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Introduction of heterologous anti–glomerular basement membrane antiserum (nephrotoxic serum, NTS) into presensitized mice triggers the production of IgG anti-NTS antibodies that are predominantly IgG2b and the glomerular deposition of pathogenic immune complexes, leading to accelerated renal disease. The pathology observed in this model is determined by the effector cell activation threshold that is established by the coexpression on infiltrating macrophages of the IgG2a/2b restricted activation receptor FcγRIV and its inhibitory receptor counterpart, FcγRIIB. Blocking FcγRIV with a specific monoclonal antibody thereby preventing IgG2b engagement or treatment with high dose intravenous γ-globulin (IVIG) to down-regulate FcγRIV while up-regulating FcγRIIB, protects mice from fatal disease. In the absence of FcγRIIB, IVIG is not protective; this indicates that reduced FcγRIV expression alone is insufficient to protect animals from pathogenic IgG2b immune complexes. These results establish the significance of specific IgG subclasses and their cognate FcγRs in renal disease.
It is now well-established that IgG antibodies mediate their effector activities in vivo through their engagement of FcγRs (1). Mice that are deficient in activation FcγRs, by virtue of the targeted disruption of the common γ chain, are protected from the pathogenic effects of cytotoxic antibodies and immune complexes in both passive and active models of inflammation (2–6). In contrast, disruption of the inhibitory Fc receptor, FcγRIIB, enhances the pathogenic activity of IgG antibodies in many of these models (7–11). Although individual subclasses of IgG display different in vivo activities (12), it has been difficult, until recently, to account for the hierarchy of these activities based on their interaction with the activation FcγRs I and III. With the identification of a third, γ chain–dependent, IgG activation FcγR, FcγRIV (13), which selectively binds to IgG2a and 2b with intermediate affinity (Kd = 10−7), a direct association between IgG subclass activity and FcγR binding could be established (13, 14). This association is based on the respective binding affinities for each subclass to its cognate activation and inhibitory FcγR, thus resulting in a ratio of activation to inhibition that is predictive of the in vivo activity of an antibody (13, 14). Thus, IgG2a has an A/I ratio of 70 and IgG2b has an A/I ratio of 7, whereas IgG1 has an A/I ratio of 0.1, which fits with the hierarchy of activities of these subclasses in vivo such that IgG2a > IgG2b IgG1. However, these activities can be modified by the relative expression levels of the activation and inhibitory receptor on a specific effector cell, thus changing the avidity of an antibody-antigen complex for a specific activation/inhibitory pair. Inflammatory cytokines, such as IFNγ and C5a, up-regulate activation receptors, while down-regulating the inhibitory receptor (15–17). In contrast, high dose intravenous γ-globulin (IVIG) has been shown to up-regulate the inhibitory receptor (18, 19). The net result of these responses is to change the thresholds required for effector cell triggering and initiation of an inflammatory cascade at a given concentration of cytotoxic antibodies or immune complexes. IgG antibodies that interact with their restricted, cognate FcRs resulting in low A/I ratios are more sensitive to these avidity considerations than antibodies which interact with FcR with high A/I ratios (13, 14).
To investigate the consequences of subclass affinity for specific FcRs and the impact of Fc receptor modulation on IgG-mediated inflammation in an active model of inflammation, we selected the murine nephrotoxic nephritis model. This model resembles human Goodpasture's Disease (20–22) by virtue of its dependence on the targeting effect of anti–glomerular basement membrane (GBM) antibodies to direct immune complex deposition to the GBM and initiate an inflammatory response. The introduction of heterologous rabbit or sheep anti-GBM antiserum (NTS) results in an acute, complement-dependent, transient phase of renal injury (day 0–4) that is characterized by mildly elevated blood urea nitrogen (BUN) and proteinuria (21–24). The development of mouse anti–rabbit or –sheep antibodies in the later, autologous stage of this model (day 4–14) results in immune complex deposition and frank glomerulonephritis with renal failure and death (21, 22, 25). This autologous phase has been shown to be FcγR dependent (25, 26); mice lacking the common γ chain are protected from renal failure and death. An accelerated model of nephrotoxic nephritis has been developed in which mice are presensitized to the heterologous anti-GBM serum by preimmunization with normal, heterologous serum at day −4, followed by introduction of the heterologous anti-GBM serum at day 0, thus initiating the autologous phase of disease concomitant with the introduction of the anti-GBM serum (25, 26). Renal disease is evident by day 4, with significant mortality by day 8 through an FcγR-dependent pathway.
