sequence determined.
Immunization with TNP–KLH
At day 0, mice were immunized i.p. with 100mg 2,4,6, trinitrophenyl hapten conjugated keyhole limpet hemocyanin (TNP–KLH) (Biosearch Technol- ogies) in alum (Merck) and boosted on day 49 with 100mg TNP–KLH without alum. Serial blood samples were collected via the retro-orbital plexus on days 21, 28, and 56 after the primary immunization. The Ab response was determined by ELISA. Plates were coated with TNP–KLH (20 mg/ml) in 0.1 M carbonate buffer, pH 9.5, overnight at 4˚C. After washing with PBS/0.1% BSA/0.05% Tween 20, plates were blocked with PBS/1%
BSA for 1 h at room temperature in a humid chamber, and samples, diluted in PBS (1:16,000), were incubated overnight at 4˚C. Plates were washed five times with PBS/0.05% Tween 20 and incubated with rabbit anti-mouse IgG HRP (1:2000) or rabbit anti-mouse IgG1 HRP (1:1000) in PBS/1% BSA/
0.01% Tween 20 for 2 h at room temperature. Plates were developed using ABTS (Sigma), and their OD was read at 405 nm. Samples were compared with a standard curve prepared from the pooled sera.
In vitro phagocytosis assay
Bone marrow-derived macrophages were cultured from mice in 15% L-cell conditioned medium (L929) for 8 d, pretreated overnight with 20 ng/ml recombinant IL-4 to increase FcgRIIB expression, and subsequently in- cubated at 37˚C for 1 h with TNP-conjugated sheep RBCs (SRBCs) opsonized with anti-TNP IgG1 Abs. The ingested SRBCs were counted using a light microscope.
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Passive cutaneous anaphylaxis
Mice were injected intradermally in the ear with 20ml of varying con- centrations (1–3mg) IgG1 anti-TNP mAb and 2 h later given an i.v. in- jection of 500mg human serum albumin (HSA)–TNP in 100ml PBS with 1% Evans blue. After 30 min, extravasation was visualized by blue staining of the ear as described previously (14).
Monitoring the development of spontaneous autoimmune disease
Mice were monitored up to the age of 12 mo, and mortality was recorded.
Moribund animals were killed before the 12 mo deadline and were counted as deceased. Blood and urine was collected periodically, and serological analyses were performed by ELISA to determine the autoantibody levels.
After sacrifice, the mice were weighed, and organs were removed. Spleens were weighed, and kidneys were fixed in Bouin’s solution (Sigma) for 4 h, transferred to ethanol 70%, and finally embedded in paraffin, whereas splenocytes were analyzed by flow cytometry.
Serological analyses
Antinucleosome Abs.Serum levels of anti-nucleosome Abs were deter- mined by ELISA. Plates were coated overnight with S2 fraction of oligonucleosomes isolated from L1210 cells. After blocking with PBS/5%
FCS/0.05% Tween 20, the plates were incubated with serially diluted serum (1:100, 1:200, 1:400) for 2 h at room temperature. Bound Abs were detected using goat anti-mouse Ig–HRP. Plates were developed with ABTS, and the OD was read at 415 nm. Titers were determined by comparison with a sample with a known concentration of mouse anti-nucleosome mAbs.
Anti-dsDNA, anti-ssDNA, anti-chromatin, and anti-histone ELISA.Levels of anti-dsDNA, anti-ssDNA, anti-chromatin, and anti-histone Abs were measured by ELISA as described previously (16). Results were expressed as arbitrary units relative to a standard positive sample derived from an MRL/Mp.lpr/lprmouse pool.
Detection of Coombs Abs.A flow cytometric assay was used to detect Coombs Abs using biotinylated rat anti-mousekchain mAb (H139.52.1.5), followed by PE-conjugated streptavidin, as described previously (17). The results are expressed as mean fluorescence intensity (MFI), analyzed with a FACSCalibur (BD Biosciences, San Jose, CA).
Assessment of renal pathology
Histological sections. Four-micrometer-thick kidney sections were pro- cessed and assessed as described previously (18).
IgG and C3 deposition. Kidneys were embedded in OCT, snap-frozen in isopentane cooled with liquid nitrogen, and stored at270˚C. Five- micrometer-thick frozen sections were processed and incubated as de- scribed previously (18). All Abs were used at a dilution of 1:200 (except anti-C3 at 1:100). For semiquantitative immunofluorescence, all sections were examined at3600 original magnification with an Olympus AX/70 Provis microscope and 2MP slider camera (Diagnostic Instruments). The mean intensity of 20 glomeruli for each sample was recorded in arbitrary fluorescence units as 0/trace, 1+, 2+, 3+, 4+.
