The relative ability of IgG subclasses to cause acute inflammation and the roles of specific effector mechanisms in this process are not clear. We explored this in an in vivo model of glomerular inflammation in the mouse. Trinitrophenol was planted on the glomerular basement membrane after conjugation to nephrotoxic Ab. The relative nephritogenicity of anti-trinitrophenol switch variant mAbs was then explored and shown to be IgG2a > IgG2b, with no disease caused by IgG1. Using knockout mice, we showed that FcγRIII was necessary for both neutrophil influx and glomerular damage induced by IgG2a and IgG2b. Surprisingly, IgG1 did not cause disease although it binds to FcγRIII. Using blocking Abs, we showed that this was explained by an additional requirement for FcγRIV, which does not bind to IgG1. IgG2a- or IgG2b-induced neutrophil influx was not affected by deficiency of either FcγRI or C3. Bone marrow chimeras were constructed to test the effect of combined deficiency of FcγRI and C3, and there was no effect on IgG2a- or IgG2b-mediated neutrophil influx. However, IgG2b-induced albuminuria and thrombosis were reduced in C3-deficient mice, showing an additional role for complement in IgG2b-mediated glomerular damage. The results show that IgG2a and IgG2b are the pathogenic subclasses in acute neutrophil-mediated glomerular inflammation, with an indispensable role for both FcγRIII and FcγRIV. Additionally, complement contributes to IgG2b-induced glomerular injury.

Several lines of evidence support a central pathogenic role for immune complexes in systemic autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis, and vasculitis. Many of these systemic diseases have glomerulonephritis (GN)3 as their most serious manifestation, and immune complexes also play a role in other forms of primary GN. Although the relative roles of cell-mediated and humoral immunity in GN continue to be debated, some key observations show a crucial role for immune complexes. Lupus-prone mice deficient in Fc receptors are almost completely protected from GN, despite the deposition of immune complexes (1). Fc receptor-deficient mice are also protected from autologous nephrotoxic nephritis (2). The latter is an induced model of GN in which disease is caused by an autologous response to a foreign Ab planted on the glomerular basement membrane.

The proinflammatory functions of IgG are largely determined by the interaction of the Fc portion of the H chain with complement and with Fc receptors. Murine IgG subclasses differ in their ability to activate complement, with IgG2a and IgG2b, but not IgG1, activating complement well (3, 4). Cell membrane receptors for the Fc portion of IgG are widely distributed on macrophages, monocytes, and neutrophils and couple immune complex formation to cellular effector mechanisms (5). Activating Fc receptors comprise FcγRI, FcγRIII, and the recently described FcγRIV (6). FcγRI is a high-affinity receptor that has been shown in vitro to bind monomeric and complexed IgG2a, with a much lower affinity for IgG2b (7). FcγRIII is a low-affinity receptor that binds to IgG2a, IgG1, and IgG2b but not to IgG3 (8). FcγRIV binds to IgG2a and IgG2b with little affinity for IgG1 and IgG3 (6).

Particular forms of GN are associated with specific subclasses of IgG in both humans and in mice. Human lupus nephritis has been associated with IgG1, IgG2, and IgG3, membranous nephropathy with IgG4, mesangiocapillary GN with IgG3, and anti-glomerular basement membrane disease with IgG1 and IgG4 (9, 10, 11). In murine lupus, earlier studies suggested that IgG2a was the dominant subclass in glomerular eluates from NZB/W mice (12, 13). In BXSB mice IgG2b was reported to be the dominant subclass in glomerular eluates, with IgG2a and IgG2b equally common in MRL/lpr (MRL/Mp-lpr/lpr) mice (13). A recent report on spontaneous lupus-like disease, in mice lacking the inhibitory receptor FcγRIIb, has also suggested that IgG2a and IgG2b are the dominant subclasses deposited in glomeruli (14). The descriptive finding of particular IgG subclasses in serum, on immunofluorescence staining, or in glomerular eluates does not directly demonstrate the pathogenicity of these subclasses. It is quite possible that a subclass deposited in small amounts is highly pathogenic. Additionally, the relative amounts in serum or glomeruli are difficult to judge in assays that use subclass-specific detecting Abs that have different affinities. To directly show the effect of an IgG subclass, the Ab needs to be passively given in vivo in a model of inflammation. Even then, the relative role of IgG subclasses cannot be reliably deduced from studies that compare random panels of mAbs. A previous study in rats compared disease that was induced by mAbs to type IV collagen in WKY rats (15). However, as well as being of different subclasses, these mAbs would have differed in affinity and specificity and this could have led to differences in pathogenicity. The most rigorous way to assess the role of subclass is through the use of switch variants that have identical heavy and L chain-variable regions and differ only in the H chain portion determining subclass.

Previous in vivo studies have explored the role of IgG subclass using switch variants in models such as hemolytic anemia (16), melanoma therapy and thrombocytopenia (6, 17), and lymphoma treatment (18). To assess the role of IgG subclass in acute glomerular inflammation we have developed a model in which disease is passively induced by monoclonal switch variants against trinitrophenol (TNP), after TNP was first planted on the glomerular basement membrane. We could show that even a high dose of IgG1 was unable to induce disease, whereas lower doses of IgG2a and IgG2b were pathogenic, with more severe disease caused by IgG2a. Using knockout mice and blocking Abs, we explored the effector mechanisms responsible. Our data show that both FcγRIII and FcγRIV were required for all of the disease manifestations caused by IgG2a and IgG2b. Additionally, complement contributed to IgG2b-mediated glomerular damage, as reflected by the development of thrombosis and proteinuria.

