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. 2006 May;144(2):309–318. doi: 10.1111/j.1365-2249.2006.03057.x

The role of Fc-receptors in murine mercury-induced systemic autoimmunity

K Martinsson 1, P Hultman 1
PMCID: PMC1809661  PMID: 16634805

Abstract

Inorganic mercury (Hg) in genetically susceptible mouse strains induces a T cell-dependent, systemic autoimmune condition (HgIA) characterized by immunostimulation, anti-nuclear antibodies (ANA) and systemic immune-complex (IC) deposits. The exact phenotypic expression of HgIA in different strains depends on H-2 and non-H-2 genes. Fc receptors (FcRs) are important in the development of many autoimmune diseases. In this study, the effect of targeted mutations for activating and inhibiting FcRs in the BALB/c model of HgIA was examined. Hg-treated BALB/c mice without mutation (wild-type, wt) showed heavy IC deposits in the renal glomerular mesangium, as well as in renal and splenic vessel walls. The renal mesangial IC deposits were severely reduced in Hg-treated BALB/c mice without the γ-chain (lack of the activating receptors FcγRI, FcγRIII and Fc∈RI), but unchanged in mice lacking the inhibitory FcγRIIB. The Hg-induced vessel wall IC deposits present in wt mice were abolished and reduced in the FcRγ and FcγRIIB strains, respectively. Hg-treated BALB/c wt mice and mice without the γ-chain showed an increase in serum IgE, while the increase in IgG1 was attenuated in the latter strain. In contrast, absence of the inhibiting FcγRIIB augmented the Hg-induced increase of both serum IgG1 and IgE. In conclusion, FcRs are important mainly for the induction of systmeic IC deposits in the HgIA model, but also affects serum IgG1 and IgE levels.

Keywords: autoimmunity, FcγR, FcγRIIB, mercury, mice

Introduction

The receptors for the constant part of immunoglobulins (Ig) (FcRs) are important in linking the cellular and the humoral immune response [1]. Activation of immune responses through FcR in mice is accomplished by binding of monomeric IgG to FcγRI and immune complexes (IC) containing IgG and IgE to the low affinity FcγRIII and FcɛRI, respectively. Activation takes place via the common γ-chain in the FcR, which has an immune receptor tyrosine-based activation motif (ITAM). Down-regulation of immune responses by FcRs is mediated via binding of the immune receptor tyrosine-based inhibitory motif (ITIM) of the FcγRIIB to an ITAM [1]. The balance between inhibitory and stimulatory FcRs may be determined by their different affinity for Ig isotypes. FcγRI shows the highest affinity to IgG2a followed by IgG3 and IgG1. Both FcγRIII and FcγRIIB bind IgG1 preferentially followed by IgG2a, IgG2b and IgG3 [2].

To maintain an appropriate immune response, the net effect of opposing activating and inhibitory signals mediated via the FcγRs need to be in balance. In autoimmune diseases the balance may be shifted towards activation due to an impairment of inhibitory signalling pathways [3]. The use of mouse strains with targeted mutations for the different FcγRs have shown that FcRs are crucial in many antibody-mediated autoimmune diseases. For example, mice lacking FcγRIII do not develop collagen-induced arthritis [4] and are resistant to autoimmune haemolytic anaemia (AIHA) [5]. The morphological damage in anti-glomerular basement membrane glomerulonephritis (GN) is dependent on recruitment of neutrophils expressing FcγRIII [6]. Although some lupus-prone mouse strains in which the γ-chain has been deleted (no expression of FcγRI, FcγRIII and FcɛRI) develop IC deposits, they are usually protected from the subsequent GN [7–9]. Mice with deficient γ-chain are also protected from allergic encephalomyelitis [10]. In contrast, mice deficient in the inhibitory FcγRIIB show an augmentation of experimental autoimmunity [10–12], with the exception of AIHA [13]. In humans there are indications that polymorphisms in FcRγs are linked to the development of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [3,14].

