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The Journal of Immunology, 2008, 180, 1948 -1953
Copyright © 2008 by The American Association of Immunologists, Inc.
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Differential Contribution of Three Activating IgG Fc Receptors (Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) to IgG2a- and IgG2b-Induced Autoimmune Hemolytic Anemia in Mice1

Lucie Baudino*, Falk Nimmerjahn{dagger}, Samareh Azeredo da Silveira*, Eduardo Martinez-Soria*, Takashi Saito{ddagger}, Michael Carroll§, Jeffrey V. Ravetch, J. Sjef Verbeek|| and Shozo Izui2,*

* Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; {dagger} Laboratory for Experimental Immunology and Immunotherapy, Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen-Nuremberg, Erlangen, Germany; {ddagger} Laboratory for Cell Signaling, Institute of Physical and Chemical Research Center for Allergy and Immunology, Yokohama, Japan; § Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA 02115; The Rockefeller University, New York, NY 10065; and || Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Murine phagocytes express three different activating IgG Fc{gamma}R: Fc{gamma}RI is specific for IgG2a; Fc{gamma}RIII for IgG1, IgG2a, and IgG2b; and Fc{gamma}RIV for IgG2a and IgG2b. Although the role of Fc{gamma}RIII in IgG1 and IgG2a anti-RBC-induced autoimmune hemolytic anemia (AIHA) is well documented, the contribution of Fc{gamma}RI and Fc{gamma}RIV to the development of IgG2a- and IgG2b-induced anemia has not yet been defined. In the present study, using mice deficient in Fc{gamma}RI, Fc{gamma}RIII, and C3, in combination with an Fc{gamma}RIV-blocking mAb, we assessed the respective roles of these three Fc{gamma}R in the development of mild and severe AIHA induced by two different doses (50 and 200 µg) of the IgG2a and IgG2b subclasses of the 34-3C anti-RBC monoclonal autoantibody. We observed that the development of mild anemia induced by a low dose of 34-3C IgG2a autoantibody was highly dependent on Fc{gamma}RIII, while Fc{gamma}RI and Fc{gamma}RIV additionally contributed to the development of severe anemia induced by a high dose of this subclass. In contrast, the development of both mild and severe anemia induced by 34-3C IgG2b was dependent on Fc{gamma}RIII and Fc{gamma}RIV. Our results indicate differential roles of the three activating Fc{gamma}R in IgG2a- and IgG2b-mediated AIHA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The New Zealand Black (NZB)3 mice spontaneously develop autoimmune hemolytic anemia (AIHA) as a result of production of Coombs’ anti-RBC autoantibodies (1). Besides the binding specificity of these autoantibodies, the Fc regions of the different Ig isotypes also play a critical role in autoantibody-mediated pathogenicity by activating IgG Fc{gamma}R-bearing effector cells, initiating the complement cascade, and inducing IgM and IgA multivalency-dependent agglutination (2, 3). The analysis of IgG class-switch variants of low affinity 4C8 and high affinity 34-3C anti-RBC monoclonal autoantibodies derived from NZB mice has demonstrated the remarkably different pathogenic potentials of four IgG subclasses, which depend on their respective capacity to interact with Fc{gamma}R and to activate complement in vivo (4, 5, 6). Moreover, these analyses revealed that Fc{gamma}R- and complement receptor (CR)-mediated erythrophagocytosis is the major pathogenic mechanism for the development of AIHA. Notably, complement-mediated intravascular hemolysis hardly plays any role in the development of AIHA, even in case of anemia induced by IgM anti-RBC mAb (3).

Different classes of Fc{gamma}R are expressed on many effector cells of the immune system and mediate various cellular responses, such as phagocytosis by macrophages, Ab-dependent cell-mediated cytotoxicity by NK cells, and degranulation of mast cells (7, 8). In the past, two classes of activating Fc{gamma}R, high affinity Fc{gamma}RI and low affinity Fc{gamma}RIII, have been identified on phagocytic effector cells in mice. Both are hetero-oligomeric complexes, in which the respective ligand-binding {alpha}-chains are associated with the common FcR {gamma}-chain (FcR{gamma}). FcR{gamma} is required for the assembly and cell surface expression of these activating Fc{gamma}R and for the triggering of their various effector functions (9). Fc{gamma}RI is capable of binding only one IgG subclass, IgG2a, with high affinity, whereas the low affinity Fc{gamma}RIII binds polymeric forms of three different IgG subclasses (IgG1, IgG2a, and IgG2b), but not IgG3 (10, 11). Most recently, a third activating receptor, Fc{gamma}RIV, which binds IgG2a- and IgG2b-immune complexes with intermediate affinity, has been identified in mice (12, 13). Fc{gamma}RIV is also composed of a specific {alpha}-chain and the common FcR{gamma}. In contrast to IgG2a and IgG2b subclasses, Fc{gamma}RIII is the sole receptor mediating IgG1-dependent phagocytosis in vivo (4, 13, 14, 15), and IgG3 is unable to trigger Fc{gamma}R-mediated phagocytosis (6, 13).