We now report the contribution of specific IgG subclasses and their cognate FcγRs to this accelerated autologous model of nephrotoxic nephritis and demonstrate the role of FcR modulation by IVIG in this active model of inflammation. The intrinsic FcγR affinities of IgG2b for the specific activating (FcγRIV) and inhibitory (FcγRIIB) receptor pair determines the pathology observed, whereas the selective receptor modulation by IVIG of both of these molecules alters their ratio on the infiltrating effector cells and contributes to the protection observed.
Presensitization of C57BL/6 mice with normal sheep IgG followed 4 d later by the introduction of sheep anti–mouse GBM serum induces acute glomerulonephritis with renal injury, as seen in Fig. 1. Elevated BUN was observed by day 4 in wild-type mice presensitized with normal sheep IgG and exposed to sheep NTS (CFA + NTS). Presensitization with sheep IgG alone, or exposure to anti-GBM serum alone did not result in elevated BUN (Fig. 1 A), despite the localization of the heterologous anti-GBM antibodies to the glomeruli (Fig. 1 C). Renal pathology was dependent, therefore, on the presence of mouse anti–sheep antibodies which colocalized to the glomeruli in the CFA + NTS–treated animals (Fig. 1 C) in a “ribbon” pattern, characteristic of Goodpasture's Disease, and was completely dependent on FcR expression. Mice deficient in the common γ chain, hence deficient in surface expression of the activation Fc receptors FcγRI, III and IV, as well as FcRI, were protected from the pathogenic consequences of CFA + NTS (Fig. 1 B).
Individual, γ chain–dependent FcRs interact with specific isotypes and subclasses of immunoglobulins (13, 14). To further define the pathology observed in the CFA + NTS model, wild-type mice were characterized for the specific subclasses of IgG anti–sheep GBM antibodies they produced at day 4 after CFA + NTS treatment. As shown in Fig. 2, only mouse IgG2b anti–sheep Ig showed a statistically significant elevation (P < 0.001) after CFA + NTS treatment. NTS alone did not induce measurable levels of mouse anti–sheep Ig at day 4 after exposure to anti-GBM serum. IgG1 anti–sheep Ig was elevated, although the level was not statistically significant over baseline or NTS alone (Fig. 2).
We recently described a novel IgG Fc receptor in the mouse with selective specificity for IgG2a and 2b and with a binding affinity ~10-fold higher than that observed for these subclasses binding to FcγRIII (13). To determine the relative contributions of these Fc receptors to the renal pathology observed in the nephrotoxic nephritis model described here, we induced disease in either common γ chain–deficient (FcRγ−/−), FcγRIII–deficient (FcγRIII−/−) or wild-type mice treated with a monoclonal anti-FcγRIV antibody shown to specifically block this activation FcγR (13, 14; Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20051900/DC1). Common γ chain–deficient mice were protected, as expected, as were mice treated with the anti-FcγRIV blocking mAb (Fig. 3, A and B, and Fig. S2). FcγRIII-deficient mice developed disease comparable with wild-type mice, whereas FcγRIIB-deficient mice displayed enhanced disease, as revealed by the accelerated mortality of this strain when treated with CFA + NTS (Fig. 3 B). We also induced nephrotoxic nephritis in FcγRI and RIII double–deficient (FcγRI/III−/−) mice, and these animals developed disease comparable with wild-type controls (Fig. 3, C and D and Fig. S2). Because no differences in the titer of mouse IgG2b anti–sheep Ig was observed for any of the genotypes studied (Fig. S3), the data indicate that the critical activation FcγR in this model is FcγRIV, which is consistent with studies in passive models of idiopathic thrombocytopenic purpura (ITP) and B cell and tumor cell clearance (13, 14; unpublished data). Staining of infiltrating macrophages with anti-FcγRIV antibody revealed accumulation of these cells in the glomeruli of CFA + NTS–treated animals, thus providing a mechanism for their engagement of IgG2b immune complex deposited on the GBM and the inflammation observed in this model (Fig. 3 E).