Induction of an autoimmune disease by metals, especially mercury, in rodents (HgIA) is a well-established and relevant model for systemic autoimmunity [15]. In the most full-blown form, in susceptible mouse strains mercuric chloride (Hg) induces antibodies to a major autoantigen, the 34-kDa nucleolar protein fibrillarin (AFA) and to a minor autoantigen, chromatin (ACA) [16–18]. Induction of AFA is dependent on CD4+ T-cells [19], the co-stimulatory molecules CD40L and CD28 [20], and interferon (IFN)-γ [21]. Susceptibility to the development of anti-nucleolar antibodies targeting fibrillarin (ANoA/AFA) is linked closely to the major histocompatibility complex (H-2) [22], and only strains of the H-2s, H-2q and H-2f haplotype will develop ANoA/AFA during Hg-treatment [16,17]. Susceptibility to development of other HgIA parameters, such as lymphoproliferation, immunostimulation, ACA and IC deposits, are not linked to H-2 [23], which is illustrated in the two H-2d strains DBA/2 and BALB/c. The former is completely resistant to HgIA, while the latter shows hypergammaglobulinaemia and systemic IC-deposits [17].

In the present study Hg-treated BALB/c mice, with deletion of the γ-chain (FcRγ) or FcγRIIB, were compared with BALB/c wild-type (wt) mice to assess the role of FcRs in HgIA. We show that FcRγs are important in the induction of HgIA in the BALB/c strain by affecting tissue IC deposition but also influence serum IgG1 levels.

Materials and methods

Animals and housing

Female mice with a BALB/c background homozygous for a targeted mutation of the γ-chain of the FcγR (FcRγ(–/–)) or the FcγRIIB (FcγRIIB(–/–)) were obtained from Taconic M&B (Georgetown, NY, USA). BALB/c mice without mutations (wt mice), were obtained from Taconic M&B (Ry, Denmark).

All mice were 11–14 weeks old at onset of the experiments. The wt mice were housed under 12-h dark−12-h light cycles in steel-wire cages and given pellets (Type R 70, Lactamin, Vadstena, Sweden) and tapwater ad libitum. The BALB/c strains with targeted mutations were kept under specific pathogen-free conditions. The local animal ethics committee approved the study.

Treatment

The two BALB/c strains with targeted mutations and the corresponding wt strain were each divided into two groups, one group for Hg treatment (10 mice) and one control group (nine mice). The FcRγ(–/–) and FcγRIIB(–/–) strains were each divided into one group of eight mice receiving Hg treatment and one control group of eight mice. Hg treatment consisted of sterilized drinking water prepared weekly with 15 mg/l HgCl2 (Fluka Chemie, Buchs, Germany) given ad libitum for 5 weeks. The control groups received sterilized water without any additions.

Blood and tissue sampling

Blood was obtained from the retro-orbital vein plexa before onset of treatment, and after 2 and 5 weeks of treatment. The blood was allowed to clot at 4°C overnight and sera were stored at −20°C. After 5 weeks the mice were killed and samples of the kidney and spleen were obtained for examination of IC deposits.

Assessment of tissue immune complex deposits

Pieces of the left kidney and the spleen were examined for IC deposits with direct immunofluorescence, as described previously [24]. Briefly, snap-frozen tissue pieces were sectioned and incubated with either a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody against all IgG isotypes (total IgG) (Sigma, St Louis, Missouri, USA) diluted 1/40–1/2,560, an anti-IgM antibody (Sigma) diluted 1/40 or an anti-C3c antibody (Organon-Technica, West Chester, PA, USA) diluted 1/320–1/10 240. Deposits of the IgG1, IgG2a, IgG2b and IgG3 isotypes were assessed using diluted FITC-conjugated goat anti-mouse antibodies to the different Ig isotypes (Southern Biotechnology, Birmingham, AL, USA). Kidneys from aged ZBWF1 mice were used as a positive control. The presence of IC deposits in the glomeruli, renal and splenic vessel walls was examined with a fluorescence microscope (Nikon, Tokyo, Japan) and recorded. The end-point titre for IgG, the IgG isotypes and C3c was defined as the highest dilution which gave a specific staining. The amount of IgM deposits in the glomeruli, as well as the total IgG, the different IgG isotypes and C3c was scored in renal and splenic vessel walls.