FcγRI- and FcγRIII-deficient mice were generated as described (19, 20). FcγRI-deficient mice were backcrossed six generations onto a C57BL/6 background (from a mixed C57BL/6/BALBc/129 background), and FcγRIII-deficient mice were backcrossed 12 generations onto C57BL/6. C3-deficient mice were originally obtained from M. C. Carroll (21) and backcrossed 11 generations onto C57BL/6. Wild-type C57BL/6 mice were obtained from Harlan U.K. Bone marrow chimeras were constructed as described previously (22). Chimerism was assessed as described using real-time PCR for the neomycin resistance gene (present in C3−/− but not FcγRI−/− mice) on genomic DNA from peripheral blood as described (22). C3 levels were measured in the serum of FcγRI−/− → C3−/− chimeras by ELISA. The coating Ab was sheep anti-mouse C3 (ICN Biomedicals), and the detecting Ab was HRP-conjugated goat anti-mouse C3 (Nordic). The standard was serial dilutions of normal mouse serum. Age-, sex-, and weight-matched mice were used for all experiments. All experiments were performed according to U.K. Home Office regulations.

Hybridomas secreting IgG1, IgG2a, and IgG2b anti-TNP switch variants were a gift from Lucien Aarden (Sanquin). An IgG1 hybridoma Hy2.15 derived from BALBc mice was originally a gift from G. Kohler. The switch variants were obtained from this hybridoma by sequential sublining in combination with isotype-specific ELISAs according to previously published methods (23). Anti-TNP hybridomas were grown at standard cell density in serum free-medium and purified using protein G chromatography from concentrated supernatant under identical conditions. Purity was confirmed at >95% by SDS gel electrophoresis. After purification, the affinities of the mAbs were compared by ELISA. Maxisorb ELISA (Nunc) plates were coated with TNP-conjugated sheep IgG (prepared as below) at 10 μg/ml. After blocking with 1% BSA, various dilutions of monoclonal anti-TNP were applied. The detecting Ab was alkaline phosphatase-conjugated polyclonal anti-kappa L chain (SouthernBiotech). FcγRIV receptor-blocking Ab 9E9 has been described previously (6) and was purified with protein G chromatography. Flow cytometric analysis 9E9 specificity was performed as described (6). Normal hamster IgG (Jackson ImmunoResearch Laboratories) was used as a control.

Nephrotoxic Ab was prepared by immunizing sheep with a kidney extract prepared as previous described (24). IgG was purified using DEAE-Sepharose and was >90% pure on SDS gel electrophoresis. This was conjugated with TNP by incubation with trinitrofluorobenzene in 0.2 M bicarbonate buffer (pH9). After dialysis into PBS, the conjugate was characterized spectrophotometrically using a described formula (25). Conjugates used had a ratio of 11 moles TNP per mole of IgG. Disease was induced by injecting 1.3 mg of conjugate i.v., followed 24 h later by anti-TNP mAb. LPS was from E. coli 0111:B4 (Sigma, catalog no. L3024).

Kidney was fixed in Bouin’s solution, and paraffin-embedded sections were stained with periodic acid-Schiff. Neutrophils per glomerular cross-section were counted and thrombosis was scored as described (22, 26). Urine was collected from mice that were housed in metabolic cages for 24 h immediately following the injection of mAb, and albuminuria was measured by radial immunodiffusion as described (22). The sensitivity of the assay was 25 μg/ml. Peripheral blood neutrophil numbers were measured as described (22).

Immunofluorescence staining was performed on material snap-frozen in isopentane. Direct immunofluorescence for TNP was performed using the IgG1 monoclonal anti-TNP, conjugated with FITC using standard techniques. Indirect immunofluorescence for complement components was performed using rat monoclonal anti-C1q, anti-C3, or anti-C4 (all from Cedarlane Laboratories) and FITC mouse anti-rat IgG (Jackson ImmunoResearch Laboratories). For assessment of complement staining, image analysis techniques could not be used due to the mesangial deposition of complement seen in normal mice. The basement membrane staining was therefore assessed by the observer (with no knowledge of sample identity) on a scale of 0–4, disregarding the mesangial staining. For FcγRIV staining, 9E9 was used with detection by goat anti-hamster IgG (Jackson ImmunoResearch Laboratories), followed by Alexa Fluor 488 goat anti-FITC (Molecular Probes).

Statistics were performed using GraphPad Prism (GraphPad Software). A Student’s t test was used where two groups were compared and a one-way ANOVA with either Tukey’s or Dunnett’s posttest where there were more than two groups. Data were analyzed after a logarithmic transformation if the variances were significantly different.

We first established a model in which glomerular inflammation was induced by anti-TNP mAb. TNP-conjugated nephrotoxic Ab was injected, followed 24 h later by monoclonal anti-TNP Ab. Immunofluorescence staining verified that TNP was deposited on the glomerular basement membrane, along with mouse IgG, as shown in Fig. 1, A and B. Preliminary experiments established that endotoxin was necessary as a co-factor, and that 15 mg/kg of IgG2a was needed to cause robust disease. When 100 ng of endotoxin was given with 15 mg/kg IgG2a anti-TNP mAb as a single i.v. injection, a significant neutrophil influx was seen, as shown in Fig. 1,C. When either mAb or endotoxin alone was given, only a mild neutrophil influx was seen, as shown in Fig. 1,D. These results established a requirement for endotoxin, and this was given with mAb at a dose of 100 ng per mouse in all subsequent experiments. As we planned to compare disease induced by the switch variants, it was important to verify identical binding to TNP of purified Ab. This was confirmed by ELISA as shown in Fig. 1 E.