Proteinuria.Albumin concentration was determined in urine samples of mice using a standard rocket immunoelectrophoresis assay. A series of diluted urine samples (1:2, 1:4, or 1:16) in PBS alongside control samples of purified mouse albumin were run for 5–6 h at 500 V/40 mA in a 1% agarose gel in the presence of polyclonal rabbit anti-mouse albumin in Gelman buffer. The gel was dried overnight, stained with Coomassie blue, and analyzed.
Flow cytometry
Single-cell suspensions of spleens were prepared by mechanical disrup- tion and labeled using specific mAbs. For flow cytometry analysis, CD4–PE (clone RM4-5; BD Biosciences), CD8b.2–FITC (clone 53-5.8; BD Bio- sciences), CD19–allophycocyanin (clone 1D3; BD Biosciences), CD62L–
allophycocyanin (clone MEL-14; BD Biosciences), CD69–FITC (clone H1.2F3; BD Biosciences), IgD–FITC (clone 11-26; eBioscience), and IgM–PE (clone eB121-15F9; eBioscience), B220–PE (BD Pharmingen), 2.4G2–FITC (BD Pharmingen), and K9.361–Alexa 488 anti-Ly17.2 (clone K9.361; B6/129-type FcgRIIB-specific) were used. Data acquisition was performed on a BD Biosciences FACScan or LSRII. For data analysis, CellQuest software was used.
Induction of arthritis
Induction and clinical evaluation of collagen-induced arthritis (CIA) and K/
B3N serum-induced arthritis was performed as described (19). In the CIA
model, on days 35 and 60 after the primary immunization, serum was collected for the measurement of anti-collagen type II (CII) Ab titers by ELISA. Because of the robust nature of the K/B3N serum-induced ar- thritis model, the relative increase in arthritis severity in the FcgRIIB2/2 mice became only apparent when a suboptimal amount of serum (7.5ml/g body weight) was injected, which induced only mild arthritis in control mice.
Induction of experimental nephritis
Induction and clinical evaluation of accelerated nephrotoxic nephritis (NTN) in mice was performed as described previously (18). Mice were assessed daily for the development of proteinuria and hematuria (Haema Combistix).
Monocytosis
PBMCs were stained with M1/70 anti-CD11b and AFS98 anti-CD115 mAb in the presence of saturating concentration of 2.4G2 anti-FcgRII/III mAb and analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA), as described previously (20). Percentages of CD11b+CD115+mono- cytes (distinguished from polymorphonuclear leukocytes by their lower granularity, as reflected in low side-light scatter) among PBMCs were determined.
Statistics
Parametric data were represented as mean6SEM. Multiple groups were compared by ANOVA and further evaluated using Bonferroni or unpaired Studentttest. Nonparametric data were represented by median and mul- tiple groups compared by using a Kruskal–Wallis test or Mann–WhitneyU test. Apvalue of 0.05 was considered statistically significant. Cumulative mortality was processed by using Gehan–Breslow–Wilcoxon test.
Results
Establishing two independent FcgRIIB
2/2C57BL/6 mouse strains, which differ in their Fcgr2b flanking regions
To investigate the contribution of the FcgRIIB-flanking chro- mosomal region to the phenotype of the FcgRIIB
2/2mice, two different FcgRIIB
2/2strains were generated, one with a 129- derived and the other with a B6-derived flanking region, Fcg- RIIB
1292/2and FcgRIIB
B62/2, respectively. The FcgRIIB
1292/2mouse was generated by gene targeting in 129-derived ES cells (13) and back-crossed for eight generations into C57BL/6/J background in our laboratory. The FcgRIIB
B62/2strain was generated by gene targeting in B6-derived Bruce4 ES cells and back-crossed for three generations into C57BL6/J background (Supplemental Fig. 1). The 129 and B6 origin of the flanking regions was confirmed by SNP analysis (Supplemental Table IA).
In both strains, the B6 origin of 17 SSLPs with linkage to SLE, on Chr 3 (16), 9, 12, and 17 (21), was confirmed as well as the B6 origin of 8 SSLPs on Chr 7, and Chr 13 in the FcgRIIB
B62/2strain (Supplemental Table IB).
Both FcgRIIB
B62/2and FcgRIIB
1292/2mice exhibit a hyperactive phenotype
FcgRIIB
B62/2and FcgRIIB
1292/2mice showed a similar increase in Ab responses after immunization, in mast cell-mediated cuta- neous anaphylaxis, and in phagocytosis of immune complex (IC) by macrophages compared with wild-type controls (Fig. 1A–C).
