Abstract
Immunoglobulins protect against disease to a considerable extent by activating complement and stimulatory immunoglobulin crystallizable fragment receptors (Ig FcRs), and aggregating microbial pathogens1,2. Yet IgG1, the predominant murine serum Ig isotype, cannot activate complement by the classical pathway, binds more avidly to an inhibitory than to stimulatory FcRs, and has limited ability to aggregate pathogens1,2,3. In these regards, it resembles human IgG4 (ref. 4). We hypothesized that limited ability to activate effector mechanisms might protect against immune complex immunopathology. Here we show that IgG1-deficient (γ1−) mice5, immunized with a potent antigen, develop lethal renal disease soon after they begin to produce antigen-specific antibody, whereas similarly immunized wild-type mice remain healthy. Surprisingly, renal disease in this model is complement and FcR independent and results from immune complex precipitation in glomerular capillaries, as in some cryoglobulinaemic humans6. IgG3, which self-associates to form large immune complexes7,8, accounts for more than 97% of the mouse Ig in this cryoglobulin; furthermore, glomerular disease develops when mice are injected with IgG3 anti-trinitrophenyl (TNP) monoclonal antibody followed by a TNP-labelled protein. Renal disease is prevented in both active and passive immunization models by antigen-specific IgG1; other isotypes are less potent at preventing disease. These observations demonstrate the adaptive significance of Ig isotypes that poorly activate effector mechanisms, reveal an immune-complex-dependent, complement- and FcR-independent nephrotoxic mechanism, and suggest that isotypes that poorly activate effector mechanisms may be useful for inhibiting immune complex immunopathology.
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Change history
21 January 2015
Spelling of author S.K.S. was corrected.
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Acknowledgements
We thank M. Wills-Karp for C3-deficient mice, D. Metzger for J-chain-deficient mice, M. Muramatsu for AID-deficient mice, S. Izui for a hybridoma that secretes mouse IgG1 anti-TNP monoclonal antibody, M. Robson and L. Aarden for switch variant hybridomas that secrete mouse IgG1, IgG2a and IgG2b anti-TNP monoclonal antibodies, B. DiPasquale for histological staining, and J. Lambris for C5aR antagonist. Research was supported by a US Department of Veterans Affairs Merit Award, National Institutes of Health R01 AI072040, and the University of Cincinnati and Cincinnati Children’s Hospital, all to F.D.F.
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R.T.S. planned, performed, interpreted and directed all experiments and participated in all drafts of the manuscript; M.K., A.M. and N.B. performed experiments; C.O.J. provided mice; J.K. provided mice and reagents and participated in planning of all complement-related studies; M.E. participated in the planning and interpretation of isotype switch variant studies; S.K.S., K.S. and D.W. performed and interpreted histological studies; M.T.P. and A.B.H. contributed to investigation of the importance of antibody hinge region length; M.M.H. performed statistical analyses; and F.D.F. designed the study, analysed data and wrote the initial draft of the paper. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 GaMD immunization of γ1− mice induces renal dysfunction and glomerular deposition of PAS+ material that includes IgG and complement.
a, Wild-type (WT) and γ1− mice (4 per group) were immunized with GaMD. Urine leukocyte esterase and blood were obtained. b, Representative photomicrographs of glomeruli stained for C3 (top) or total mouse IgG (bottom) from wild-type (right) and γ1−/− mice (left), 12 days after GaMD immunization (3 mice per group). c, Deposition of amorphous PAS+ material in glomeruli of γ1−, but not wild type begins ∼7 days after GaMD immunization and leads to glomerular destruction by day 9. Note the scarcity of inflammatory cells in glomeruli. Representative data from 6 mice per group. Original magnification, ×400 for b, ×200 for c.