Light microscopy

Renal and splenic tissues from three to four randomly selected animals in each strain treated with Hg or controls were obtained at the end of the study, and examined by light microscopy using paraffin-embedded sections as described previously [25]. The type and degree of glomerular cell proliferation was assessed without knowledge of treatment or other data, and graded as follows: 0, no difference compared with reference sections from young untreated mice; 1, slight proliferation; 2, moderate proliferation; and 3, severe proliferation. The presence of histological alterations in the glomeruli or other renal and splenic tissues were searched for.

Serum IgG1 concentration assessed by enzyme-linked immunosorbent assay (ELISA)

The method used has been described previously [24]. Microtitre wells (Nunc, Copenhagen, Denmark) were coated with purified rat anti-mouse IgG1 monoclonal antibody (mAb) (1 mg/ml) (LO-IMEX, Brussels, Belgium) overnight at 4°C. The wells were washed, blocked and incubated with diluted sera. Bound IgG1 was detected using a horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG1 mAb (LO-IMEX). After incubation the substrate was added. The reaction was stopped with 2 M H2SO4, the optical density measured at 450 nm and the background values subtracted. To obtain the actual concentration a standard curve, using mouse myeloma protein of the IgG1 (LO-IMEX) isotype, was used.

Serum IgE concentration assessed by ELISA

The method described by Havarinasab et al. [24] was used for determining the IgE concentration. The microtitre wells (Nunc) were coated with a rat anti-mouse IgE mAb (Southern Biotechnology), incubated overnight, washed, blocked and diluted serum added. After incubation a HRP-conjugated goat anti-mouse IgE antibody (Nordic Immunological Laboratory, Tilburg, the Netherlands) was added. Following washes the substrate was added and the reaction stopped with 0·5 M citric acid. The optical density was measured at 450 nm and the background values were subtracted. A monoclonal anti-dinitrophenyl-substituted human albumin (DNP) antibody of IgE type (SP-7) (Sigma) was used as a standard to derive the actual IgE concentration in the samples.

Serum IgG2a, IgG2b and IgG3 concentration assessed by ELISA

The serum IgG2a, IgG2b and IgG3 concentrations were measured using kits from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Microtitre wells (Nunc) were coated with capture antibody, washed, blocked and diluted serum added. Bound Igs were detected with an HRP-conjugated detection antibody. The substrate was added, the reaction stopped with 2 M H2SO4, the optical density read at 450 nm and the background values were subtracted. To obtain the actual IgG2a, IgG2b or IgG3 concentrations, standards supplied with the kits were used.

Serum anti-nuclear antibodies analysed by indirect immunofluorescence

Antibodies to nuclear antigens were detected by indirect immunofluorescence, as described previously [26]. HEp-2 slides (Binding Site Ltd, Birmingham, UK) were used as the antigenic substrate. For screening, serum was diluted 1/40 and bound anti-nuclear antibodies were detected with a FITC-conjugated goat anti-mouse IgG antibody (Sigma) diluted 1/50. The slides were analysed using a fluorescence microscope (Nikon). Sera showing nuclear staining at a dilution of 1/40 were diluted urther. The highest dilution of the serum, which resulted in specific nuclear staining, was defined as the titre and documented.

Serum anti-nuclear antibodies assessed by immunoblotting

The specificity of the anti-nuclear antibodies in the serum was assessed by immunoblotting as described previously [27], with minor modifications. Mouse liver nuclei were isolated [28], separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad Laboratory, Hercules, CA, USA). The nitrocellulose strips were blocked with 5% fat-free milk, incubated with diluted sera, and the bound antibody was detected with HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotechnology) followed by enhanced chemiluminescence (ECL Western blotting detection reagents; Amersham, Stockholm, Sweden).