FcgRIIB
B62/2mice were susceptible to CIA (Fig. 1D, 1E), al- though incidence was lower and onset was slightly delayed com- pared with incidence and onset of CIA in FcgRIIB
1292/2mice (22). An explanation might be that FcgRIIB
1292/2mice developed higher anti-CII titers compared with those of FcgRIIB
B62/2mice.
However, the difference is not significant due to the strong vari- ation in anti-CII titers in FcgRIIB
1292/2mice, which might be attributed to their mixed 129/B6 background (Supplemental Fig.
2). In the passive K/B3N serum-induced arthritis model, which depends exclusively on downstream Ab effector pathways, both FcgRIIB KO strains developed more severe disease compared with
1306 FcgRIIB IS A MODIFIER OF AUTOIMMUNE SUSCEPTIBILITY
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FIGURE 1. FcgRIIBB62/2mice exhibit a hyperactive phenotype.A, Increased Ag-specific serum IgG titers after immunization with TNP–KLH. Repre- sentative results from two independent experiments (10 mice per group). *,#p,0.05,##p,0.01, ***,###p,0.001 (Student unpairedttest).B, Enhanced IgG- mediated passive cutaneous anaphylaxis. Mice were intradermally injected with anti-TNP IgG1 Ab and subsequently challenged by i.v. injection of HSA–TNP in Evans blue. Anaphylaxis was visualized by extravasation of the blue dye. Data are representative of three independent experiments (two to five mice per group). Original magnification31.C, Enhanced phagocytic activity of macrophages. Bone marrow-derived macrophages were incubated with mouse IgG1 anti-TNP opsonized SRBC–TNP and ingested SRBCs counted under a light microscope. Data are representative of two independent experiments (six mice per group). ***p,0.001 (Bonferroni’s multiple comparison test).DandE, Susceptible to CIA. Combined data from three independent experiments (10–25 mice per group). CIA incidence (D) in FcgRIIB1292/2and FcgRIIBB62/2mice was significantly higher compared with B6 mice,p,0.001 andp= 0.012, re- spectively (Fisher’s exact test). End point incidence in FcgRIIBB62/2mice was lower compared with that of FcgRIIB1292/2mice,p,0.01 (Fisher’s exact test).
CIA severity score (E).F–J, Enhanced accelerated NTN. Results of two independent experiments (8–10 mice per group). Experiment 1: 1:10 dilution of nephrotoxic serum inducing severe disease. Survival curve of mice with NTN (F). Experiment 2: 1:20 dilution of nephrotoxic serum inducing moderate disease.
Urinary albumin (G) was measured by radial immunodiffusion in urine collected overnight from mice in metabolic cages. Glomerular thrombosis (H). Kidneys were fixed in Bouin’s solution and stained with periodic acid–Schiff (PAS) stain. Deposition of IgG (I) or C3 (J). Immunohistochemistry of snap-frozen kidneys presented as arbitrary fluorescence units (AFU). *p,0.05, **p,0.01, ***p,0.001 (Kruskal–Wallis test).
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wild-type B6 mice. However, severity was significantly higher in FcgRIIB
1292/2mice compared with FcgRIIB
B62/2mice (Sup- plemental Fig. 3). Both strains showed enhanced accelerated NTN
compared with wild-type controls. However, onset of the disease was somewhat delayed and severity was lower in the FcgRIIB
B62/2mice compared with the onset and severity of NTN in FcgRIIB
1292/2mice (Fig. 1F–J). Similar to CIA, in NTN the increased severity in FcgRIIB
1292/2mice compared with that in FcgRIIB
B62/2mice might be explained partially by higher spe- cific Ab titers (Table I). Taken together, both FcgRIIB
B62/2and FcgRIIB
1292/2mice display a hyperactive phenotype, which is more pronounced in FcgRIIB
1292/2mice, suggesting a significant contribution of the 129-derived Fcgr2b flanking region (Sle16) to this phenotype both in the efferent and afferent phase.