Extended Data Figure 2 The development of kidney disease in GaMD-immunized γ1− mice is independent of IFN-γ, IgG2a, C3 and FcRγ.
a–d, BALB/c wild-type (WT) and γ1− mice (5 per group) were immunized with GaMD on day 0 and injected with 1 mg of either anti-IFN-γ or control monoclonal antibody (mAb) on days 0 and 5. a, Total levels of all Ig isotypes were determined in 24 h culture supernatants of spleen cells harvested on the days shown. b, GaMD-immunized γ1−, γ1−/FcRγ−, γ1−/C3−, C3−/FcRγ− and γ1−/C3−/FcγR− mice (5 per group) had their urine tested for leukocyte esterase and blood on the days shown. c, d, BALB/c wild-type and γ1− mice (5 per group) were immunized with GaMD on day 0 and injected with 1 mg of either anti-IFN-γ or control monoclonal antibody on days 0 and 5. c, Urine obtained on days indicated was assayed for protein, leukocyte esterase and blood. d, BUN levels were determined before and 10 days after GaMD immunization. *P < 0.05.
Extended Data Figure 3 Neither complement nor stimulatory FcRs are required for renal disease development in GaMD-immunized γ1− mice.
Mixed background γ1− and γ1−/C3−/FcRγ− mice (4 per group) were immunized with GaMD plus or minus C5aR antagonist. Urinalyses were obtained at baseline and daily starting on day 6. LE, leukocyte esterase.
Extended Data Figure 4 Delayed antigen elimination does not account for renal disease in GaMD-immunized γ1− mice.
a, BALB/c wild-type (WT) and γ1− mice (10 per group) were immunized s.c. with GaMD. Sera obtained 5, 6, 7 and 9 days later were evaluated by gel double diffusion for the presence of goat IgG. b–e, BALB/c wild-type mice (4 or 5 per group) were injected s.c. with a total of 0.2 ml of different mixtures of GaMD and goat anti-KLH antisera. b, c, Mouse sera collected 9 days later were assayed for BUN (b) and IgG1 anti-goat IgG antibody (c). d, Sera obtained 6–13 days post-immunization were evaluated by gel double diffusion for the presence of goat IgG. e, Urine samples collected 4–12 days post-immunization were analysed for protein. *P < 0.05, **P < 0.005.
Extended Data Figure 5 IgG3 immune complexes persist and accumulate in the glomeruli of GaMD-immunized γ1− mice.
BALB/c wild-type (WT) and γ1− mice were left untreated or were immunized with GaMD. Kidney sections were stained for mouse IgG1, IgG2a, IgG2b, IgG3 and IgM and 12 days after immunization. Representative photomicrographs from three GaMD-immunized mice are shown. Insets show magnified views. No staining was observed with sections from unimmunized mice (data not shown). Original magnification, ×400.
Extended Data Figure 6 Severe renal disease develops in GaMD-immunized γ−/J-chain− mice.
a–c, BALB/c γ1− (12 mice), J-chain− (9 mice) and γ1−/J-chain− (12 mice) mice were injected s.c. with GaMD. a, Urinalysis was performed on the indicated days. b, BUN levels on day 0 and 11. The difference between γ1− and γ1− × J-chain− mice was not consistently observed. c, Survival of GaMD-immunized mice. *P < 0.05.
Extended Data Figure 7 IgG1 inhibits IgG3-induced cryoglobulin kidney disease independent of complement and FcγRIIB and better than IgG2a and IgG2b.