Serum anti-chromatin antibodies assessed by ELISA

ACA were measured using the method of Burlingame and Rubin [29], with minor modifications [24]. Microtitre wells (Nunc) were coated overnight at 4°C with chromatin (1·25 µg/ml), blocked with gelatine (Bio-Rad Laboratories, Richmond, CA, USA), and diluted sera added. Pooled sera from differently aged MRLlpr/lpr mice were used as highly and moderately positive controls. Pooled sera from young mice in non-autoimmune strains were used as negative controls. The wells were incubated with alkaline phosphatase (ALP)-conjugated goat anti-mouse IgG antibody (Sigma), washed, and the substrate added. The optical density was read at 405 nm (Multiscan, Thermolabsystems, Helsinki, Finland) and the background values were subtracted.

Serum anti-ssDNA antibodies assessed by ELISA

The method used for measuring anti-single stranded (ss)DNA antibodies has been described previously [30]. Microtitre wells (Nunc) were coated overnight at 4°C with ssDNA, washed, blocked, and diluted serum added. The positive control was a pool of sera from aged MRLlpr/lpr mice and the negative control a pool of sera from non-autoimmune young mice. After incubation the wells were washed, and ALP-conjugated goat anti-mouse Ig-antibodies reacting with IgG, IgA and IgM (Sigma) were added. Following incubation the substrate was added, and the reaction was stopped by adding NaOH (3 M) when the controls reached their predetermined values. The optical density was measured at 405 nm and background values were subtracted.

Serum anti-DNP antibodies assessed by ELISA

The method used has been described previously [30]. Microtitre wells (Nunc) were coated overnight at 4°C with 2 µg/ml DNP. The wells were washed and diluted serum added. The same sera as in the anti-ssDNA antibody assay were used as positive and negative controls. Following repeated washes the anti-DNP antibodies were detected with ALP-conjugated goat anti-mouse Ig-antibody (Sigma), as in the anti-ssDNA method. The substrate was added and the reaction stopped with 3 M NaOH when the controls reached their predetermined values. The optical density was measured at 405 nm and the background values were subtracted.

Statistical methods

The differences between the groups were analysed with the non-parametric Mann–Whitney test except for the presence or absence of IC deposits in the vessel walls, where Fisher's exact test were used. P< 0·05 was considered statistically significant.

Results

Animal health

None of the mice showed any signs of disease during the experiment. At pretreatment a single FcγRIIB(–/–) mouse showed serum anti-DNP antibody and IgE values which were higher than mean ± 3 standard deviations (s.d.) in all mice. Data from this mouse were therefore not included further in the results.

Renal glomerular immune-complex deposits and histology

After 5 weeks’ Hg treatment wt mice showed a significantly higher titre of granular IgG deposits in the renal glomerular mesangium (Fig. 1a) compared to controls (P< 0·001) (Table 1). The titre of IgG deposits in the mesangium of Hg-treated FcRγ(–/–) mice (Fig. 1b) was also higher and significantly different from the strain-specific controls (P< 0·01), but the titre was substantially lower than in Hg-treated wt mice (P< 0·001) (Table 1). The FcγRIIB(–/–) mice showed a significant increase in mesangial IgG deposits after Hg treatment (Fig. 1c) compared to their controls (P< 0·001); the increase was identical in magnitude compared with wt mice (Table 1).

Fig. 1.

Fig. 1

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Direct immunofluorescence on cryostate sections showing glomeruli (a–c) and splenic vessel walls (d–f) after incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibodies, from female BALB/c wild-type (a,d), FcRγ(–/–) (b,e) and FcγRIIB(–/–) (c,f) mice treated with 15 mg/l HgCl2 in the drinking water for 5 weeks. Granular mesangial deposits in (a) and (c) but not in (b). Granular splenic vessel wall deposits in (d) and (f) but not in (e).

Table 1.

Renal mesangial immune-complex deposits and histology after 5 weeks in female BALB/c mice with or without mutation of the Fcγ-chain or the FcγRIIB.