FcgRIIB
B62/2mice do not develop spontaneous autoimmunity Because it has been reported that FcgRIIB
1292/2mice develop high ANA titers spontaneously, with high frequency with age (1), the presence of these autoantibodies in the blood of cohorts of about twenty 10-mo-old FcgRIIB
B62/2and FcgRIIB
1292/2fe- male mice was analyzed. Whereas most of the FcgRIIB
1292/2mice had high serum titers of autoantibodies specific for dsDNA and ssDNA, chromatin and histone, confirming the published phenotype, in the serum of FcgRIIB
B62/2mice hardly any of these autoantibodies were detectable (Fig. 2A–D). This lack of autoantibodies was neither caused by a delay in onset nor re- stricted to ANA. At the age of 12 mo, at which the mice were sacrificed, ANA Abs were still undetectable (data not shown), whereas anti-erythrocyte Abs present in FcgRIIB
1292/2mice were also absent in FcgRIIB
B62/2mice (Fig. 2E). Moreover, FcgRIIB
B62/2mice did not show other signs of systemic auto- immunity, such as splenomegaly (Fig. 2F) or an increase in the proportion of activated T and B cells (Table II). These re- sults indicate that, despite their strongly increased susceptibility to CIA, FcgRIIB
B62/2mice fail to develop spontaneous auto- immunity, whereas under the same experimental conditions FcgRIIB
1292/2mice do. This suggests that epistatic interactions between the 129-derived Sle16 locus, present in FcgRIIB
1292/2mice, and the B6 genome are decisive for the development of spontaneous autoimmunity in FcgRIIB
1292/2mice.
Mild nonlethal glomerular damage in FcgRIIB
B62/2mice The presence of ANA has been strongly associated with the de- velopment of immune-complex glomerulonephritis resulting in kidney damage. A first sign of kidney failure is proteinuria. At the age of 8 mo, despite the absence of autoantibodies, 30% of the FcgRIIB
B62/2mice showed a small but significant increase in the urinary albumin level (between 10 and 30 mg protein/g creatinine) compared with wild-type control mice (Fig. 3A). However, more substantial urinary albumin levels between 50 and 175 mg protein/
g creatinine were detected in 22% of the FcgRIIB
1292/2mice.
Careful examination of kidney sections of 12-mo-old mice revealed that all genotypes developed kidney pathology ranging from very mild in wild-type B6 controls, to moderate in
Table I. Anti-sheep IgG titers in NTN modelC57BL/6 (n= 10)
FcgRIIB1292/2
(n= 10)
FcgRIIBB62/2
(n= 10) Total IgG 0.21460.04 0.62661.06 0.24360.04 IgG1 0.17160.03 0.23960.09 0.18560.02 IgG2a 0.84460.07 1.13760.29* 0.95560.13 IgG2b 0.12760.05 0.43060.82 0.14560.03 IgG3 0.15560.03 0.23360.17 0.23460.24 Mean OD6SD for anti-sheep total IgG, IgG1, IgG2a, IgG2b, and IgG3.
*p,0.001 (one-way ANOVA, compared with C57BL/6).
FIGURE 2. FcgRIIBB62/2 mice do not develop spontaneous autoim- munity.A–D, Anti-chromatin (A), anti-dsDNA (B), anti-ssDNA (C), and anti-histone (D) autoantibody titers in FcgRIIBB62/2, FcgRIIB1292/2, and C57BL/6 female mice at the age of 10 mo as determined by ELISA and presented in arbitrary ELISA units (AEU). Data are representative of two independent experiments (15–20 mice per group). *p,0.05, ***p, 0.001 (Kruskal–Wallis test). E, Antierythrocyte (Coombs) autoantibody titers in 12-mo-old FcgRIIB1292/2, FcgRIIBB62/2, and C57BL/6 mice as determined by flow cytometric assay and expressed as mean fluorescence intensity (8–15 mice per group). ***p,0.001 (Kruskal–Wallis test).F, Spleen weights of FcgRIIBB62/2, FcgRIIB1292/2, and C57BL/6 female mice at the age of 12 mo. Data are representative of two independent experiments (15–20 mice per group). ***p,0.001 (Kruskal–Wallis test).
Horizontal bar represents median.
Table II. No increase in proportion of activated T and B cells in FcgRIIBB62/2mice
C57BL/6 FcgRIIB1292/2 FcgRIIBB62/2
CD8+CD62Llow 20.261.2 55.469.1* 16.964.2 CD4+CD62Llow 47.264.5 89.561.4*** 6664.9 CD4+CD69high 19.361.3 46.763.9*** 26.962.6*
CD19+CD69high 1.960.1 3.961.0*** 1.460.2 CD19+IgMlowIgDlow 2.460.2 5.561.1 5.660.7 Percentage of splenic T and B cell subpopulations as determined by flow cytom- etry. Results represent mean6SEM of 12-mo-old mice.
*p,0.05, ***p,0.01 (significance compared with C57BL/6 mice; statistical significance was analyzed by ANOVA and Bonferroni test).