We have previously demonstrated in passive models of ITP and arthritis that IVIG is able to protect from the pathogenic consequences of cytotoxic antibodies or immune complex deposition by induction of the inhibitory receptor, FcγRIIB (18, 19). Similarly, IVIG treatment attenuates the renal pathology observed in CFA + NTS nephrotoxic nephritis and is dependent on the Fc fragment for its activity (Fig. 4 A). This protection results in extended survival for IVIG-treated animals (Fig. 4 B). IVIG treatment did not modify the development of mouse IgG2b anti–sheep Ig antibodies (Fig. S3) or the deposition of either sheep anti-GBM antibodies, total mouse IgG or IgG2b antibodies, or complement C3 in the glomeruli (Fig. 5). To determine the mechanism by which IVIG protects CFA + NTS–treated mice from developing renal pathology, we examined the expression of FcγRIIB and FcγRIV on macrophages infiltrating the kidney in this disease. As shown in Fig. 6 A, IVIG significantly induces the surface expression of FcγRIIB on infiltrating macrophages (P = 0.004), as we have previously described for splenic (18) and infiltrating synovial macrophages (19). However, and in contrast to these previous studies, IVIG treatment also resulted in reduced surface expression of FcγRIV (Fig. 6 B and Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20051900/DC1). FcγRIII expression was unchanged (unpublished data) as was FcγRIIB and FcγRIV expression on granulocytes (unpublished data). The combined effect of reduced FcγRIV and elevated FcγRIIB alters the activation threshold for IgG2b engagement of FcγRIV and protects mice from the pathological consequences of IgG2b immune complex deposition in the glomeruli.
Because IgG2b can bind to both FcγRIII and FcγRIV, albeit with significantly different affinities, the relative contributions of FcγRIIB, FcγRIII and FcγRIV modulation to the protective effect of IVIG was examined. As we have observed previously (18, 19), IVIG protection was ablated in FcγRIIB-deficient mice (Fig. 7 and Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20051900/DC1), which is consistent with FcγRIIB expression contributing to the protective effect of IVIG. Thus, the down-regulation of FcγRIV by IVIG treatment was not sufficient to provide protection, and thus required additional modulation of FcγRIIB. CFA + NTS–treated FcγRIIB-deficient mice were protected by anti-FcγRIV, indicating that FcγRIV is the sole activation FcR involved in the pathology of this disease, despite the ability of IgG2b to engage FcγRIII. Consistent with this observation, IVIG protection was not affected by FcγRIII deficiency (Fig. 7).
The selective engagement of IgG subclasses with specific FcγRs is a determining factor in the pathophysiology of a variety of antibody-mediated inflammatory disorders and antibody based immunotherapeutics. Simultaneous engagement of both activating (FcγRIII and FcγRIV in the mouse model; FcγRIIA and FcγRIIIA in the human model) and inhibitory receptors (FcγRIIB in both mouse and human models) determines the threshold for triggering activation of effector cells such as macrophages, mast cells, and neutrophils. Detailed studies performed in the murine system have revealed the specifics of these interactions. Thus, IgG1 antibodies mediate their in vivo activity through simultaneous engagement of the low-affinity receptors FcγRIIB and FcγRIII, whereas IgG2a and IgG2b are dependent on the FcγRIIB and FcγRIV pair. Several studies have confirmed the role of FcγRIIB and FcγRIII in IgG1-mediated inflammation in vivo, based on studies performed in experimental models of cytotoxic antibody–triggered clearance including ITP (13), hemolytic anemia (27, 28) B cell clearance (29) and tumor metastases (9); and immune complex–mediated diseases, such and the cutaneous and alveolar Arthus reactions (30) and KRNxNOD serum-induced arthritis (19). Passive models of IgG2a- and 2b-mediated clearance, although sensitive to deletion of the common γ chain and hence deficient in FcγRI, III and RIV, were not sensitive to either FcγRI or RIII deletion (13, 14). Instead, blockade of FcγRIV abrogates IgG2a- or 2b-mediated effector responses in vivo in passive models of ITP, B cell clearance; and tumor metastasis (13, 14; unpublished data). Thus, despite the ability of IgG2a to bind with high affinity to the activating receptor FcγRI (31), this receptor has not been shown to be central to the mechanism by which IgG2a mediates its biological activity in vivo (28, 32). This general lack of FcγRI involvement is likely to result from the occupancy of this receptor by circulating immunoglobulin during the steady state, thereby rendering it unavailable for specific IgG2a-mediated cross-linking.