We have previously demonstrated that the development of mild anemia induced by low affinity 4C8 and high affinity 34-3C IgG2a anti-RBC mAb is highly dependent on the expression of Fc{gamma}RIII (4, 15), indicating a predominant role for this receptor in AIHA. However, because of the specific recognition by Fc{gamma}RI of IgG2a and by Fc{gamma}RIV of IgG2a and IgG2b, it is of importance to define the respective contributions of Fc{gamma}RI and Fc{gamma}RIV (in comparison with Fc{gamma}RIII) to IgG2a- and IgG2b-dependent AIHA. Indeed, we have observed that depending on the affinity and the concentrations of the IgG2a and IgG2b anti-RBC mAb used, Fc{gamma}RIII-deficient mice were not always fully resistant under conditions where FcR{gamma} knockout mice were (4, 6, 15, 16). Therefore, in the present study, using different mutant mice deficient in Fc{gamma}RI, Fc{gamma}RIII, FcR{gamma}, and/or C3, in combination with an Fc{gamma}RIV-blocking mAb, we assessed the respective contributions of all FcR{gamma}-associated activating Fc{gamma}R (i.e., Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) to the development of mild and severe anemia induced by 34-3C IgG2a and IgG2b class-switch variants. Our results show that Fc{gamma}RIII plays a major role in the development of mild anemia induced by IgG2a 34-3C anti-RBC mAb, and that Fc{gamma}RI and Fc{gamma}RIV additionally contribute to the development of severe anemia induced by a high dose of this subclass. In contrast, both Fc{gamma}RIII and Fc{gamma}RIV were involved in the development of mild and severe forms of anemia induced by IgG2b 34-3C anti-RBC mAb.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Fc{gamma}RI–/–, Fc{gamma}RIII–/–, and C3–/– mice were generated by gene targeting in 129-derived embryonic stem cells, whereas FcR{gamma} –/– mice were generated with C57BL/6 (B6)-derived embryonic stem cells, as described previously (14, 16, 17, 18). Fc{gamma}RIII–/– mice were backcrossed for seven generations on a BALB/c background and C3–/– mice for five generations with a B6 background. BALB/c and B6 mice were purchased from The Jackson Laboratory. Fc{gamma}RIII/C3–/–, Fc{gamma}RI/Fc{gamma}RIII/C3–/–, and FcR{gamma}/C3–/– mice were obtained through intercross between corresponding deficient mice. FcR{gamma}/C3–/– mice carry a B6 background, whereas Fc{gamma}RIII/C3–/– and Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice bear a mixed BALB/c and B6 background. The Fc{gamma}RI, Fc{gamma}RIII, and FcR{gamma} genotypes were determined by PCR analysis using the following sets of primers. Fc{gamma}RI: wild type (WT)-specific sense primer (5'-GTTTGCTGTGGTTTGAGACC-3'), mutant-specific sense primer (5'-TCGCCGATAGTGGAAACCGAC-3'), and common antisense primer (5'-TCCTTCTGGAAAATACTGACC-3'); Fc{gamma}RIII: WT-specific sense primer (5'-TCCATCTCTCTAGTCTGGTACC-3'), mutant-specific sense primer (5'-ACTTGTGTAGCGCCAAGTGCCA-3'), and common antisense primer (5'-AAAAGTTGCTGCTGCCACC-3'); and FcR{gamma}: WT-specific sense primer (5'-TGCTGTCCTGTTTTTGTATGG-3'), mutant-specific sense primer (5'-CCAACGCTATGTCCTGATAG-3'), and common antisense primer (5'-GCTGCCTTTCGGACCTGGAT-3'). C3-deficient mice were identified by the absence of serum C3, as determined by ELISA (6).