1308 FcgRIIB IS A MODIFIER OF AUTOIMMUNE SUSCEPTIBILITY
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FcgRIIB
B62/2mice, to severe in FcgRIIB
1292/2mice (Fig. 3B, 3C). The pathology score in arbitrary categories from 0 to 4 correlated with increased mortality. Twenty-two percent of the FcgRIIB
1292/2mice developed fatal disease between the ages of 8 and 12 mo, whereas neither the FcgRIIB
B62/2nor the wild-type control mice developed fatal disease even at the age of 12 mo (Fig.
3D). These results show that the Sle16-driven nonlethal autoim- mune features culminate in fatal lupus in the absence of func- tional FcgRIIB, suggesting epistatic interactions between the Sle16 locus and the Fcgr2b gene. Moreover, the significant increase of IgG and C3 depositions in the kidneys of 12-mo-old FcgRIIB
B62/2mice (Fig. 3B, Table III) in the absence of substantial autoantibody titers suggests that in the absence of FcgRIIB, the clearance of IgG–IC is impaired. This is in agreement with our previous observations in a model of Ag-induced arthritis (23) and might point to an important role of FcgRIIB on resident mesangial cells, on which FcgRIIB is the most abundantly expressed FcgR (24), in the clearance of IgG–IC in the kidney.
Spontaneous autoimmunity in FcgRIII
1292/2mice with 129-derived Sle16 locus
To confirm further the contribution of the Sle16 locus to lowering the threshold for breaking tolerance in B6 mice, the development
FIGURE 3. Mild nonlethal glomerular damage in FcgRIIBB62/2mice.A, Proteinuria in 8-mo-old FcgRIIB1292/2and FcgRIIBB62/2mice as determined with a standard rocket immunoelectrophoresis assay (20 mice per group). ***p,0.001 (Kruskal–Wallis test).B,Top row, Representative PAS-stained kidney sections from 12-mo-old female mice;left panel(C57BL/6) shows normal glomeruli; themiddle panel(FcgRIIB1292/2) shows marked glomerular enlargement and global hypercellularity; and theright panel(FcgRIIBB62/2) shows slight glomerular enlargement with moderate hypercellularity (original magnification340).Middleandbottom rows, Immunohistochemistry of IgG and C3 deposition in snap-frozen kidney sections of 12-mo-old female mice (original magnification3600).C, Histological score of kidney pathology in PAS-stained kidney sections. Glomerular hypercellularity was graded on a 0–4 scale as follows: grade 0, normal; grade 1, segmental hypercellularity in 10–25% of the glomeruli; grade 2, hypercellularity involving.50% of the glomerular tuft in 25–50% of glomeruli; grade 3, hypercellularity involving.50% of the glomerular tuft in 50–75% of glomeruli; grade 4, glomerular cellularity in.75% or crescents in.25% of glomeruli. Values are averages of 20 glomeruli of 12-mo-old mice per group (13–19 mice per group) (ANOVA, Bonferroni).D, Cumulative mortality of 20 female mice of each genotype (FcgRIIBB62/2; FcgRIIB1292/2; C57BL/6) up to the age of 12 mo (Gehan–Breslow–Wilcoxon test). Horizontal bar represents median. *p,0.05, **p,0.01, ***p,0.001.
Table III. IgG and C3 deposition in kidneys of FcgRIIB1292/2and FcgRIIBB62/2mice
Genotype Mouse No. Pathology Score IgG C3
C57BL/6 5 0 + 2/+
8 0 + +
9 0 ++ ++
12 2 ++ +
17 2 +++ +
FcgRIIB1292/2 22 4 +++ ++++
23 3 +++ +++
24 4 +++ ++++
25 4 +++ +++
26 3 ++ ++++
29 3 ++ ++++
40 3 + ++
FcgRIIBB62/2 44 2 ++ +++
46 0 ++ +
49 4 +++ ++++
50 2 +++ +++
52 3 + +++
53 2 ++ +++
56 3 +++ ++
IgG and C3 deposition was graded on a scale from negative (2) up to strong positive (++++).
