Murine phagocytes express three different activating IgG FcγR: FcγRI is specific for IgG2a; FcγRIII for IgG1, IgG2a, and IgG2b; and FcγRIV for IgG2a and IgG2b. Although the role of FcγRIII in IgG1 and IgG2a anti-RBC-induced autoimmune hemolytic anemia (AIHA) is well documented, the contribution of FcγRI and FcγRIV to the development of IgG2a- and IgG2b-induced anemia has not yet been defined. In the present study, using mice deficient in FcγRI, FcγRIII, and C3, in combination with an FcγRIV-blocking mAb, we assessed the respective roles of these three FcγR 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 FcγRIII, while FcγRI and FcγRIV 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 FcγRIII and FcγRIV. Our results indicate differential roles of the three activating FcγR in IgG2a- and IgG2b-mediated AIHA.
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 FcγR-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 FcγR and to activate complement in vivo (4, 5, 6). Moreover, these analyses revealed that FcγR- 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 FcγR 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 FcγR, high affinity FcγRI and low affinity FcγRIII, have been identified on phagocytic effector cells in mice. Both are hetero-oligomeric complexes, in which the respective ligand-binding α-chains are associated with the common FcR γ-chain (FcRγ). FcRγ is required for the assembly and cell surface expression of these activating FcγR and for the triggering of their various effector functions (9). FcγRI is capable of binding only one IgG subclass, IgG2a, with high affinity, whereas the low affinity FcγRIII binds polymeric forms of three different IgG subclasses (IgG1, IgG2a, and IgG2b), but not IgG3 (10, 11). Most recently, a third activating receptor, FcγRIV, which binds IgG2a- and IgG2b-immune complexes with intermediate affinity, has been identified in mice (12, 13). FcγRIV is also composed of a specific α-chain and the common FcRγ. In contrast to IgG2a and IgG2b subclasses, FcγRIII is the sole receptor mediating IgG1-dependent phagocytosis in vivo (4, 13, 14, 15), and IgG3 is unable to trigger FcγR-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 FcγRIII (4, 15), indicating a predominant role for this receptor in AIHA. However, because of the specific recognition by FcγRI of IgG2a and by FcγRIV of IgG2a and IgG2b, it is of importance to define the respective contributions of FcγRI and FcγRIV (in comparison with FcγRIII) 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, FcγRIII-deficient mice were not always fully resistant under conditions where FcRγ knockout mice were (4, 6, 15, 16). Therefore, in the present study, using different mutant mice deficient in FcγRI, FcγRIII, FcRγ, and/or C3, in combination with an FcγRIV-blocking mAb, we assessed the respective contributions of all FcRγ-associated activating FcγR (i.e., FcγRI, FcγRIII, and FcγRIV) to the development of mild and severe anemia induced by 34-3C IgG2a and IgG2b class-switch variants. Our results show that FcγRIII plays a major role in the development of mild anemia induced by IgG2a 34-3C anti-RBC mAb, and that FcγRI and FcγRIV additionally contribute to the development of severe anemia induced by a high dose of this subclass. In contrast, both FcγRIII and FcγRIV were involved in the development of mild and severe forms of anemia induced by IgG2b 34-3C anti-RBC mAb.