Monoclonal Ab

Hybridoma secreting the 34-3C IgG2a anti-RBC monoclonal autoantibody was derived from unmanipulated NZB mice (2). The generation of the IgG2b class-switch variant of the 34-3C mAb was described previously (5, 6). 34-3C IgG2a VDJ34-3C-C{gamma}2a (L235E) mAb mutant at position 235 (leucine to glutamic acid), thus lacking the high affinity binding motif (LEGGP instead of LLGGP) for Fc{gamma}RI in the CH2 domain (19), was generated by transfecting a 34-3C H chain loss cell line with a L235E mutant plasmid, which was generated by oligonucleotide-directed mutagenesis, as described (20). Notably, the wild-type (WT) 34-3C IgG2a and the IgG2aL235E mutant exhibited a comparable mouse RBC-binding activity in vitro, as assessed by a flow cytometric analysis using a biotinylated rat anti-mouse {kappa}-chain mAb (H139.52.1.5), followed by PE-conjugated streptavidin (5, 6). Hamster IgG 9E9 Fc{gamma}RIV-blocking mAb was described previously (13). IgG mAb were purified from culture supernatants by protein A or G column chromatography. The purity of IgG was >90% as documented by SDS/PAGE.

Experimental AIHA

AIHA was induced by a single i.v. injection of purified anti-RBC mAb into 2- to 3-mo-old mice. The injection of mAb was controlled 24 h later by assessing the level of Ab opsonization of circulating RBC by flow cytometric analysis using biotinylated rat anti-mouse {kappa}-chain mAb, as described (5). Blood samples were collected into heparinized microhematocrit tubes every 2 days after the injection, and hematocrit (Ht) values were directly determined after centrifugation. To block Fc{gamma}RIV, mice were treated with 400 µg of 9E9 anti-Fc{gamma}RIV mAb 30 min before and 2 days after administration of the 34-3C mAb. As a control, mice were treated with polyclonal hamster IgG (Jackson ImmunoResearch Laboratories). Livers, obtained 8 days after injection of mAb, were processed for histological examination, and the extent of in vivo RBC destruction by Kupffer cell-mediated phagocytosis was determined by Perls iron staining.

Flow cytometric analysis of in vitro binding of IgG on macrophages

Bone marrow cells were obtained from femurs of WT and Fc{gamma}RI–/– BALB/c mice and cultured in DMEM with 30% L cell-conditioned medium for 7 days, according to the procedure by Vairo and Hamilton (21). Bone marrow-derived macrophages were then incubated with biotinylated 34-3C IgG2a or IgG2aL235E mAb and FITC-labeled anti-CD11b mAb in the presence of saturating concentrations of 2.4G2 anti-Fc{gamma}RII/III and 9E9 anti-Fc{gamma}RIV mAb, followed by PE-conjugated streptavidin, and the extent of IgG2a binding by Fc{gamma}RI on CD11b+ macrophages was analyzed with a FACSCalibur (BD Biosciences).

Surface plasmon resonance analysis

A Biacore 3000 biosensor system was used to determine the interaction of soluble murine Fc{gamma}R (Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) with 34-3C IgG2a and IgG2aL235E, as described previously (13). Briefly, soluble versions of murine Fc{gamma}R were injected through flow cells containing immobilized Abs at five different concentrations (0.25, 0.5, 1, 2, and 4 µg/ml). Background binding to a reference flow cell containing immobilized BSA was subtracted.

Statistical analysis

Statistical analysis was performed with the Wilcoxon two-sample test. Probability values <5% were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Predominant role of Fc{gamma}RIII in the development of mild anemia induced by 34-3C IgG2a mAb

To define the respective roles of Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV in the development of IgG2a-induced anemia, the pathogenic effect of 34-3C IgG2a mAb was assessed in BALB/c mice deficient in either Fc{gamma}RI or Fc{gamma}RIII. A single injection of 50 µg of 34-3C IgG2a anti-RBC mAb provoked mild anemia, with the most pronounced drop in Ht values (mean ± SD: 34.7 ± 1.6%) 4 days after the injection in WT BALB/c mice (Fig. 1A). Fc{gamma}RIII–/– mice were protected from the development of mild anemia induced by this dose of 34-3C IgG2a mAb (mean Ht values at day 4: 44.4 ± 1.6%; p < 0.01). However, no such protection was observed in Fc{gamma}RI–/– mice (Fig. 1A) or in WT BALB/c mice treated with 9E9 Fc{gamma}RIV-blocking mAb (Fig. 1B). These results indicated a major role of Fc{gamma}RIII in the development of mild anemia caused by 34-3C IgG2a mAb.