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of spontaneous autoimmunity was analyzed in 12-mo-old female mice of a KO strain generated by targeting the Fcgr3 gene, closely linked to the Fcgr2b gene, in 129-derived ES cells (FcgRIII
1292/2mice) and back-crossed 13 generations into B6 background. The presence of the 129-derived flanking Sle16 region was confirmed by SNP analysis (data not shown). FcgRIII
1292/2mice developed a high incidence of autoantibodies specific for dsDNA and ssDNA, nucleosome, chromatin, and histone (Fig. 4A–E, Table IV) and showed other signs of systemic autoimmunity, such as splenomegaly (Fig. 4F). In addition, in FcgRIII
1292/2mice the proportion of activated T and B cells was increased (Table V), which is characteristic for the highly autoimmune FcgRIIB
1292/2mouse. These results demonstrate the strong contribution of the 129-derived Sle16 region in the development of spontaneous au- toimmunity in B6 background (12). However, increased mortality was not observed. The absence of FcgRIII, one of the activating FcgR expressed on circulating myeloid effector cells, which are indispensable for the development of glomerulonephritis (25), most likely increases resistance to lupus.
The 129-derived FcgRIIB flanking Sle16 region is a dominant lupus susceptibility locus
To investigate whether the 129-derived Sle16 locus is a recessive or dominant lupus susceptibility locus in B6 mice deficient for FcgRIIB, the development of lupus was analyzed in F1 offspring from the cross between FcgRIIB
1292/2and FcgRIIB
B62/2mice.
The F1 offspring (FcgRIIB
1293B62/2), containing one copy of the 129-derived flanking region (Sle16) and one copy of the B6- derived flanking region, developed splenomegaly and high ANA titers (Fig. 5A–E), although titers and incidence were decreased
compared with titers and incidence in FcgRIIB
1292/2mice (Figs.
2A–D, 5A–D) suggesting a gene dose effect. This is in agreement with observations in Sle1 congenic strains. Kidney pathology was significantly increased in F1 mice compared with wild-type B6 mice (Fig. 5F, 5G), whereas mortality was comparable between F1 and FcgRIIB
1292/2mice; 25 and 22%, respectively, died be- fore the age of 1 y (Figs. 3D, 5H). These results show that the 129- derived Sle16 locus is a dominant lupus susceptibility locus and explain the finding that heterozygosity of FcgRIIB
1292/2mice is sufficient for the development of rheumatoid factor (26). In ad- dition, it demonstrates that the lack of autoimmune disease in FcgRIIB
B62/2mice is not caused by the presence of an un- identified, dominant SLE-suppressor locus.
Positive epistasis between the FcgRIIB
B62/2allele and the Yaa locus in lupus
FcgRIIB
1292/2male mice carrying the Yaa locus develop severe autoimmune disease characterized by striking splenomegaly, pro- teinuria with 90% penetrance, and a median survival of only 4 mo (21). This severe autoimmune phenotype has been explained by strong synergism between the Yaa locus and the Fcgr2b KO allele.
However, synergism between the 129-derived flanking Sle16 re- gion, with its NZW haplotype, and the B6 genome could not be excluded because strong synergism between Sle1
NZWand Yaa has been demonstrated (27). To investigate the putative synergism between the Yaa and Fcgr2b KO allele in the absence of the 129- derived Sle16 locus, the FcgRIIB
B62/2strain was crossed with a Yaa B6 congenic strain, and the development of autoimmune disease in the FcgRIIB
B62/2.Yaa male offspring was analyzed. At the age of 10 mo, the male mice of the Yaa B6 congenic strain developed low but significantly increased anti-chromatin and anti- dsDNA autoantibody titers compared with those of males of the B6 control and FcgRIIB
B62/2strain (Fig. 6A, 6B). The penetrance and the titers were further increased in the FcgRIIB
B62/2.Yaa male mice (Fig. 6A, 6B), indicating that the absence of FcgRIIB enhanced the mild autoimmune phenotype of the C57BL/6.Yaa males. The FcgRIIB
B62/2.Yaa males displayed a moderate lupus phenotype with age characterized by splenomegaly, monocytosis, and substantial kidney pathology, but only 30% died between the ages of 10 and 12 mo (Fig. 6C–F, Table VI). These results confirm
FIGURE 4. Spontaneous autoim-munity in FcgRIII1292/2 mice with 129-derivedSle16 locus. A–E, Anti- chromatin (A), anti-dsDNA (B), anti- ssDNA (C), anti-histone (D), and anti- nucleosome (E) autoantibody titers in aged (6–12 mo old) FcgRIII1292/2
female mice and 12-mo-old C57BL/6 female control mice as determined by ELISA and presented in arbitrary ELISA units (AEU).F, Spleen weights of aged female FcgRIII1292/2 and C57BL/6 control mice. Horizontal bar indicates median (four to eight mice per group). *p , 0.05, **p , 0.01, ***p,0.001 (unpairedttest or Mann–WhitneyUtest).