Strain No Treatment Total IgGa IgMb C3ca Glom. prolif.c
Wt 10 Hg 1920 ± 675d 2·1 ± 0·3e 4608 ± 1079d 1·5 ± 0·6f
Wt 9 C 9 ± 27 1·2 ± 0·4 462 ± 169 0·3 ± 0·5f
FcRγ(–/–) 8 Hg 550 ± 507g,h 1·8 ± 0·5 800 ± 296h,i 1·3 ± 0·6j
FcRγ(–/–) 8 C 20 ± 37 1·4 ± 0·5 420 ± 382 0·3 ± 0·6j
FcγRIIB(–/–) 7 Hg 1920 ± 684k 1·4 ± 0·5 3200 ± 1185k 1·5 ± 0·6f
FcγRIIB(–/–) 8 C 6 ± 15 1·1 ± 0·4 600 ± 317 0·3 ± 0·5f
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Hg 15 mg/l HgCl2 in the drinking water. C, controls given water without additions. Wt, wild-type. FcRγ(–/–), targeted mutation of the gene for the Fcγ-chain.FcγRIIB(–/–), targeted mutation of the FcγRIIB gene.

a

Reciprocal titre ± s.d.

b

Scoring: 0, no specific staining; 1, slight staining; 2, moderate staining and 3, strong staining. Figures are mean ± s.d.

c

Scoring of glomerular proliferation: 0, no difference compared with reference sections from young untreated mice; 1, slight proliferation; 2, moderate proliferation; 3, severe proliferation. Figures are mean ± s.d.

d

Hg-treated wt mice significantly different from the strain-specific controls (Mann–Whitney test P < 0·001).

e

Hg-treated wt mice significantly different from the strain-specific controls (Mann–Whitney test P < 0·01).

f

Four mice examined.

g

Hg-treated FcRγ(–/–) mice significantly different from the strain-specific controls (Mann–Whitney test P < 0·01).

h

Significantly different from wt mice (Mann–Whitney test P < 0·001).

i

Hg-treated FcRγ(–/–) mice significantly different from the strain-specific controls (Mann–Whitney test P < 0·05).

j

Thee mice examined.

k

Hg-treated FcγRIIB(–/–) mice significantly different from the strain-specific controls (Mann–Whitney test P < 0·001).

Staining for the different IgG isotypes in the mesangium of the controls in the three strains was either absent or not higher than 1 : 160, with the exception of a single FcγRIIB(–/–) mouse, which showed an IgG1 titre of 1 : 640 (Fig. 2). The control animals in the three strains showed no significant difference in the IgG isotype titre. After Hg treatment, the titre of the IgG isotypes in the mesangium was increased significantly in the wt and FcγRIIB(–/–) strains compared with the controls (P< 0·01 or P < 0·001). The order of increase in titre was IgG1 >> IgG2b ≈ IgG3 > IgG2a, and similar in the wt and FcγRIIB(–/–) strains (Fig. 2). The FcRγ(–/–) mice showed increased mesangial deposits of the different IgG isotypes after Hg-treatment compared to controls (P< 0·05 or P < 0·001). However, the titre of mesangial IgG1, IgG2b and IgG3 deposits was lower than in Hg-treated wt mice (P< 0·001, P < 0·001 and P < 0·05, respectively).

Fig. 2.

Fig. 2

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Renal mesangial immune-complex deposits. Granular deposits of IgG1, IgG2a, IgG2b and IgG3 in the glomeruli of female BALB/c wild-type, FcRγ(–/–) and FcγRIIB(–/–) mice treated for 5 weeks with 15 mg/l HgCl2 in the drinking water. End titres of serially diluted fluorescein isothiocyanate (FITC)-conjugated goat anti-Ig incubated on cryostate section. Horizontal bars denote median values. **P< 0·01, ***P< 0·001 (Mann–Whitney test). Controls showed no or low titres (1 : 40–1 : 160).

All controls showed a low titre of IgM in the glomerular mesangium, which was not significantly different among the three strains (P > 0·05) (Table 1). The IgM titre increased significantly after Hg treatment in wt mice compared to controls (P< 0·01), while no significant increase was seen in FcRγ(–/–) or FcγRIIB(–/–) mice. The titre of C3c deposits in the renal mesangium of the control mice was moderate and not significantly different among the three strains (P > 0·05) (Table 1). After Hg treatment wt, FcγRIIB(–/–) and FcRγ(–/–) mice showed a significant increase in mesangial C3c titre compared to controls (P< 0·001, P < 0·001 and P < 0·05, respectively). However, the titre of C3c in Hg-treated FcRγ(–/–) mice was significantly lower than in Hg-treated wt mice (P< 0·001).