Materials and Methods
FcγRI−/−, FcγRIII−/−, and C3−/− mice were generated by gene targeting in 129-derived embryonic stem cells, whereas FcRγ −/− mice were generated with C57BL/6 (B6)-derived embryonic stem cells, as described previously (14, 16, 17, 18). FcγRIII−/− 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. FcγRIII/C3−/−, FcγRI/FcγRIII/C3−/−, and FcRγ/C3−/− mice were obtained through intercross between corresponding deficient mice. FcRγ/C3−/− mice carry a B6 background, whereas FcγRIII/C3−/− and FcγRI/FcγRIII/C3−/− mice bear a mixed BALB/c and B6 background. The FcγRI, FcγRIII, and FcRγ genotypes were determined by PCR analysis using the following sets of primers. FcγRI: wild type (WT)-specific sense primer (5′-GTTTGCTGTGGTTTGAGACC-3′), mutant-specific sense primer (5′-TCGCCGATAGTGGAAACCGAC-3′), and common antisense primer (5′-TCCTTCTGGAAAATACTGACC-3′); FcγRIII: WT-specific sense primer (5′-TCCATCTCTCTAGTCTGGTACC-3′), mutant-specific sense primer (5′-ACTTGTGTAGCGCCAAGTGCCA-3′), and common antisense primer (5′-AAAAGTTGCTGCTGCCACC-3′); and FcRγ: 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).
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-Cγ2a (L235E) mAb mutant at position 235 (leucine to glutamic acid), thus lacking the high affinity binding motif (LEGGP instead of LLGGP) for FcγRI 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 κ-chain mAb (H18.104.22.168), followed by PE-conjugated streptavidin (5, 6). Hamster IgG 9E9 FcγRIV-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.
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 κ-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 FcγRIV, mice were treated with 400 μg of 9E9 anti-FcγRIV 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 FcγRI−/− 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-FcγRII/III and 9E9 anti-FcγRIV mAb, followed by PE-conjugated streptavidin, and the extent of IgG2a binding by FcγRI 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 FcγR (FcγRI, FcγRIII, and FcγRIV) with 34-3C IgG2a and IgG2aL235E, as described previously (13). Briefly, soluble versions of murine FcγR 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 was performed with the Wilcoxon two-sample test. Probability values <5% were considered significant.
Predominant role of FcγRIII in the development of mild anemia induced by 34-3C IgG2a mAb
To define the respective roles of FcγRI, FcγRIII, and FcγRIV in the development of IgG2a-induced anemia, the pathogenic effect of 34-3C IgG2a mAb was assessed in BALB/c mice deficient in either FcγRI or FcγRIII. 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. 1,A). FcγRIII−/− 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 FcγRI−/− mice (Fig. 1,A) or in WT BALB/c mice treated with 9E9 FcγRIV-blocking mAb (Fig. 1 B). These results indicated a major role of FcγRIII in the development of mild anemia caused by 34-3C IgG2a mAb.
Contribution of FcγRI and FcγRIV 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 FcRγ but failed to do so in FcRγ/C3−/− mice, indicating that FcγR- and CR-mediated erythrophagocytosis acted in an additive fashion to promote the development of severe anemia (6). Because FcRγ/C3−/− mice lack the functional expression of FcγRI, FcγRIII, and FcγRIV, we generated FcγRIII/C3−/− and FcγRI/FcγRIII/C3−/− mice to determine the possible contribution of FcγRI and FcγRIV to the development of severe anemia in this experimental setting. When 200 μg of 34-3C IgG2a mAb was injected, both FcγRIII/C3−/− and FcγRI/FcγRIII/C3−/− mice still developed highly significant anemia. However, the extent of anemia occurring in FcγRI/FcγRIII/C3−/− mice (mean Ht values at day 4: 29.2 ± 1.8%) was less severe than that of FcγRIII/C3−/− mice (20.7 ± 2.6%; p < 0.05; Fig. 2 A), suggesting the contribution of FcγRI 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 FcRγ/C3−/− mice failed to develop anemia (43.6 ± 0.8%; p < 0.05), these results suggested the involvement of FcγRI and/or FcγRIV in the development of severe anemia induced by IgG2a anti-RBC mAb.
To better define the contribution of FcγRI and/or FcγRIV to the development of IgG2a-induced severe anemia, FcγRIII/C3−/− and FcγRI/FcγRIII/C3−/− mice were treated with either 9E9 FcγRIV-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 FcγRIII/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. 2,B). In contrast, FcγRI/FcγRIII/C3−/− mice treated with 9E9 FcγRIV-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. 2,B). Histological analysis confirmed the complete absence of iron deposits by Kupffer cells in FcγRI/FcγRIII/C3−/− mice treated with 9E9 FcγRIV-blocking mAb, which contrasted to the presence of substantial levels of erythrophagocytosis in 9E9-treated FcγRIII/C3−/− mice (Fig. 3).