a, Wild-type (WT) mice (4 per group) were injected i.v. with 4 mg of mouse IgG1, IgG2a, IgG2b or IgG3 anti-TNP monoclonal antibody and s.c. with 100 µl of TNP-goat serum on days 0 and 1. Urine leukocyte esterase and blood was measured before injections and on day 1 and day 2. b, Urine leukocyte esterase (LE) and blood for BALB/c wild-type and C3− mice (4 per group) injected i.v. with 4 mg of IgG3 anti-TNP monoclonal antibody and s.c. with 400 µl of TNP-goat serum on day 0 and day 1. c, Wild-type and FcγRIIB-deficient (FcγRIIB−) mice (4 per group) were injected s.c with 100 µl of TNP-goat serum and i.v. with 4 mg of IgG3 anti-TNP plus or minus 5 mg of IgG1 anti-TNP on day 0 and day 1. Urinalysis on days 0, 1 and 2. d, BALB/c mice were injected i.v. with 4 mg of IgG3 anti-TNP and s.c. with 1.4 mg of TNP-BSA on days 0 and 1. Some mice were also injected with 0.625, 1.25, 2.5 or 5 mg of switch variants of IgG1, IgG2a or IgG2b anti-TNP monoclonal antibodies on days 0 and 1. Urine protein was determined on day 0 (data not shown), day 1 (top) and day 2 (bottom). Results are pooled from a total of seven experiments. Group size was as follows. IgG3 alone: 19 mice; 0.625 mg of IgG1, IgG2a or IgG2b: 4 mice; 1.25 mg of IgG1, IgG2a or IgG2b: 8 mice; 2.5 mg of IgG1, IgG2a or IgG2b: 6 mice; 5 mg of IgG1, IgG2a or IgG2b: 8 or 9 mice. The significance of differences between treatment groups was determined as described in the legend to Fig. 4f. *P < 0.05 as compared to IgG3 alone; †P < 0.05 as compared to IgG2b plus IgG3; e, Binding of the Ig isotype switch variants to ELISA wells coated with TNP-BSA, reported as percentage of maximal binding. f, Binding of the Ig isotype switch variants to ELISA wells coated with IgG3 anti-TNP monoclonal antibody. g, Binding of IgG3 and the Ig isotype switch variants to ELISA wells coated with themselves.
Extended Data Figure 8 GaMD-immunized γ1+/− mice generate large IgG3 responses but develop mild renal disease.
a, b, BALB/c mice homozygous (γ1+/+), heterozygous (γ1+/−) and null (γ1−/−) for a functional γ1 allele (6 per group) were injected s.c. with GaMD. a, Sera were titred for goat IgG-specific IgG1, IgG2a and IgG3 0, 8 and 12 days later. Day 0 titres were zero for all Ig isotypes (data not shown). b, Urine samples from the same mice were assayed for protein and leukocyte esterase. ND, none detected.
Extended Data Figure 9 GaMD immune serum from wild-type mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1− mice by 5 days after immunization.
a, BALB/c γ1− mice (4 or 8 per group) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized wild-type mice (GaMD immune WT serum) or unimmunized wild-type mice (non-immune serum), starting 4, 5 or 6 days after GaMD immunization. Day 7 urine samples were analysed. LE, leukocyte esterase; ND, none detected. b, BALB/c γ1− mice (4 or 8 per group) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized wild-type mice (GaMD immune WT serum) or unimmunized wild-type mice (non-immune serum), 5, 6 and 7 days after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and IgG3 on day 0 (unimmunized) and 8 days after GaMD immunization. ND, none detected. *P < 0.05, **P < 0.005 (both as compared to day 6 only in a and unimmunized in b).
Extended Data Figure 10 Antigen-specific IgG1 can prevent IgG3 immune complex glomerular deposition.
a, b, BALB/c wild-type (WT) mice were injected i.v. with mouse IgG1 and/or IgG3 anti-TNP monoclonal antibody with or without s.c. injection of TNP-BSA on days 0 and 1. a, Kidneys were stained with PAS on day 2. Representative micrographs from 3 mice per group are shown. b, Kidney serial sections were stained with PAS or for IgG3 or IgG1 (brown pigment). Representative micrographs from 4 mice per group are shown. Original magnification, ×200 for a, b.
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Strait, R., Posgai, M., Mahler, A. et al. IgG1 protects against renal disease in a mouse model of cryoglobulinaemia. Nature 517, 501–504 (2015). https://doi.org/10.1038/nature13868
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DOI: https://doi.org/10.1038/nature13868
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