FIGURE 1.

Glomerular inflammation induced by switch variant mAbs. A, Immunofluorescence staining for TNP in mice given nephrotoxic Ab conjugated with TNP. This shows a linear glomerular basement membrane pattern. B, Immunofluorescence staining for mouse IgG in mice given anti-TNP mAb 24 h after an injection of TNP-conjugated nephrotoxic Ab, again showing a linear glomerular basement membrane pattern. C, Histological changes observed 2 h after the injection of anti-TNP mAb given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. This shows a prominent neutrophil influx (identified as irregular lobulated nuclei). D, Endotoxin is required for the neutrophil influx. A significant neutrophil influx is seen when both endotoxin (100 ng) and 15 mg/kg IgG2a anti-TNP mAb are given together, but not when either is given alone. GCS, Glomerular cross-section. E, Switch variant anti-TNP monoclonals have identical binding to TNP as shown by ELISA.

FIGURE 1.

Glomerular inflammation induced by switch variant mAbs. A, Immunofluorescence staining for TNP in mice given nephrotoxic Ab conjugated with TNP. This shows a linear glomerular basement membrane pattern. B, Immunofluorescence staining for mouse IgG in mice given anti-TNP mAb 24 h after an injection of TNP-conjugated nephrotoxic Ab, again showing a linear glomerular basement membrane pattern. C, Histological changes observed 2 h after the injection of anti-TNP mAb given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. This shows a prominent neutrophil influx (identified as irregular lobulated nuclei). D, Endotoxin is required for the neutrophil influx. A significant neutrophil influx is seen when both endotoxin (100 ng) and 15 mg/kg IgG2a anti-TNP mAb are given together, but not when either is given alone. GCS, Glomerular cross-section. E, Switch variant anti-TNP monoclonals have identical binding to TNP as shown by ELISA.

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As we had established that 15 mg/kg of IgG2a was necessary to induce disease, we compared disease induced by this dose of IgG1, IgG2a, and IgG2b anti-TNP switch variants. Results at 2 h are shown in Fig. 2,A, with representative histology in Fig. 3. IgG2a induced a greater neutrophil influx into glomeruli than did IgG2b or IgG1. IgG2b induced a greater neutrophil influx into glomeruli than did IgG1. IgG1 did not cause a neutrophil influx, since neutrophil numbers in glomeruli after IgG1 administration were not significantly different from those seen after endotoxin alone. Note that in this and later experiments, the control group given endotoxin alone was given TNP-conjugated nephrotoxic Ab 24 h earlier, as were the other mice. We then assessed whether a higher dose of IgG1 could induce a neutrophil influx. Even 100 mg/kg of IgG1 did not result in a neutrophil influx greater than that seen after endotoxin alone. IgG2a induced more severe albuminuria than did IgG2b, as shown in Fig. 2,B. Even after 100 mg/kg of IgG1, albuminuria was seen only at trace levels (<70 μg/24 h in all mice). At 24 h, assessment of glomerular thrombosis gave results that mirrored those for albuminuria as shown in Fig. 2,B, with representative histology shown in Fig. 3. There was significantly more thrombosis after IgG2a than IgG2b, with none seen even after 100 mg/kg of IgG1. These data established the relative pathogenicity of IgG subclasses as IgG2a > IgG2b ≫ IgG1.

FIGURE 2.

The relative pathogenicity of switch variant mAbs. A, Numbers of neutrophils infiltrating glomeruli at 2 h in mice given the same i.v. dose of switch variant anti-TNP mAbs, with endotoxin 100 ng, 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The lps group are mice given endotoxin 100 ng alone in the second injection. Numbers for IgG2a were significantly greater than for IgG2b, and numbers for IgG2b were significantly greater than for IgG1. IgG1 did not cause a neutrophil influx above that seen with endotoxin alone. B, Even a higher dose of IgG1 did not cause a neutrophil influx above that seen with endotoxin alone. C, Albuminuria in the first 24 h after anti-TNP mAb given with endotoxin 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. IgG2a induced a greater albuminuria than did IgG2b. Even after a much higher dose of IgG1, albuminuria was minimal. D, Quantitation of glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

FIGURE 2.

The relative pathogenicity of switch variant mAbs. A, Numbers of neutrophils infiltrating glomeruli at 2 h in mice given the same i.v. dose of switch variant anti-TNP mAbs, with endotoxin 100 ng, 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The lps group are mice given endotoxin 100 ng alone in the second injection. Numbers for IgG2a were significantly greater than for IgG2b, and numbers for IgG2b were significantly greater than for IgG1. IgG1 did not cause a neutrophil influx above that seen with endotoxin alone. B, Even a higher dose of IgG1 did not cause a neutrophil influx above that seen with endotoxin alone. C, Albuminuria in the first 24 h after anti-TNP mAb given with endotoxin 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. IgG2a induced a greater albuminuria than did IgG2b. Even after a much higher dose of IgG1, albuminuria was minimal. D, Quantitation of glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

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FIGURE 3.