To confirm the involvement of FcγRI 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 FcγRI, as shown by the analysis with human FcγRI (19). Flow cytometric analysis confirmed the lack of binding of the IgG2aL235E mutant to FcγRI on bone marrow-derived macrophages (Fig. 4,A). The incapacity of the IgG2aL235E mutant to bind FcγRI was further confirmed by surface plasmon resonance analysis, while it binds to FcγRIV 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. 4,B). Thus, if FcγRI 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 FcγRIII/C3−/− mice and be unable to cause anemia when these mice were treated with 9E9 FcγRIV-blocking mAb. This was the case, since FcγRIII/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. 2,C), as compared with WT IgG2a (p < 0.05; Fig. 2,B). Notably, the treatment with 9E9 FcγRIV-blocking mAb completely abolished the development of anemia in IgG2aL235E-injected FcγRIII/C3−/− mice (45.4 ± 0.8%; p < 0.05; Fig. 2,C). Histological analysis confirmed the protective effect of 9E9 FcγRIV-blocking mAb on the induction of anemia induced by 34-3C IgG2aL235E mutant in FcγRIII/C3−/− mice (Fig. 3). Taken together, these results indicated a significant role of both FcγRI and FcγRIV in the development of severe anemia induced by 200 μg of 34-3C IgG2a mAb.
|mAb .||FcγRI .||FcγRIII .||FcγRIV .|
|IgG2a||3.9 × 107||6.0 × 105||1.4 × 107|
|IgG2aL235E||ND||1.7 × 105||1.8 × 107|
|mAb .||FcγRI .||FcγRIII .||FcγRIV .|
|IgG2a||3.9 × 107||6.0 × 105||1.4 × 107|
|IgG2aL235E||ND||1.7 × 105||1.8 × 107|
Results are expressed as association constant (Ka) (1/M); ND, not detectable.
Contribution of FcγRIV to the development of mild and severe anemia induced by 34-3C IgG2b mAb
We also evaluated the respective roles of FcγRIII and FcγRIV 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 FcγRIII−/− BALB/c mice (44.4 ± 1.9%; p < 0.01; Fig. 5,A). However, unlike after injection of 34-3C IgG2a mAb (Fig. 1,B), the development of mild anemia was also prevented in BALB/c mice treated with 9E9 FcγRIV-blocking mAb (9E9-treated mice, 43.4 ± 2.8%; control IgG-treated mice, 36.8 ± 1.3%; p < 0.05; Fig. 5,B). As in the case of 34-3C IgG2a mAb, at a highly pathogenic dose (200 μg) of 34-3C IgG2b mAb, FcγRIII/C3−/− mice still developed significant anemia (31.1 ± 2.2%), whereas FcRγ/C3−/− mice were resistant (44.9 ± 1.6%; p < 0.05; Fig. 5,C). The contribution of FcγRIV to the development of IgG2b-mediated severe anemia was confirmed by the failure of FcγRIII/C3−/− mice treated with 9E9 FcγRIV-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. 5,D). Notably, these mice failed to show any sign of erythrophagocytosis by Kupffer cells (Fig. 3).