Figure 1
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  		FIGURE 1. Predominant role of Fc{gamma}RIII in the development of mild anemia induced by a low dose of 34-3C IgG2a mAb. A, A total of 50 µg of 34-3C IgG2a mAb was injected i.v. into WT, Fc{gamma}RI–/– (I–/–), or Fc{gamma}RIII–/– (III–/–) BALB/c mice. B, A total of 50 µg of 34-3C IgG2a mAb was injected i.v. into WT BALB/c mice, which were treated with 400 µg of 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG (Ctr) 30 min before and 2 days after administration of the 34-3C IgG2a mAb. Mean Ht values are indicated by horizontal lines. The normal range of Ht values (mean ± 3 SD) of 2- to 3-mo-old BALB/c mice is represented as shaded areas.

 
Contribution of Fc{gamma}RI and Fc{gamma}RIV to the development of severe anemia induced by 34-3C IgG2a mAb

We have previously shown that the injection of a high dose (200 µg) of 34-3C IgG2a anti-RBC mAb induced significant anemia in mice deficient in the common FcR{gamma} but failed to do so in FcR{gamma}/C3–/– mice, indicating that Fc{gamma}R- and CR-mediated erythrophagocytosis acted in an additive fashion to promote the development of severe anemia (6). Because FcR{gamma}/C3–/– mice lack the functional expression of Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV, we generated Fc{gamma}RIII/C3–/– and Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice to determine the possible contribution of Fc{gamma}RI and Fc{gamma}RIV to the development of severe anemia in this experimental setting. When 200 µg of 34-3C IgG2a mAb was injected, both Fc{gamma}RIII/C3–/– and Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice still developed highly significant anemia. However, the extent of anemia occurring in Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice (mean Ht values at day 4: 29.2 ± 1.8%) was less severe than that of Fc{gamma}RIII/C3–/– mice (20.7 ± 2.6%; p < 0.05; Fig. 2A), suggesting the contribution of Fc{gamma}RI to the severe form of anemia. Although these double- and triple-deficient mice carry a mixed BALB/c and B6 background, it is unlikely that the observed differences between them were due to differences in their genetic backgrounds, since B6, BALB/c, and their F1 hybrid mice developed equally severe anemia by this high dose of 34-3C IgG2a mAb (data not shown). Since FcR{gamma}/C3–/– mice failed to develop anemia (43.6 ± 0.8%; p < 0.05), these results suggested the involvement of Fc{gamma}RI and/or Fc{gamma}RIV in the development of severe anemia induced by IgG2a anti-RBC mAb.


Figure 2
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  		FIGURE 2. Contribution of Fc{gamma}RI and Fc{gamma}RIV to the development of severe anemia induced by a high dose of 34-3C IgG2a or IgG2aL235E mutant. A, A total of 200 µg of 34-3C IgG2a mAb was injected i.v. into Fc{gamma}RIII/C3–/– (III,C3–/–), Fc{gamma}RI/Fc{gamma}RIII/C3–/– (I,III,C3–/–), or FcR{gamma}/C3–/– ({gamma},C3–/–) mice. B, A total of 200 µg of 34-3C IgG2a mAb was injected i.v. into Fc{gamma}RIII/C3–/– (III,C3–/–) or Fc{gamma}RI/Fc{gamma}RIII/C3–/– (I,III,C3–/–) mice, which were treated with 400 µg of 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG (Ctr) 30 min before and 2 days after administration of the 34-3C IgG2a mAb. C, A total of 200 µg of 34-3C IgG2aL235E mutant was injected i.v. into Fc{gamma}RIII/C3–/– mice, which were treated with 400 µg of 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG. Ht values of individual mice measured 4 days after i.v. injection of 34-3C mAb are shown. Mean Ht values are indicated by horizontal lines. The normal range of Ht values (mean ± 3 SD) of 2- to 3-mo-old BALB/c mice is represented as shaded areas.