The relative pathogenicity of switch variant mAbs. Histology after anti-TNP mAb, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. Left panels, Representative glomeruli 2 h after 15 mg/kg anti-TNP mAb. Neutrophils are identified by lobulated nuclei, with a greater number seen after IgG2a than after IgG2b, and none shown after IgG1. Right panels, Representative glomeruli 24 h after anti-TNP mAb. There is glomerular thrombosis after IgG2a, with less after IgG2b and none after IgG1.

FIGURE 3.

The relative pathogenicity of switch variant mAbs. Histology after anti-TNP mAb, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. Left panels, Representative glomeruli 2 h after 15 mg/kg anti-TNP mAb. Neutrophils are identified by lobulated nuclei, with a greater number seen after IgG2a than after IgG2b, and none shown after IgG1. Right panels, Representative glomeruli 24 h after anti-TNP mAb. There is glomerular thrombosis after IgG2a, with less after IgG2b and none after IgG1.

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To assess the relative ability of the switch variants to activate complement, we stained kidney sections for C1q, C4, and C3. This was performed on samples from the experiment shown in Fig. 2,A in which 15 mg/kg of IgG1, IgG2a, or IgG2b had been given. C1q was detected in a linear pattern on the glomerular capillary wall after IgG2a and IgG2b but not after IgG1. In mice that were given endotoxin alone and no mAb, faint mesangial C3, and strong mesangial C4 staining was seen, at similar levels to those seen in untreated mice. However, superimposed on this pattern, there was linear capillary wall staining after IgG2a and IgG2b but not after IgG1. Capillary wall staining for both C3 and C4 was stronger for IgG2b than for IgG2a. Representative glomeruli are shown in Fig. 4,A and data in Fig. 4 B. These data established that IgG2b was a stronger activator of the classical pathway than was IgG2a, with no activation by IgG1. They also established that IgG1 did not bind C1q.

FIGURE 4.

Complement activation by switch variant mAbs. A, Immunofluorescence staining of glomeruli for C1q, C4, and C3 after 15 mg/kg anti-TNP monoclonals with endotoxin, or endotoxin alone (control), given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. In the control group there was no C1q present, with sparse mesangial C3 and prominent mesangial C4. After IgG2a or IgG2b linear capillary wall, Clq, C3, and C4 staining was present. In the case of C3 and C4, this was superimposed on the mesangial staining, with more C3 and C4 after IgG2b. There was no C1q, C3, or C4 deposited after injection of IgG1 anti-TNP. B, Quantitative data confirming the above findings.

FIGURE 4.

Complement activation by switch variant mAbs. A, Immunofluorescence staining of glomeruli for C1q, C4, and C3 after 15 mg/kg anti-TNP monoclonals with endotoxin, or endotoxin alone (control), given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. In the control group there was no C1q present, with sparse mesangial C3 and prominent mesangial C4. After IgG2a or IgG2b linear capillary wall, Clq, C3, and C4 staining was present. In the case of C3 and C4, this was superimposed on the mesangial staining, with more C3 and C4 after IgG2b. There was no C1q, C3, or C4 deposited after injection of IgG1 anti-TNP. B, Quantitative data confirming the above findings.

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We then proceeded to explore the effector mechanisms responsible for disease induced by IgG2a and IgG2b using knockout mice. We compared disease induced by 15 mg/kg IgG2a or IgG2b in wild-type mice with that seen in FcγRI-, FcγRIII-, and C3-deficient mice. Figs. 5,A and 6,A show that FcγRIII is absolutely required for the neutrophil influx due to IgG2a and IgG2b, respectively. In the absence of FcγRIII, neutrophil numbers were the same as those seen after endotoxin alone in both cases. We did not demonstrate any role for FcγRI or complement in the neutrophil influx due to either IgG2a or IgG2b, as neutrophil numbers in FcγRI- and C3-deficient mice were equivalent to those seen in wild-type mice in both cases. As shown in Fig. 5,B, albuminuria due to IgG2a was significantly decreased in FcγRIII-deficient mice but not in C3-deficient mice compared with wild-type controls. However, IgG2b induced significantly less albuminuria in both FcγRIII- and C3-deficient mice than in wild-type controls, as shown in Fig. 6,B. The results for glomerular thrombosis mirrored these data, as shown in Figs. 5,C and 6 C. These data established that FcγRIII, but not FcγRI or C3, was indispensable for the glomerular neutrophil influx due to either IgG2a or IgG2b. However, glomerular damage (as shown by thrombosis and albuminuria) due to either IgG2a or IgG2b depended on interaction with FcγRIII, with an additional role for complement in the case of IgG2b but not IgG2a.

FIGURE 5.

Effector mechanisms of IgG2a-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAb with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The wild-type control groups are wild-type mice given endotoxin alone in the second injection. There were significantly less neutrophils in FcγRIII−/− mice, with neutrophil numbers reduced to those seen after endotoxin alone. There was no significant difference in neutrophil numbers for wild-type compared with Fcγ RI−/− or C3−/− mice. B, Albuminuria in the first 24 h after IgG2a with endotoxin given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. There was significantly less albuminuria in FcγRIII−/− mice compared with wild-type mice. There was no significant difference in albuminuria for wild-type compared with C3−/− mice. C, Quantitation of the glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

FIGURE 5.