Several active models of antibody-mediated inflammation have suggested that skewing of the IgG subclass response occurs in response to a variety of inflammatory stimuli. Thus, in spontaneous murine lupus models, anti-DNA antibodies of the IgG2a or 2b class appear to dominate (unpublished data). Similarly, active antiviral protection models have observed a dominant role for IgG2a antiviral antibodies (33–35). Our present study reveals that skewing to IgG2b is prominent in accelerated anti-GBM nephritis, perhaps as a result of the contribution of TGFβ to the glomerular injury seen in this model (36). TGFβ up-regulation has been shown to occur in a variety of conditions of renal injury (37, 38) and has also been shown to induce class switching to IgG2b in vitro (39, 40). It is likely that skewing of IgG subclasses will be a common feature of inflammatory conditions, in response to the specific cytokine milieu present in these states. Although previous studies of accelerated nephrotoxic nephritis all demonstrated a dependence on FcγRs by virtue of the protection observed in the common γ chain–deficient strain, these studies differed on the role of FcγRIII in this model. Fujii et al. (41) demonstrated that renal disease was substantially attenuated in a rabbit anti-GBM model in FcγRIII-deficient mice, although to a lesser degree than FcRγ chain deficiency for some parameters of renal injury. These investigators did not define the IgG subclasses involved in their model, which could account for the differences with our study. It is tempting to speculate that the mouse anti–rabbit GBM antibody response is dominated by IgG1 antibodies, hence FcγRIII dependent, in contrast to the mouse anti–sheep GBM antibody response that is IgG2b and FcγRIV dependent. Tarzi et al. (42), using a sheep anti-GBM model of nephrotoxic nephritis, similar to that used in this study, observed protection in FcRγ-deficient mice, but not FcγRIII-deficient animals, similar to our observations. They did not investigate FcγRI-deficient mice, but did report attenuation in the double FcγRI/III-deficient strain. They, however, used a fivefold lower dose of sheep GBM antiserum in their experiments and mainly histological changes as readout. Moreover, the specific IgG subclasses were not described. In contrast, we observe severe BUN elevation and almost 50% mortality under our experimental conditions.
Consistent with our previous studies (18, 19), we demonstrate that high dose intravenous IgG protects mice from nephrotoxic nephritis and this protection is dependent on FcγRIIB. FcγRIIB-deficient mice are not protected by IVIG, because one activity of IVIG is to up-regulate FcγRIIB on infiltrating macrophages. This mechanism has also been observed in the serum transfer arthritis model, KRNxNOD (19), which is an IgG1-mediated immune complex–driven model of arthritis and results in an elevated threshold for FcγRIII-driven inflammation. However, in contrast to that FcγRIII-driven model, the effect of IVIG in this study is to both up-regulate FcγRIIB and down-regulate FcγRIV. Because IgG2b has an activation/inhibitory affinity ratio 70 times that of IgG1, resulting from the higher affinity of IgG2b for FcγRIV (13), up-regulation of FcγRIIB alone would not be sufficient to raise the threshold required to prevent macrophage triggering by IgG2b immune complexes. Thus, the mechanism of protection by IVIG differs for IgG subclasses and suggests that the normal physiological mechanism underlying this activity reflects an in vivo feedback inhibition pathway that is subclass specific and has evolved to prevent sustained and inappropriate effector cell activation after resolution of an inflammatory state.