The present study was designed to define the contribution of FcγRI, FcγRIII, and FcγRIV) 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 FcγRI, FcγRIII, FcRγ, and/or C3, in combination with 9E9 FcγRIV-blocking mAb, revealed differential roles of the three activating FcγR in the development of mild and severe anemia induced by IgG2a and IgG2b anti-RBC mAb. Our results demonstrate a major role for FcγRIII in the development of IgG2a-induced mild anemia, an additional contribution of FcγRI and FcγRIV to the development of IgG2a-induced severe anemia, and the involvement of both FcγRIII and FcγRIV 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 FcγRIII 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 FcγRIV-blocking mAb as well as FcγRI−/− mice clearly indicates that neither FcγRI nor FcγRIV plays a significant role in the development of mild anemia caused by the IgG2a subclass of anti-RBC autoantibodies. In contrast, both FcγRI and FcγRIV 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 FcγRIII/C3−/− mice than in FcγRI/FcγRIII/C3−/− mice; second, the IgG2aE235L mutant, which fails to interact with FcγRI, induced less severe anemia in FcγRIII/C3−/− mice, as compared with WT 34-3C IgG2a; and third, treatment with FcγRIV-blocking mAb partially and completely inhibited the development of WT IgG2a-induced anemia in FcγRIII/C3−/− and FcγRI/FcγRIII/C3−/− mice, respectively. Hence, the lack of involvement of both FcγRI and FcγRIV in the development of mild anemia induced by a low dose of 34-3C IgG2a mAb suggests that FcγRI- and FcγRIV-mediated erythrophagocytosis requires more extensive opsonization of RBC with IgG2a Abs in vivo, as compared with FcγRIII-dependent erythrophagocytosis.
It is noteworthy that FcγRI contributes to the development of IgG2a-mediated AIHA, since it has been considered that the high affinity FcγRI 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 FcγRI binding on phagocytes, thereby participating in erythrophagocytosis. This suggests that FcγRI 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 FcγRI 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 FcγRI−/− mice (16).
In contrast to the observations made with the IgG2a, both FcγRIII and FcγRIV 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 FcγRIII−/− mice but also in WT mice treated with FcγRIV blocking mAb. This suggests that neither FcγRIII nor FcγRIV 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 FcγRIII to polymeric forms of IgG2a and IgG2b. Apparently, RBC weakly opsonized with the IgG2b subclass require an additional involvement of FcγRIV to optimally trigger FcγR-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 FcγRIII-mediated erythrophagocytosis (4). In addition, a synergistic cooperation of FcγR 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 FcγRIII/C3−/− mice developed anemia as a result of FcγRIV-mediated erythrophagocytosis, as documented by the protection from anemia due to treatment with FcγRIV 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 FcγRIII and FcγRIV, thus inducing FcγRIII- and FcγRIV-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 FcγRIV in the development of mild anemia induced by IgG2b anti-RBC mAb is in agreement with the finding that FcγRIV 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 FcγR in the development of AIHA, in which the usage of different FcγR depends on the affinity, dose, and IgG subclass of anti-RBC autoantibodies (4, 6). The supplementary contribution of FcγRI and FcγRIV (i.e., in addition to FcγRIII) 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 FcγR and CR (26, 27, 28). It has been established that activating FcγRI and FcγRIII contribute to the development of various IgG immune complex-mediated inflammatory reactions (14, 16, 29, 30, 31). In view of the contribution of FcγRIV to the development of autoimmune thrombocytopenia (13, 24), nephrotoxic nephritis, (25) and AIHA (this report), FcγRIV, too, plays a significant role in the pathogenesis of diverse inflammatory diseases mediated by autoantibodies and immune complexes. Further analyses in mice deficient in FcγRI, FcγRIII, and FcγRIV will provide a better comprehension of the respective roles of individual FcγR in IgG Ab-mediated pathology. Finally, a further understanding of FcγRI in immune clearance, in addition to FcγRIII and FcγRIV, should provide useful guiding principles for the engineering of mAb for in vivo applications.
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.
The authors have no financial conflict of interest.
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.
This work was supported by a grant from the Swiss National Foundation for Scientific Research.
Abbreviations used in this paper: NZB, New Zealand Black; AIHA, autoimmune hemolytic anemia; CR, complement receptor; B6, C57BL/6; WT, wild type; Ht, hematocrit.