Effector mechanisms of IgG2a-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAb with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The wild-type control groups are wild-type mice given endotoxin alone in the second injection. There were significantly less neutrophils in FcγRIII−/− mice, with neutrophil numbers reduced to those seen after endotoxin alone. There was no significant difference in neutrophil numbers for wild-type compared with Fcγ RI−/− or C3−/− mice. B, Albuminuria in the first 24 h after IgG2a with endotoxin given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. There was significantly less albuminuria in FcγRIII−/− mice compared with wild-type mice. There was no significant difference in albuminuria for wild-type compared with C3−/− mice. C, Quantitation of the glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

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FIGURE 6.

Effector mechanisms of IgG2b-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2b anti-TNP mAb with endotoxin given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The wild-type control groups are wild-type mice given endotoxin alone in the second injection. There were significantly less neutrophils in FcγRIII−/− mice, with neutrophil numbers reduced to those seen after endotoxin alone. There was no significant difference in neutrophil numbers for wild-type compared with FcγRI−/− or C3−/− mice. B, Albuminuria in the first 24 h after IgG2b with endotoxin was significantly less in FcγRIII−/− and C3−/− mice compared with wild-type mice. C, Quantitation of the glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

FIGURE 6.

Effector mechanisms of IgG2b-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2b anti-TNP mAb with endotoxin given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The wild-type control groups are wild-type mice given endotoxin alone in the second injection. There were significantly less neutrophils in FcγRIII−/− mice, with neutrophil numbers reduced to those seen after endotoxin alone. There was no significant difference in neutrophil numbers for wild-type compared with FcγRI−/− or C3−/− mice. B, Albuminuria in the first 24 h after IgG2b with endotoxin was significantly less in FcγRIII−/− and C3−/− mice compared with wild-type mice. C, Quantitation of the glomerular thrombosis, with results that mirror those for albuminuria. GCS, Glomerular cross-section.

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FcγRIV is also required, with no role for combined C3 and FcγRI deficiency. Our experiments so far had shown that IgG2a and IgG2b but not IgG1 was pathogenic, with a dominant role for FcγRIII. This presented a paradox since IgG1 is known to interact with FcγRIII. It remained possible that FcγRI and C3 were important for the neutrophil influx in IgG2a- and IgG2b-mediated disease, but that this was not apparent in mice deficient in only one of these molecules. To explore the effect of a combined deficiency, we generated bone marrow chimeras by transplanting FcγRI-deficient marrow into C3-deficient mice. These animals lacked FcγRI on neutrophils and circulating C3. Analysis of genomic DNA from peripheral blood in eight chimeric mice showed 94.89 ± 5.11% donor-derived bone marrow cells. We confirmed that circulating levels of C3 were <1% of those found in wild-type mice in all seven chimeras tested. These animals were compared with sham chimeric mice (wild-type donor and recipient). As shown in Fig. 7, there were no differences in neutrophil numbers when disease was induced by 15 mg/kg IgG2a or IgG2b (with endotoxin) in these mice. These data established that FcγRI and C3 did not play a role in the neutrophil influx in this model, even in mice rendered doubly deficient. A further possible explanation for the lack of a pathogenic role of IgG1 is that the development of disease requires not only activation of FcγRIII but also of the recently described FcγRIV, which does not bind IgG1. To explore this we used the blocking mAb 9E9. A previous publication has shown specificity data for another mAb, 9G8.1 (6), and in Fig. 8,A we show similar data for 9E9. Data showing that cells expressed the relevant transfected Fc receptor in these experiments are in this previous publication (Fig. 2,B). We also performed immunofluorescence staining using 9E9. As shown in Fig. 8 B, this confirmed that FcγRIV was expressed in glomeruli of mice with disease and a glomerular neutrophil influx, but not in glomeruli from normal mice. FcγRIV was also expressed in the interstitium, but this was also present in normal mice and probably represents resident dendritic cells or macrophages.

FIGURE 7.

The effect of combined FcγRI and C3 deficiency in IgG2a- and IgG2b-induced disease. FcγRI−/− → C3−/− bone marrow chimeras were constructed and compared with wild-type (WT) → WT sham chimera controls. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAb with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The WT → WT control groups are WT → WT mice given endotoxin alone in the second injection. There were no significant differences. B, Shows a similar experiment in which disease was induced by IgG2b anti-TNP. GCS, Glomerular cross-section.

FIGURE 7.

The effect of combined FcγRI and C3 deficiency in IgG2a- and IgG2b-induced disease. FcγRI−/− → C3−/− bone marrow chimeras were constructed and compared with wild-type (WT) → WT sham chimera controls. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAb with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab. The WT → WT control groups are WT → WT mice given endotoxin alone in the second injection. There were no significant differences. B, Shows a similar experiment in which disease was induced by IgG2b anti-TNP. GCS, Glomerular cross-section.

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FIGURE 8.

Specificity of 9E9 and localization of FcγRIV in diseased kidney. A, Characterization of the FcγRIV-specific mAb 9E9. Chinese hamster ovary cells were stably transfected with the indicated Fc receptor (which was FLAG tagged). In the case of FcγRI, FcγRIIII, and FcγRIV the common γ chain was cotransfected. Shaded histograms are 9E9 staining, and open histograms are isotype-matched control Ab staining. Data showing similar expression of all Fc receptors, using a FLAG-specific Ab, is in Ref. 6 . B, Immunofluorescence staining using 9E9 to show FcγRIV in inflamed kidney. A representative section from a mouse in which a neutrophil influx was induced 2 h previously is shown. The arrow indicates a glomerulus (darker then the surrounding tubules) with positive staining that is not seen in normal mice. The interstitium has some positive cells, which are also seen in normal mice.