Our present study highlights the importance of specific IgG subclasses and their cognate Fc receptors in defining the pathophysiology of disease states. This knowledge is of importance in identifying the molecular pathways involved and thereby selecting the appropriate targets for intervention.
C57BL/6 mice were purchased from the Jackson Laboratory. FcRγ−/− (2), FcγRIIB−/− (8), and FcγRIII−/− mice were generated in our laboratory and backcrossed for 12 generations to the C57BL/6 background. FcγRI/III−/− mice on a hybrid C57BL/6/129F2 background were compared with a genetically matched control line. Female mice at 6–10 wk of age were used for all experiments and maintained at the Rockefeller University animal facility. All experiments were done in compliance with federal laws and institutional guidelines and have been approved by the Rockefeller University.
Human IVIG (5% in 10% maltose, chromatography purified) was purchased from Octapharma. Digestion of human IVIG was performed as described previously (19). In brief, IVIG was digested by 0.5 mg/ml papain for 1 h at 37°C, and stopped by the addition of 2.5 mg/ml iodocasetamide. The resulting Fab and Fc fragments were separated from nondigested IVIG on a HiPrep 26/60 S-200HR column (GE Healthcare), and then Fc fragments were separated from Fab fragments using a protein G column (GE Healthcare) and a protein L column (Pierce Chemical Co.). Fragment purity was checked by immunoblotting using anti–human IgG Fab- or Fc-specific antibodies (Jackson ImmunoResearch Laboratories). Purity was judged to be >99%. Antibodies 2.4G2, Mac-1, Gr-1 were purchased from BD Biosciences. The anti-FLAG antibody was purchased from Sigma-Aldrich. The F4/80 and CD68 antibody was obtained from Serotec. The Ly 17.2 antibody was purchased from Caltag. Hamster monoclonal antibodies against mouse FcγRIV, 9E9, and 9G8.1 were generated in our laboratory (13). 200 μg of the 9E9 antibody, which interferes with immune complex binding to FcγRIV, was injected intravenously every other day from day 0 to 14. Hamster IgG was used as an isotype control. The 9G8.1 antibody was conjugated with Alexa 647 (Invitrogen) and used for flow cytometric analysis. Chinese hamster ovary cells stably transfected with FLAG-tagged murine versions of FcγRI, FcγRIIB, FcγRIII, and FcγRIV were generated in our laboratory (13).
Sheep anti-GBM antigen was prepared as described previously (43). In brief, glomeruli were isolated from normal C57BL/6 mouse kidneys by differential sieving, washed extensively, and used to hyperimmunize sheep as described previously (43). The hyperimmune serum was heat inactivated, and then absorbed with excess amount of murine red blood cells.
Mice were preimmunized intraperitoneally with 200 μg of sheep IgG (Serotec) in CFA, followed by intravenous injection of sheep NTS (2.5 μl of serum per gram of mouse) 4 d later. IVIG (1 g/kg) or its vehicle alone was injected 1 h before anti-GBM antiserum injection.
Kidneys were removed, fixed in 10% buffered formalin, and embedded in paraffin. 4 μm paraffin sections were stained with periodic acid-Schiff (PAS) and evaluated by light microscopy in a blind manner as previously described (44). In brief, the clinical scores of glomerular injury were graded into five grades: 0 (normal), 1 (mild increase in cellularity), 2 (definite glomerular enlargement, focal hypercellularity and mild increase of matrix), 3 (focal hypercellularity and proliferation in >50% of glomeruli), and 4 (diffuse proliferative change with crescents and sclerosis in >50% of glomeruli). Tubulointerstitial lesions were also graded from 0 to 4 according to the severity of inflammatory cell infiltration. Crescent formation was evaluated as the number of the crescents per 10 glomeruli. The mean number of macrophages infiltrating in the glomeruli were counted in 50 glomeruli in each sample.