 
To better define the contribution of Fc{gamma}RI and/or Fc{gamma}RIV to the development of IgG2a-induced severe anemia, Fc{gamma}RIII/C3–/– and Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice were treated with either 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG and then injected with 200 µg of 34-3C IgG2a mAb. Treatment with 9E9 mAb significantly, but not completely, inhibited the development of anemia compared with hamster IgG-treated Fc{gamma}RIII/C3–/– control mice (mean Ht values at day 4: 9E9-treated mice, 35.8 ± 5.1%; control IgG-treated mice, 22.0 ± 1.1%; p < 0.05; Fig. 2B). In contrast, Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice treated with 9E9 Fc{gamma}RIV-blocking mAb became totally resistant to the pathogenic effect of 200 µg of 34-3C IgG2a mAb (9E9-treated mice, 41.8 ± 1.9%; control IgG-treated mice, 31.3 ± 1.5%; p < 0.05; Fig. 2B). Histological analysis confirmed the complete absence of iron deposits by Kupffer cells in Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice treated with 9E9 Fc{gamma}RIV-blocking mAb, which contrasted to the presence of substantial levels of erythrophagocytosis in 9E9-treated Fc{gamma}RIII/C3–/– mice (Fig. 3).


Figure 3
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  		FIGURE 3. Representative histological appearance of iron deposits in Kupffer cells from mice after injection of 34-3C IgG2a, IgG2aL235E, or IgG2b. A total of 200 µg of 34-3C IgG2a, IgG2aL235E, or IgG2b mAb was injected i.v. into Fc{gamma}RIII/C3–/– (III,C3–/–) or Fc{gamma}RI/Fc{gamma}RIII/C3–/– (I,III,C3–/–) mice, which were treated with 400 µg of 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG 30 min before and 2 days after administration of the 34-3C mAb. Mice were sacrificed at day 8, and extent of in vivo erythrophagocytosis was determined histologically by coloration of liver sections with Perls iron staining (original magnifications: x200).

 
To confirm the involvement of Fc{gamma}RI in the development of anemia induced by 200 µg of 34-3C IgG2a mAb, we generated an IgG2aL235E mutant of the 34-3C mAb. The replacement of leucine by glutamic acid at position 235 was expected to result in the loss of the high affinity interaction of IgG2a with Fc{gamma}RI, as shown by the analysis with human Fc{gamma}RI (19). Flow cytometric analysis confirmed the lack of binding of the IgG2aL235E mutant to Fc{gamma}RI on bone marrow-derived macrophages (Fig. 4A). The incapacity of the IgG2aL235E mutant to bind Fc{gamma}RI was further confirmed by surface plasmon resonance analysis, while it binds to Fc{gamma}RIV as efficiently as WT IgG2a mAb (Table I). Notably, the IgG2aL235E mutant and WT IgG2a displayed a comparable mouse RBC-binding activity in vivo, when analyzed by a flow cytometric assay 24 h after a single i.v. injection into BALB/c mice (Fig. 4B). Thus, if Fc{gamma}RI was indeed involved in the development of anemia induced by a high dose of IgG2a, the 34-3C IgG2aL235E mutant should induce less severe anemia in Fc{gamma}RIII/C3–/– mice and be unable to cause anemia when these mice were treated with 9E9 Fc{gamma}RIV-blocking mAb. This was the case, since Fc{gamma}RIII/C3–/– mice developed less severe anemia with 200 µg of the 34-3C IgG2aL235E mutant (mean Ht values at day 4: 30.9 ± 1.7%; Fig. 2C), as compared with WT IgG2a (p < 0.05; Fig. 2B). Notably, the treatment with 9E9 Fc{gamma}RIV-blocking mAb completely abolished the development of anemia in IgG2aL235E-injected Fc{gamma}RIII/C3–/– mice (45.4 ± 0.8%; p < 0.05; Fig. 2C). Histological analysis confirmed the protective effect of 9E9 Fc{gamma}RIV-blocking mAb on the induction of anemia induced by 34-3C IgG2aL235E mutant in Fc{gamma}RIII/C3–/– mice (Fig. 3). Taken together, these results indicated a significant role of both Fc{gamma}RI and Fc{gamma}RIV in the development of severe anemia induced by 200 µg of 34-3C IgG2a mAb.