FIGURE 8.

Specificity of 9E9 and localization of FcγRIV in diseased kidney. A, Characterization of the FcγRIV-specific mAb 9E9. Chinese hamster ovary cells were stably transfected with the indicated Fc receptor (which was FLAG tagged). In the case of FcγRI, FcγRIIII, and FcγRIV the common γ chain was cotransfected. Shaded histograms are 9E9 staining, and open histograms are isotype-matched control Ab staining. Data showing similar expression of all Fc receptors, using a FLAG-specific Ab, is in Ref. 6 . B, Immunofluorescence staining using 9E9 to show FcγRIV in inflamed kidney. A representative section from a mouse in which a neutrophil influx was induced 2 h previously is shown. The arrow indicates a glomerulus (darker then the surrounding tubules) with positive staining that is not seen in normal mice. The interstitium has some positive cells, which are also seen in normal mice.

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We performed experiments in which 20 mg/kg FcγRIV blocking Ab or control was given to mice 90 min before disease induction. As shown in Fig. 9, A and B, there were significantly fewer neutrophils infiltrating glomeruli after FcγRIV blockade in both IgG2a- and IgG2b-mediated disease. Levels were similar to those seen after endotoxin alone (with mice given TNP-conjugated nephrotoxic Ab, then control hamster IgG, followed by endotoxin alone in this control group). Albuminuria (Fig. 9, C and D) and thrombosis (Fig. 9, E and F) were also significantly decreased after FcγRIV blockade. These data established that, in addition to FcγRIII, FcγRIV was required for both glomerular neutrophil influx and glomerular damage in both IgG2a- and IgG2b-mediated disease.

FIGURE 9.

The role of FcγRIV in IgG2a- and IgG2b-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAbs with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab and 90 min after 20 mg/kg of anti-FcγRIV or control Ab. The control group (left group of each graph) is wild-type mice given normal hamster IgG in the second injection and endotoxin alone in the third injection. B, A similar experiment in which disease was induced by IgG2b anti-TNP. For both IgG2a- and IgG2b-induced disease, there were significantly fewer neutrophils in mice given anti-FcγRIV Ab compared with control, with neutrophil numbers reduced to those seen after endotoxin alone. C, Albuminuria in disease induced by IgG2a with endotoxin was significantly less after anti-FcγRIV compared with control Ab. D, Albuminuria in disease induced by IgG2b with endotoxin was significantly less after anti-FcγRIV compared with control Ab. E, Quantitation of the glomerular thrombosis induced by IgG2a, with results that mirror those for albuminuria. F, Thrombosis was also decreased by anti-FcγRIV Ab in IgG2b-induced disease. GCS, Glomerular cross-section.

FIGURE 9.

The role of FcγRIV in IgG2a- and IgG2b-induced disease. A, Numbers of neutrophils infiltrating glomeruli 2 h after IgG2a anti-TNP mAbs with endotoxin, given 24 h after an i.v. injection of TNP-conjugated nephrotoxic Ab and 90 min after 20 mg/kg of anti-FcγRIV or control Ab. The control group (left group of each graph) is wild-type mice given normal hamster IgG in the second injection and endotoxin alone in the third injection. B, A similar experiment in which disease was induced by IgG2b anti-TNP. For both IgG2a- and IgG2b-induced disease, there were significantly fewer neutrophils in mice given anti-FcγRIV Ab compared with control, with neutrophil numbers reduced to those seen after endotoxin alone. C, Albuminuria in disease induced by IgG2a with endotoxin was significantly less after anti-FcγRIV compared with control Ab. D, Albuminuria in disease induced by IgG2b with endotoxin was significantly less after anti-FcγRIV compared with control Ab. E, Quantitation of the glomerular thrombosis induced by IgG2a, with results that mirror those for albuminuria. F, Thrombosis was also decreased by anti-FcγRIV Ab in IgG2b-induced disease. GCS, Glomerular cross-section.

Close modal

It was possible that differences in glomerular neutrophil numbers simply reflected differences in peripheral blood neutrophils. To explore this possibility, we measured peripheral blood neutrophil numbers at 2 h, as this was the time at which we assessed glomerular neutrophil numbers. As shown in Table I, no differences were seen between groups in experiments with either IgG2a or IgG2b. Our findings regarding the role of FcγRIII, FcγRI, FcγRIV, and C3 in IgG2a- or IgG2b-mediated disease could therefore not be explained by differences in peripheral blood neutrophil numbers.

Table I.

Peripheral blood neutrophil counts 2 h after disease inductiona

FcγRIII−/− (WT)FcγRI−/− (WT)C3−/− (WT)FcγRI−/− → C3−/− Chimeras (WT → WT)bAnti-FcγRIV (control IgG)c
IgG2a 266 ± 115 (355 ± 170) 340 ± 69 (355 ± 170) 169 ± 29 (222 ± 49) 337 ± 87 (305 ± 42) 179 ± 30 (199 ± 77) 
      
IgG2b 425 ± 100 (378 ± 88) 346 ± 39 (311 ± 50) 443 ± 48 (459 ± 11) 181 ± 21 (255 ± 34) 187 ± 47 (133 ± 11) 
FcγRIII−/− (WT)FcγRI−/− (WT)C3−/− (WT)FcγRI−/− → C3−/− Chimeras (WT → WT)bAnti-FcγRIV (control IgG)c
IgG2a 266 ± 115 (355 ± 170) 340 ± 69 (355 ± 170) 169 ± 29 (222 ± 49) 337 ± 87 (305 ± 42) 179 ± 30 (199 ± 77) 
      