Histological examination showed a mild glomerular endocapillary cell proliferation accompanied by slight widening of the mesangium in Hg-treated mice of all three strains compared with the untreated controls. Proliferation varied slightly among the three strains, but the limited number of tissue samples did not allow any conclusions regarding statistically significant differences among the strains (Table 1).

Neither extracapillary cell proliferation nor inflammation was present in the glomeruli of any of the mice.

Renal vessel wall immune-complex deposits and histology

Deposits containing IgG, including the different IgG isotypes or C3c, were not observed in the renal vessel walls of any of the control mice from the three strains (data not showed). Wt mice treated with Hg developed a significantly higher titre of total IgG deposits in the renal vessel walls compared to controls (0·5 ± 0·3 versus 0 ± 0, mean ± s.d., P < 0·001). The deposits consisted of IgG1 only with the exception of a single mouse, which also showed slight IgG2a deposits (Table 2). There were no deposits of any of the IgG isotypes in the renal vessel walls of Hg-treated FcRγ(–/–) mice except in a single mouse, which showed traces of IgG3 deposits. A few of the Hg-treated FcγRIIB(–/–) mice showed a low titre of IgG1 deposits in the renal vessel walls. Except for traces of C3c in a single Hg-treated FcγRIIB(–/–) mouse, none of the mice in the three strains showed C3c deposits in the renal vessel walls (Table 2). Light microscopy revealed no signs of vasculitis in any of the examined animals.

Table 2.

Renal and splenic vessel walls immune-complex deposits in HgCl2-treated BALB/c mice and controls after 5 weeks.

Kidney Spleen
Strain No Treatment IgG1 IgG2a IgG2b IgG3 C3c IgG1 IgG2a IgG2b IgG3 C3c
Wt 10 Hg 60a 10 0 0 0 100 20 20 60 70
(0·5 ± 0)b (1·0 ± 0) (3·3 ± 0·5) (1·0 ± 0) (2·0 ± 0) (1·2 ± 0·4) (1·0 ± 0·4)
FcRγ(–/–) 8 Hg 0c 0 0 13 0 50c 0 0 0c 0c
(0·5 ± 0) (1·5 ± 0·6)
FcγRIIB(–/–) 7 Hg 29 0 0 14 14 100 14 14 71 57
(0·8 ± 0·4) (1·0 ± 0) (0·5 ± 0) (3·1 ± 1·0) (1·0 ± 0) (1·0 ± 0) (1·2 ± 0·4) (0·9 ± 0·3)
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Hg 15 mg/l HgCl2 in the drinking water. Wt, wild-type. FcRγ(–/–), targeted mutation of the gene for the Fc γ-chain. FcγRIIB(–/–), targeted mutation of the gene FcγRIIB.

a

Fraction of mice with immune-complex deposits (%).

b

Grading, 0–4: figures denote mean ± s.d. in mice with deposits.

c

Hg-treated FcRγ(–/–) mice significantly different from wt mice (Fisher's exact test P < 0·05).

Splenic vessel wall immune-complex deposits and histology

While the controls showed no IgG or C3c deposits in the vessel walls of the spleen, all Hg-treated wt mice developed a high titre of IgG deposits (Fig. 1d), and 70% showed C3c deposits. The IgG deposits consisted mainly of IgG1, but six of 10 mice also showed IgG3 deposits and two mice showed IgG2a and IgG2b deposits (Table 2). After Hg treatment only four of eight FcRγ(–/–) mice showed IC deposits in the splenic vessel walls (Fig. 1e), and they all consisted of IgG1 (Table 2); the titre was moderate and significantly lower compared to Hg-treated wt mice (P< 0·05). All Hg-treated FcγRIIB(–/–) mice developed splenic vessel wall IC deposits (Fig. 1f) consisting of IgG1; five mice showed IgG3 deposits and four mice showed C3c deposits (Table 2). Light microscopy revealed no signs of vasculitis in any of the examined animals.