Table IV. Penetrance of autoantibody titers in FcgRIII1292/2mice
Autoantibody % C57BL/6 (n) % FcgRIII1292/2(n)
Anti-nucleosome 0 (0/8) 83.3 (5/6)
Anti-ssDNA 0 (0/4) 71.4 (5/7)
Anti-dsDNA 0 (0/4) 57.1 (4/7)
Anti-chromatin 0 (0/4) 71.4 (5/7)
Anti-histone 0 (0/4) 85.7 (6/7)
Penetrance based on cutoff mean + 23SD of C57BL/6 females at 12 mo.
1310 FcgRIIB IS A MODIFIER OF AUTOIMMUNE SUSCEPTIBILITY
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synergism between the Fcgr2b KO allele and the Yaa locus, which in B6 mice is sufficient for the development of fatal lupus in a minority of the male mice after the age of 10 mo. However, FcgRIIB
B62/2.Yaa male mice show a moderate lupus phenotype compared with the published highly severe phenotype of the FcgRIIB
1292/2.Yaa male mice. This indicates that in the latter one, in addition to synergism between the Fcgr2b KO allele and the Yaa locus, synergism between the Yaa locus and the 129- derived Sle16 locus and the Sle16 locus and the B6 genome strongly contributes to its very severe autoimmune phenotype.
Discussion
Our comparison of two FcgRIIB KO strains on B6 background, one with 129-derived (FcgRIIB
1292/2) and the other with B6-
derived (FcgRIIB
B62/2) Fcgr2b flanking regions, revealed that in full B6 mice, FcgRIIB deficiency is not sufficient to cause lupus despite its pleiotropic effect affecting not only B cells and mac- rophages but also dendritic cells (28, 29). In FcgRIIB
1292/2mice, the development of lupus is initiated by epistatic interactions between the 129-derived Fcgr2b flanking Sle16 region and the B6 genome, causing loss of tolerance resulting in the development of autoreactive B cells. This is confirmed by the nonlethal autoim- mune phenotype of FcgRIII KO mice back-crossed into B6 back- ground, which contain the same Sle16 region. In FcgRIIB
1292/2mice, the contribution of these autoreactive B cells to the disease process is amplified by the loss of FcgRIIB turning the nonlethal autoimmune phenotype into a fatal lupus phenotype. Our obser- vations support genetic studies based on congenic dissection of the
FIGURE 5. The 129-derived Sle16 locus is a dominant lupus susceptibility locus. A–D, Anti- chromatin (A), anti-dsDNA (B), anti-ssDNA (C), and anti-histone (D) autoantibody titers as de- termined by ELISA and presented in arbitrary ELISA units (AEU) at the age of 10 mo.EandF, Spleen weight at the age of 12 mo (E) and pro- teinuria at the age of 9 mo (F) in FcgRIIB1293B62/2
and C57BL/6 female mice.G, Histological score of kidney pathology in PAS-stained kidney sections (as described in the legend of Fig. 3) of 12-mo- old FcgRIIB1293B62/2and C57BL/6 female mice.
*p,0.05, **p,0.01, ***p,0.001 (unpaired t test or Mann–Whitney U test).H, Cumulative mortality of 20 female FcgRIIB1293B62/2 and C57BL/6J mice up to the age of 12 mo. *p,0.05 (Gehan–Breslow–Wilcoxon Test). Horizontal bar represents median (15–17 mice per group).
Table V. Analysis of splenocyte populations of FcgRIII1292/2mice
FcgRIIBB6fl/fl FcgRIII1292/2 pValue
CD8+CD62Llow 25.7060.4 65.1064.9 ** (p= 0.052)
CD4+CD62Llow 29.6560.25 88.0862.6 *** (p,0.0001)
CD4+CD69high 10.5560.75 32.3063.4 * (p= 0.026)
CD19+CD69high 0.7960.22 1.9860.22 * (p= 0.029)
CD19+IgMlowIgDlow 1.4960.24 6.6861.5 p= 0.094
Percentages of splenic T and B cells. Results represent mean6SEM of 10-mo-old mice (M/F).
*p,0.05, **p,0.01, ***p,0.001 (significance compared with FcgRIIBB6fl/flmice; statistical significance was analyzed by unpairedttest).
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Sle1 locus derived from the autoimmune-prone NZW strain showing that Sle1b
NZWcongenic B6 mice develop ANA inde- pendently from the presence of the Fcgr2b
NZWallele, whereas Fcgr2b
NZWcongenic B6 mice do not develop autoimmunity de- spite impaired FcgRIIB expression. In contrast, the absence of autoimmunity in FcgRIIB
B62/2mice seems to be conflicting with the results of a recent functional study using Ig gene cloning from single isolated B cells (30). However, because the FcgRIIB
1292/2mouse was used, most likely Sle16-dependent mechanisms are responsible for the observed development of autoreactive B cells.