IgG2b 425 ± 100 (378 ± 88) 346 ± 39 (311 ± 50) 443 ± 48 (459 ± 11) 181 ± 21 (255 ± 34) 187 ± 47 (133 ± 11) 
a

Data are shown for the experiments in which 15 mg/kg IgG2a or IgG2b anti-TNP Ab, given 24 h after TNP-conjugated nephrotoxic Ab, was given to mice as shown in Figs. 5–8. Peripheral blood neutrophil numbers were assessed at 2 h after disease induction. The first three columns are from experiments in which IgG2a or IgG2b was given to knockout or wild-type (WT) mice. Values for WT mice from each experiment are shown in parentheses beneath values for the knockout mice (mean ± SEM) in each case. There were no significant differences in peripheral blood neutrophil counts at 2 h in any of these experiments.

b

Data from experiments in which FcγRI−/− → C3−/− bone marrow chimeras were compared with WT → WT controls (the latter shown in parentheses).

c

Data from experiments in which mice given anti-FcγRIV blocking Ab were compared with control-treated mice (shown in parentheses).

The major finding in these studies is that IgG2a and IgG2b are the pathogenic IgG subclasses in acute glomerular inflammation (IgG2a > IgG2b) and that IgG1 is not pathogenic. Both FcγRIII and FcγRIV are required for neutrophil influx and glomerular damage mediated by IgG2a or IgG2b. We further demonstrated an additional role for complement in proteinuria and thrombosis due to IgG2b but not IgG2a.

The relative potency of switch variants of IgG subclasses has been assessed in studies of passively induced cellular depletion or cytotoxicity such as hemolytic anemia (16, 27), B cell depletion (18), thrombocytopenia, and melanoma therapy (17). These studies agreed that IgG2a and IgG2b were the most pathogenic in experimental situations where high-affinity Abs are used. A recent further study documented that FcγRI, FcγRIII, and FcγRIV all contributed to IgG2a-mediated anemia with FcγRIII and FcγRIV important for IgG2b-mediated disease (28). Our study differs from these previous experiments in that we employ switch variants to explore the role of IgG subclass in a model of tissue-specific immune complex-mediated inflammation. This is the first such in vivo study.

A previous study in the heterologous nephrotoxic nephritis model has suggested an important role for FcγRIII (29). Our experimental system is derived from this model, in which acute glomerular inflammation is induced by a single dose of foreign polyclonal Ab. Our finding of a dominant role for FcγRIII is consistent with this previous work and extends it by showing that FcγRIII is essential regardless of the IgG subclass initiating disease. IgG1 is known to bind to FcγRIII, although with a lower affinity than IgG2a and IgG2b (6). This lower affinity for FcγRIII may contribute to the lack of disease due to IgG1. However, a more convincing explanation is given by the additional requirement that we have shown for FcγRIV, which does not significantly bind to IgG1 (6). Our experiments did not explore the contribution of binding to the inhibitory receptor FcγRIIb in this model. Recent data have shown that pathogenicity is determined by the relative balance of interactions with activating and inhibiting Fcγ receptors (17). IgG2b has a greater affinity than does IgG2a for FcγRIIb, and it also has a lower affinity for FcγRIV (6). These differences may explain the lesser pathogenicity of IgG2b compared with IgG2a. IgG1 binds to FcγRIIb with higher affinity than IgG2a and a similar affinity to IgG2b. This would also tip the balance toward a lack of pathogenicity for IgG1.

There are at present limited in vivo data on the role of FcγRIV. In addition to the role in IgG2a- or IgG2b-mediated anemia described above (28), FcγRIV blockade inhibited IgG2a- or IgG2b-mediated thrombocytopenia. In the anemia study, the effect was seen in wild-type mice only for IgG2b-mediated disease. For IgG2a-mediated disease, FcγRIV blockade only had an effect in mice deficient in FcγRIII (28). In the thrombocytopenia studies the effect of FcγRIV blockade was moderate and led to a partial inhibition of platelet reduction (6, 17). FcγRIV is also important for tumor destruction, although again the effect of FcγRIV blockade was partial (17). The autologous phase of nephrotoxic nephritis is a model in which progressive GN is mediated by a humoral and cellular immune response to a foreign Ab planted on the glomerular basement membrane. One study suggested a dominant role for FcγRIV in this model (30), although other studies have supported a role for FcγRI and FcγRIII (31). This model is a different experimental system from the present study, however, and interactions of Fc receptors on both neutrophils and macrophages may be important. In summary, previous data have shown a contribution of FcγRIV to tissue injury, but in none has there been an absolute requirement for this receptor, as shown in the experiments presented herein. The requirement for both FcγRIII and FcγRIV is similar to that seen when hemolytic anemia is induced by IgG2b (28). This suggests that in both of these experimental models, ligation of both of these receptors is required to reach the threshold required for disease.