Serum immunoglobulins

After 2 and 5 weeks Hg treatment the FcγRIIB(–/–) mice showed a 38% and 44% increase in the mean serum IgG1 concentration, respectively, when compared with the level in pretreatment samples. The IgG1 concentration was significantly higher compared with controls after 2 and 5 weeks (Fig. 3) (P< 0·05 and P < 0·01, respectively). Serum IgG1 was also significantly higher in FcγRIIB(–/–) mice compared to wt mice after 2 and 5 weeks of Hg treatment (P< 0·05 and P< 0·01, respectively). In contrast, the IgG1 concentration decreased 16% in FcRγ(–/–) mice during 5 weeks’ Hg treatment (P< 0·01) (Fig. 3). The wt mice developed a 41% increase in mean serum IgG1 concentration during Hg treatment, and serum IgG1 was significantly higher compared to controls after 2 and 5 weeks (P< 0·01).

Fig. 3.

Fig. 3

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Serum concentration of IgG1 in BALB/c female wild-type, FcRg(–/–) and FcγRIIB(–/–) mice after 2 and 5 weeks of Hg treatment in drinking water (15 mg/l HgCl2). *P< 0·05, **P< 0·01 (Mann–Whitney test).

All three strains responded to Hg treatment with a significant increase of serum IgE after 2 and 5 weeks compared to controls (P< 0·001) (Fig. 4). The serum level of IgE in the FcγRIIB(–/–) strain was significantly higher compared with the wt strain after 5 weeks’ Hg treatment (P< 0·05).

Fig. 4.

Fig. 4

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Serum concentration of IgE in BALB/c female wild-type, FcRγ(–/–), and FcγRIIB(–/–) mice after 2 and 5 weeks of Hg treatment in drinking water (15 mg/l HgCl2). *P< 0·05, ***P< 0·001 (Mann–Whitney test).

Neither wt nor FcRγ(–/–) mice showed any significant difference in serum IgG2a, IgG2b or IgG3 concentrations between Hg-treated and control mice at any time-point. However, FcγRIIB(–/–) mice showed a significant increase in IgG2a after 5 weeks’ Hg treatment (P< 0·05), and a significant increase in serum IgG2b (P< 0·01) after 2 week’ treatment compared with controls (data not shown).

Serum anti-nuclear antibodies

ANA with a finely speckled pattern, including distinct staining of the nucleolar membrane and the condensed chromosomes in dividing cells, was seen after 2 weeks of Hg treatment using an anti-IgG antibody detecting all isotypes of IgG (IgG ANA). In wt mice the IgG ANA titre was 180 ± 105 (mean ± s.d.) after 2 weeks, and increased to 1280 ± 0 after 5 weeks of Hg-treatment (Fig. 5). The use of IgG isotype-specific detecting antibodies revealed that the IgG ANA consisted of IgG1 and IgG2a with a significantly higher titre of IgG1 than of IgG2a (P< 0·05) (data not shown). None of the wt controls showed any ANA. No consistent reactivity to mouse liver nuclear proteins was detected in finely speckled positive sera using immunoblotting.

Fig. 5.

Fig. 5

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Reciprocal titre of serum IgG anti-nuclear antibodies in BALB/c mice treated with 15 mg/l HgCl2 in the drinking water and controls as determined by indirect immunofluorescence using Hep-2 cells as substrate. Horizontal bars denote median values.

After 2 weeks all Hg-treated FcRγ(–/–) mice had developed IgG ANA; the titre was 360 ± 113, which was significantly higher than the IgG ANA titre of the wt mice (P< 0·01). After 5 weeks’ Hg treatment the IgG ANA titre had increased to 1200 ± 226, which was similar to the titre in the wt mice, and consisted of a significantly higher titre of IgG1 than of IgG2a (P< 0·01). The titre of ANA of the IgG1 isotype in FcRγ(–/–) mice was significantly higher (P< 0·05) than in the Hg-treated wt mice. In the FcRγ(–/–) controls two mice showed traces of ANA before onset of the study and one mouse after 5 weeks.