Taken together, our results show that FcgRIIB is an autoimmune disease modifier and not, as has been postulated, a “single over- riding factor,” which may ultimately dictate whether the disease progresses or not (31). Defining accurately the relative contribu- tion of an individual disease modifier, such as FcgRIIB, to the development of a complex autoimmune disease is very difficult.
The model of “threshold liability” can explain a substantial part of the highly severe phenotype of FcgRIIB
1292/2.Yaa male mice.
The penetrance of the autoimmune phenotype increases in relation to the increasing number of the susceptibility genes, Yaa, Sle16, and Fcgr2b
2/2, present in the genome of these male mice.
However, in addition to the inheritance in a simplistic additive fashion, epistatic interactions might modify the inheritance of the autoimmune phenotype in a complex fashion that may not be additive (2). This is demonstrated by our observations that in B6 mice, besides the reported positive epistatic interactions between the Sle1b and Yaa loci (27), also epistatic interactions between Fcgr2b and yaa resulted in the development of lethal lupus.
According to this “multiplicative model,” the interactions of all susceptibility and suppressor alleles present in its genome de- termine the susceptibility of an individual. As a consequence, the contribution of an individual gene to the development of auto- immune disease can vary depending on all other susceptibility and suppressor loci present in the genome. In addition, environmental conditions also play an important role, as illustrated by the signif- icant differences in kidney pathology between the two genetically
FIGURE 6. Positive epistasis between FcgRIIBB62/2and theYaalocus.AandB, Anti-chromatin (A) and anti-dsDNA (B) autoantibody titers as de- termined by ELISA and presented in arbitrary units/ml (U/ml) (ANOVA, Bonferroni).C, Spleen weights (unpairedttest or Mann–WhitneyUtest).D, Monocytosis in FcgRIIBB62/2.Yaaand C57BL/6/Yaa+male mice, FcgRIIBB62/2female mice, and C57BL/6 control mice (ANOVA, Bonferroni).E, Histological score of kidney pathology (unpairedttest or Mann–WhitneyUtest).F, Cumulative mortality at the age of 12 mo. Horizontal bar indicates median (8–15 mice per group). *p,0.05, ***p,0.001 (Gehan–Breslow–Wilcoxon test).Table VI. Splenic phenotype of FcgRIIBB62/2.Yaa
FcgRIIBB62/2 FcgRIIBB62/2.Yaa pValue
CD4+ 14.462.0 14.361.0 0.92
CD4+CD62L+ 37.769.5 17.269.7* 0.02 CD4+CD69+ 17.863.9 35.7610.1* 0.02
CD8+ 9.862.0 8.262.7 0.37
CD8+CD62L+ 75.267.6 72.167.7 0.59
B220+ 53.068.8 58.664.7 0.31
B220+IgM+ 42.967.0 40.065.4 0.54
B220+CD69+ 2.160.6 6.563.1* 0.03
Percentages of splenic cell populations. Results represent mean6SEM of 10- mo-old mice (M/F) (n= 4).
*p ,0.05 (significance compared with FcgRIIBB6fl/flmice; statistical signifi- cance was analyzed by unpairedttest).
1312 FcgRIIB IS A MODIFIER OF AUTOIMMUNE SUSCEPTIBILITY
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identical aging cohorts of FcgRIIB
B62/2mice maintained in dif- ferent facilities (Figs. 3C, 6E).
The absence of autoimmunity in FcgRIIB
B62/2mice together with the increase in IgG and C3 deposition and pathology in the kidney independent from the development of autoantibodies suggest a minor role of FcgRIIB in maintaining tolerance in the efferent phase but an important role in regulating downstream Ab effector pathways. In humans, autoantibodies develop long before the clinical onset of SLE, suggesting that unknown additional changes are required to trigger the development of the disease (32). We hypothesize that in FcgRIIB
1292/2mice, FcgRIIB de- ficiency results in impaired IC clearance and enhanced myeloid effector cell responses, which lower the threshold for the induction of fatal glomerulonephritis by autoantibodies.
Acknowledgments
We thank Dr. R. Woltenbeek for support with statistical calculations, Dr.
A. Snijders and Dr. C.E. van der Poel for critically reading the manuscript, M. Szajna and M. Alvarez for the microsatellite analysis, and O. Demirbacak, Y. Allen, and all the staff of Proefdiercentrum for excellent animal care.
Disclosures
The authors have no financial conflicts of interest.
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