In the present work and in these other studies, the role of FcγRIV has been explored using blocking Abs. It is unlikely that our data are complicated by an interaction of the Fc portion of 9E9 with FcγRIII. Data in Fig. 8 A show no interaction of 9E9 with FcγRIII. Additionally, previous published work shows that 9E9 does not block IgG1 immune complex activity and hence does not bind to FcγRIII in vivo (17). The current data extend the in vivo role of FcγRIV to a model of neutrophil-mediated inflammation. They show a complete requirement for FcγRIV in disease due to either IgG2a or IgG2b. A recent study concluded that FcγRIII and FcγRIV both contributed to murine neutrophil activation by immobilized immune complexes in vitro (32). A moderate effect was seen with deficiency or blockade of one of these receptors, with complete abrogation of responses if both are deficient or blocked. Our in vivo study is consistent with this in vitro work in showing a role for both FcγRIII and FcγRIV. However, it differs from the in vitro findings in showing that both receptors are required, and that deficiency in either one is sufficient to prevent disease.

We also demonstrated a role for complement in IgG2b- but not IgG2a-mediated glomerular damage. This is consistent with our immunofluorescence findings that IgG2b is a stronger activator of the classical pathway than is IgG2a. Complement activation due to different IgG subclasses was assessed by immunofluorescence of frozen tissue sections. Glomerular C1q staining was detectable at similar levels in mice given IgG2a and IgG2b following TNP-conjugated nephrotoxic Ab. No deposition of C1q, C4, or C3 was seen in our study after IgG1. In vitro studies have suggested that IgG1 does not activate complement, but they have not identified differences between the other subclasses (3, 4, 27). In the present study, however, we have shown a difference between IgG2b and IgG2a, with greater complement activation for the former. We found similar fluorescence staining for C1q with IgG2a and IgG2b, with greater C4 and C3 staining for IgG2b. This could suggest that both subclasses bind C1q similarly, but that IgG2b is better able than IgG2a to activate C4 and the rest of the classical pathway. However, immunofluorescence staining may not differentiate between biologically important differences in C1q binding, and this must be interpreted with caution. We found that IgG1 was not able to activate complement, as there was no glomerular C3 or C4 deposition. This could have resulted from two possibilities. First, IgG1 may have been unable to bind C1q, and second, IgG1 may have been unable to activate the remainder of the classical pathway after C1q binding. Our demonstration that C1q is not deposited in the glomerulus with IgG1 shows that the first option is the case. Note that recent data have shown that C5 may be activated independently of C3 (33). This means that the lack of effect due to C3 deficiency in IgG2a-mediated disease does not rule out the possibility of a role for terminal complement components.

Previous work in the model of heterologous nephrotoxic nephritis has given conflicting results on the role of complement in acute Ab-mediated glomerular inflammation. In this model disease is induced by a single pathogenic dose of foreign nephrotoxic Ab. Some studies have shown a role for complement in both neutrophil influx and proteinuria, some a role in proteinuria but not neutrophil influx, and others a role in neither one nor the other (34, 35, 36, 37). The current data suggest a possible reason for these discrepancies. The polyclonal Abs used in these studies would have differed in subclass composition, with differing abilities to activate mouse complement and bind to mouse Fc receptors. We have shown that complement activation may be required for proteinuria with some but not other subclasses. Additionally, we did not show a role for complement in causing neutrophil influx in any circumstances. However, it is possible that with heterologous Ab that activates complement well, but binds Fc receptors poorly, such an effect may occur. In the current model and also in heterologous nephrotoxic nephritis, LPS is required as a cofactor with Ab to induce disease. We have previously shown that stimulation of TLR4 (the LPS receptor) on both renal cells and leukocytes is required for disease induction in heterologous nephrotoxic nephritis (38). The mechanism of the effects on neutrophils in these models could include modulation of Fc receptor expression. Indeed, previous data have shown that both FcγRIII and FcγRIV are up-regulated by LPS on myeloid cells (6, 39).

In the model used in the present study, disease develops within hours of deposition of Ab on the glomerular basement membrane and is characterized by a glomerular neutrophil influx. Therefore, our findings concerning Fc receptors can be considered to apply to neutrophil Fc receptors in the context of acute glomerular inflammation, and to reflect the early stages of immune complex-mediated GN. However, in recent years, a neutrophil glomerulitis has been recognized as a feature of humorally mediated renal transplant rejection (40). Although the Abs in this context will be against HLA molecules, the principles regarding the mechanisms by which Ab recruits and activates neutrophils in the glomerulus may be similar. This will therefore widen the potential relevance of our findings. In many forms of glomerular inflammation, humoral and cell-mediated immunity combine to cause glomerular inflammation. We have isolated the component due to humoral immunity in a simplified passive model to study in detail IgG effector mechanisms. In an active immune response the processes we have described will occur in parallel with cell-mediated mechanisms. It has been proposed that proliferative crescentic GN is largely mediated by Th1-type immune responses, although the relative roles of humoral and cell-mediated processes are not clear (41). In agreement with this, we have shown that IgG2a and IgG2b subclasses, associated with Th1-type immune response, are most pathogenic. It is likely that the immune complex-mediated acute inflammation increases T cell recruitment to the glomerulus and subsequent cell-mediated processes. The manner in which humoral and cell-mediated processes interact in glomerular and other inflammatory diseases is an area in need of further study.

We are grateful to Lucien Aarden for providing hybridomas for the switch variants, to Ram Abuknesha for help with TNP conjugation, and to Mike Carroll for C3-deficient mice.

The authors have no financial conflicts 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.

1

This work was funded by the Wellcome Trust.

3

Abbreviations used in this paper: GN, glomerulonephritis; TNP, trinitrophenol.

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