Figure 4
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  		FIGURE 4. Lack of binding of 34-3C IgG2aL235E mutant to Fc{gamma}RI on bone marrow-derived macrophages. A, Bone marrow-derived macrophages prepared from WT or Fc{gamma}RI–/– BALB/c mice were incubated with biotinylated 34-3C IgG2aL235E mutant or WT mAb and FITC-labeled anti-CD11b mAb in the presence of a saturating concentration of 2.4G2 anti-Fc{gamma}RII/III and 9E9 anti-Fc{gamma}RIV mAb, followed by PE-conjugated streptavidin. Fluorescence intensities of IgG2a WT or IgG2aE235L on Fc{gamma}RI-deficient (shaded) and Fc{gamma}RI-sufficient CD11b+ macrophages are shown. B, Mouse RBC were obtained 24 h after i.v. injection of 50 µg of 34-3C IgG2aL235E mutant or WT mAb into BALB/c mice and then stained with biotinylated rat anti-mouse {kappa}-chain mAb, followed by PE-conjugated streptavidin. Shaded histogram indicates the control staining of noninjected mice.

 

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  		Table I. Affinities of Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV for 34-3C IgG2a and IgG2aL235E anti-RBC mAba

 
Contribution of Fc{gamma}RIV to the development of mild and severe anemia induced by 34-3C IgG2b mAb

We also evaluated the respective roles of Fc{gamma}RIII and Fc{gamma}RIV in the development of anemia induced by 34-3C IgG2b mAb. The development of mild anemia occurring in WT BALB/c mice injected with 50 µg of 34-3C IgG2b mAb (mean Ht values at day 4: 36.9 ± 1.9%) was prevented in Fc{gamma}RIII–/– BALB/c mice (44.4 ± 1.9%; p < 0.01; Fig. 5A). However, unlike after injection of 34-3C IgG2a mAb (Fig. 1B), the development of mild anemia was also prevented in BALB/c mice treated with 9E9 Fc{gamma}RIV-blocking mAb (9E9-treated mice, 43.4 ± 2.8%; control IgG-treated mice, 36.8 ± 1.3%; p < 0.05; Fig. 5B). As in the case of 34-3C IgG2a mAb, at a highly pathogenic dose (200 µg) of 34-3C IgG2b mAb, Fc{gamma}RIII/C3–/– mice still developed significant anemia (31.1 ± 2.2%), whereas FcR{gamma}/C3–/– mice were resistant (44.9 ± 1.6%; p < 0.05; Fig. 5C). The contribution of Fc{gamma}RIV to the development of IgG2b-mediated severe anemia was confirmed by the failure of Fc{gamma}RIII/C3–/– mice treated with 9E9 Fc{gamma}RIV-blocking mAb to develop anemia after the injection of 200 µg of 34-3C IgG2b mAb (9E9-treated mice, 45.8 ± 1.2%; control IgG-treated mice, 32.5 ± 2.0%; p < 0.05; Fig. 5D). Notably, these mice failed to show any sign of erythrophagocytosis by Kupffer cells (Fig. 3).


Figure 5
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  		FIGURE 5. Contribution of Fc{gamma}RIII and Fc{gamma}RIV to the development of mild and severe anemia induced by 34-3C IgG2b mAb. A, A total of 50 µg of 34-3C IgG2b mAb was injected i.v. into WT or Fc{gamma}RIII–/– (III–/–) BALB/c mice. B, A total of 50 µg of 34-3C IgG2b mAb was injected i.v. into WT BALB/c mice, which were treated with 9E9 anti-Fc{gamma}RIV mAb or control hamster IgG. C, A total of 200 µg of 34-3C IgG2b mAb was injected i.v. into Fc{gamma}RIII/C3–/– (III,C3–/–) or FcR{gamma}/C3–/– ({gamma},C3–/–) mice. D, A total of 200 µg of 34-3C IgG2b mAb was injected i.v. into Fc{gamma}RIII/C3–/– (III,C3–/–) mice, which were treated with 400 µg of 9E9 Fc{gamma}RIV-blocking mAb or control hamster IgG 30 min before and 2 days after administration of the 34-3C IgG2b mAb. Ht values of individual mice measured 4 days after i.v. injection of 34-3C mAb are shown. Mean Ht values are indicated by horizontal lines. The normal range of Ht values (mean ± 3 SD) of 2- to 3-mo-old BALB/c mice is represented as hatched areas.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study was designed to define the contribution of Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV) to the development of AIHA induced by IgG2a or IgG2b class-switch variant of 34-3C anti-RBC mAb. The analysis of mice deficient in Fc{gamma}RI, Fc{gamma}RIII, FcR{gamma}, and/or C3, in combination with 9E9 Fc{gamma}RIV-blocking mAb, revealed differential roles of the three activating Fc{gamma}R in the development of mild and severe anemia induced by IgG2a and IgG2b anti-RBC mAb. Our results demonstrate a major role for Fc{gamma}RIII in the development of IgG2a-induced mild anemia, an additional contribution of Fc{gamma}RI and Fc{gamma}RIV to the development of IgG2a-induced severe anemia, and the involvement of both Fc{gamma}RIII and Fc{gamma}RIV in the development of mild as well as severe anemia induced by IgG2b anti-RBC mAb.