4-μm frozen sections were fixed in acetone and stained with FITC-conjugated anti–sheep IgG, Cy3-conjugated anti–mouse IgG (Jackson ImmunoResearch), FITC-conjugated anti–mouse C3 (Cedarlane), and FITC-conjugated anti–mouse IgG2b (BD Biosciences) antibody, respectively. For quantitative immunofluorescence, blinded sections were examined at 100 magnification using MetaMorph version 6.1 (Molecular Devices). The mean intensity of 20 glomeruli for each sample was measured for evaluation. For immunohistochemical analysis, frozen sections were incubated with 5 μg/ml of biotinylated anti-FcγRIV monoclonal antibody (clone 9E9) after blocking with 500 μg/ml of hamster IgG in 5% goat serum and biotin blocking (DakoCytomation). After blocking of endogenous peroxidase was performed, HRP-conjugated rabbit antibiotin antibody ( DakoCytomation) was used as the secondary antibody and 3–3′-diaminobenzidine was used for visualization. Biotinylated anti-CD68 antibody (clone FA11) was used to detect macrophages in the kidney.
96-well ELISA plates coated with 5 μg/ml of sheep IgG were incubated with 1:500 diluted test sera after blocking with 5% bovine serum albumin. After washing with PBS containing 0.05% Tween 20, the plates were incubated with HRP-conjugated anti–mouse IgG1, IgG2c (Igh 1b allele of IgG2a), IgG2b, or IgG3 antibody (Bethyl Laboratories). For the color development, 3,3′,5,5′-tetramethylbenzidine was used.
BUN in sera was measured by the urease–indophenol method with an Enzymatic Urea Nitrogen kit (Stambio Laboratory).
Kidneys were pressed through a mesh, and the cells were resuspended in PBS. After washing, the cells were suspended in 33% Percoll solution and centrifuged 2,000 rpm for 20 min at room temperature. After red blood cell lysis, the cells were stained with the indicated monoclonal antibodies, and were subjected to flow cytometric analysis. 2,000 F4/80-positive cells infiltrating in the kidneys of each mouse were evaluated for mean fluorescence intensity of FITC-conjugated Ly 17.2 or Alexa 647–conjugated 9G8.1 antibody.
Statistical differences in each group for the BUN, circulating IgG levels, and mean fluorescent intensity, were calculated with Student's t test. Mann-Whitney U test was used for histological analysis. Survival rate was analyzed with Kaplan-Meier estimates, and groups were compared with the log rank test. P < 0.05 was considered significant. SPSS version 11.0 for Windows (SPSS Inc.) was used for statistical analysis.
Fig. S1 shows specificity of anti-FcγRIV monoclonal antibody 9E9. Fig. S2 demonstrates histological evaluation of kidney sections from wild-type C57BL/6 mice, anti-FcγRIV antibody–treated or isotype-matched antibody–treated wild-type C57BL/6 mice, FcγRI/III−/− mice or its genetically matched control (129/B6) mice on day 8 from NTS injection. Fig. S3 contains serum titer of each isotype of autologous mouse anti–sheep IgG-specific IgG. Fig. S4 shows histograms of FcγRIIB and FcγRIV expression on F4/80-positive cells infiltrating the kidney on day 2. Fig. S5 demonstrates histological evaluation of kidney sections from wild-type C57BL/6 mice and FcγRIIB−/− mice treated with IVIG or its vehicle on day 8 from NTS injection. Supplemental figures are available at http://www.jem.org/cgi/content/full/jem.20051900/DC1.
We thank all the members of the Ravetch laboratory for expert technical assistance, helpful discussions, and suggestions.
Y. Kaneko was supported by fellowships from the Naito Foundation, Kanae Foundation for Life and Social-medical Science, and the Uehara Memorial Foundation. F. Nimmerjahn was supported by a grant form the Cancer Research Institute. These studies were supported in part by grants to J.V. Ravetch from the National Institutes of Health.
The authors have no conflicting financial interests.
Abbreviations used: BUN, blood urea nitrogen; GBM, glomerular basement membrane; ITP, idiopathic thrombocytopenic purpura; IVIG, intravenous γ-globulin; NTS, anti-GBM antiserum; PAS, periodic acid-Schiff.