Studies with a low dose (50 µg) of high affinity 34-3C IgG2a mAb confirmed a critical role of Fc{gamma}RIII in the development of mild AIHA, as is the case with 200 µg of low affinity 4C8 IgG2a mAb (4). The fact that the pathogenic effect of a low dose of 34-3C IgG2a mAb was unchanged in WT mice treated with Fc{gamma}RIV-blocking mAb as well as Fc{gamma}RI–/– mice clearly indicates that neither Fc{gamma}RI nor Fc{gamma}RIV plays a significant role in the development of mild anemia caused by the IgG2a subclass of anti-RBC autoantibodies. In contrast, both Fc{gamma}RI and Fc{gamma}RIV additionally contribute to the development of severe anemia induced by a high dose (200 µg) of 34-3C IgG2a mAb. This was documented by the following findings: first, the injection of this dose of 34-3C IgG2a mAb provoked a more severe anemia in Fc{gamma}RIII/C3–/– mice than in Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice; second, the IgG2aE235L mutant, which fails to interact with Fc{gamma}RI, induced less severe anemia in Fc{gamma}RIII/C3–/– mice, as compared with WT 34-3C IgG2a; and third, treatment with Fc{gamma}RIV-blocking mAb partially and completely inhibited the development of WT IgG2a-induced anemia in Fc{gamma}RIII/C3–/– and Fc{gamma}RI/Fc{gamma}RIII/C3–/– mice, respectively. Hence, the lack of involvement of both Fc{gamma}RI and Fc{gamma}RIV in the development of mild anemia induced by a low dose of 34-3C IgG2a mAb suggests that Fc{gamma}RI- and Fc{gamma}RIV-mediated erythrophagocytosis requires more extensive opsonization of RBC with IgG2a Abs in vivo, as compared with Fc{gamma}RIII-dependent erythrophagocytosis.

It is noteworthy that Fc{gamma}RI contributes to the development of IgG2a-mediated AIHA, since it has been considered that the high affinity Fc{gamma}RI plays a limited role in immune complex-mediated pathology, because of the competition of circulating monomeric IgG2a for its binding site. Nevertheless, our data suggest that higher densities of IgG2a bound to RBC in mice injected with a high dose of 34-3C IgG2a mAb can efficiently compete with circulating monomeric IgG2a for Fc{gamma}RI binding on phagocytes, thereby participating in erythrophagocytosis. This suggests that Fc{gamma}RI may play a particularly important role in immune clearance of pathogens and tumor cells present in the circulating blood, as well as in tissues. Indeed, Ab therapeutic approaches in mice revealed a considerable contribution of Fc{gamma}RI to the elimination of melanoma cells and blood B lymphocytes (16, 22, 23), and the clearance of Bordetella pertussis was shown to be markedly impaired in Fc{gamma}RI–/– mice (16).

In contrast to the observations made with the IgG2a, both Fc{gamma}RIII and Fc{gamma}RIV contributed to the development of mild and severe anemia induced by the IgG2b subclass. However, the way these two receptors trigger erythrophagocytosis is apparently different between anemia induced by low vs high doses of this subclass. The development of mild anemia after injection of a low dose (50 µg) was prevented not only in Fc{gamma}RIII–/– mice but also in WT mice treated with Fc{gamma}RIV blocking mAb. This suggests that neither Fc{gamma}RIII nor Fc{gamma}RIV alone is capable of triggering phagocytosis of RBC opsonized weakly with IgG2b, in contrast to those opsonized with IgG2a, which may be due to possible differences in the avidity of Fc{gamma}RIII to polymeric forms of IgG2a and IgG2b. Apparently, RBC weakly opsonized with the IgG2b subclass require an additional involvement of Fc{gamma}RIV to optimally trigger Fc{gamma}R-dependent phagocytosis. This interpretation is consistent with the previous finding that the IgG2b subclass of the low affinity 4C8 anti-RBC mAb was hardly pathogenic, whereas its IgG2a variant induced anemia as a result of Fc{gamma}RIII-mediated erythrophagocytosis (4). In addition, a synergistic cooperation of Fc{gamma}R and CR was required to promote efficient erythrophagocytosis and provoke anemia after injection of a low dose (50 µg) of 34-3C IgG2b but not 34-3C IgG2a mAb (5, 6). However, this restriction was no longer observed after administration of a high dose (200 µg) of 34-3C IgG2b in the present study, since Fc{gamma}RIII/C3–/– mice developed anemia as a result of Fc{gamma}RIV-mediated erythrophagocytosis, as documented by the protection from anemia due to treatment with Fc{gamma}RIV blocking mAb. Thus, it is possible that the extensive opsonization resulting from the injection of the high dose could overcome the low-avidity interaction of IgG2b with Fc{gamma}RIII and Fc{gamma}RIV, thus inducing Fc{gamma}RIII- and Fc{gamma}RIV-mediated phagocytosis in an independent manner. Notably, a similar scenario was proposed for the triggering of CR-mediated erythrophagocytosis in mice injected with a high dose of 34-3C IgG2a or IgG2b mAb (5, 6). In addition, our demonstration of a critical role of Fc{gamma}RIV in the development of mild anemia induced by IgG2b anti-RBC mAb is in agreement with the finding that Fc{gamma}RIV plays a remarkable role in IgG2b-mediated autoimmune thrombocytopenia, nephrotoxic nephritis, and immune depletion of B lymphocytes (13, 23, 24, 25).

Collectively, our present results have defined the understanding of the respective roles of the three known activating Fc{gamma}R in the development of AIHA, in which the usage of different Fc{gamma}R depends on the affinity, dose, and IgG subclass of anti-RBC autoantibodies (4, 6). The supplementary contribution of Fc{gamma}RI and Fc{gamma}RIV (i.e., in addition to Fc{gamma}RIII) to the development of severe anemia induced by IgG2a anti-RBC mAb is consistent with the idea that the development of severe tissue and cellular injury caused by IgG-immune complexes or autoantibodies is likely to be promoted through the involvement of multiple receptors, such as the activating Fc{gamma}R and CR (26, 27, 28). It has been established that activating Fc{gamma}RI and Fc{gamma}RIII contribute to the development of various IgG immune complex-mediated inflammatory reactions (14, 16, 29, 30, 31). In view of the contribution of Fc{gamma}RIV to the development of autoimmune thrombocytopenia (13, 24), nephrotoxic nephritis, (25) and AIHA (this report), Fc{gamma}RIV, too, plays a significant role in the pathogenesis of diverse inflammatory diseases mediated by autoantibodies and immune complexes. Further analyses in mice deficient in Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV will provide a better comprehension of the respective roles of individual Fc{gamma}R in IgG Ab-mediated pathology. Finally, a further understanding of Fc{gamma}RI in immune clearance, in addition to Fc{gamma}RIII and Fc{gamma}RIV, should provide useful guiding principles for the engineering of mAb for in vivo applications.


   Acknowledgments
 
We thank Dr. T. Moll for critical reading of the manuscript and G. Celetta, G. Brighouse, G. Sealy, and T. Le Minh for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by a grant from the Swiss National Foundation for Scientific Research. Back

2 Address correspondence and reprint requests to Dr. Shozo Izui, Department of Pathology and Immunology, Centre Médicale Universitaire, 1211 Geneva 4, Switzerland. E-mail address: Shozo.Izui{at}medecine.unige.ch Back

3 Abbreviations used in this paper: NZB, New Zealand Black; AIHA, autoimmune hemolytic anemia; CR, complement receptor; B6, C57BL/6; WT, wild type; Ht, hematocrit. Back

Received for publication September 28, 2007. Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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