All but one FcγRIIB(–/–) mouse had developed IgG ANA after 2 weeks’ Hg treatment and the titre was 149 ± 132. After 5 weeks all Hg-treated FcγRIIB(–/–) mice showed IgG ANA with a significantly higher titre (1189 ± 242) (P< 0·001) than controls. The IgG1 titre was significantly higher than that of IgG2a (P< 0·01), and the IgG2a titre was significantly (P< 0·05) lower in the FcγRIIB(–/–) mice compared with the wt mice. One of the FcγRIIB(–/–) controls showed ANA with a titre of 1 : 320 after 2 and 5 weeks.

Serum anti-chromatin antibodies

There was no statistically significant increase in ACA between Hg-treated and control mice either in wt or in FcRγ(–/–) mice (data not shown). The Hg-treated FcγRIIB(–/–) mice showed a significant increase (P< 0·01) in ACA titre compared to the controls after 2 and 5 weeks of treatment, but the increase from 0·011 ± 0·002 to 0·019 ± 0·005 after 2 weeks and from 0·012 ± 0·003 to 0·023 ± 0·012 after 5 weeks (mean ± s.d., OD 405) is unlikely to be biologically significant.

Serum markers of polyclonal B-cell activation

After 2 weeks of Hg treatment the wt strain showed no increase in anti-DNP antibodies, but a significant increase in anti-ssDNA antibodies (P< 0·05) (data not shown). However, the increase from 0·141 ± 0·021 to 0·157 ± 0·022 (means.d., OD 405) is unlikely to be biologically significant. The FcRγ(–/–) strain showed no significant increase in anti-ssDNA or anti-DNP antibodies during Hg treatment compared to the controls. After 5 weeks of Hg treatment the FcγRIIB(–/–) mice showed a significant anti-DNP antibody response (P< 0·05) compared to controls, from 0·105 ± 0·030 to 0·180 ± 0·103, while there was no significant increase in anti-ssDNA antibodies during Hg treatment.

Discussion

In this study we show that FcRs are of profound importance for induction of systemic tissue IC deposits in an experimental model for systemic autoimmunity, HgIA. Genetically Hg-susceptible mice lacking the FcR γ-chain (absence of the activating FcRs FcγRI, FcγRIII and FcɛRI) did not develop the IC deposits observed in wt mice. Because the tissue IC deposits in the wt mice are composed mainly of IgG, particularly IgG1, the loss of IC deposits in the Fcγ-chain-deficient mice could be due hypothetically to the lack of a global IgG1 response in the FcRγ-deficient strain (see below). As BALB/c mice develop circulating IC (CIC) following Hg treatment [31], another explanation for the loss of tissue IC deposits in the FcRγ(–/–) strain might be that FcRs containing the γ-chain are necessary for the development of tissue deposits [2]. The deposits were composed predominantly of IgG1, which binds preferentially to FcγRIII [2]; this receptor might be the most important for development of tissue deposits in HgIA. Future studies developing FcγRIII-deficient mice with a BALB/c background may answer this question.

While, in the present study, we show that activating FcγRs are important for the formation of tissue IC deposits, the spontaneously developing IC deposits in ZBWF1 mice [7] and the anti-glomerular basement membrane-induced deposits in C57BL/6 mice [8] are preserved even if activating FcγRs are deleted. Furthermore, FcγRIIB-deficient C57BL/6 mice but not BALB/c mice develop IC deposits spontaneously [12]. This shows that the genetic background determines the effect of FcR with regard to the formation of tissue deposits. However, in the HgIA model the FcγRIIB-deficient mice showed the same amount of IC deposits as the wt mice, which indicates that FcγRIIB does not regulate the development of IC deposits in HgIA.

Even in the presence of IC deposits in the glomeruli, the histological damage in the form of GN is heavily dependent on genetic factors. For example, following induction of IC deposits, Fcγ-chain-deficient MRLlpr/lpr mice [9] but not ZBWF1 [7] mice developed GN. The histological damage in murine HgIA on the BALB/c background consisted of mild glomerular endocapillary proliferation and slight expansion of the mesangium [25,32], a feature which was neither aggravated nor attenuated in FcγRIIB(–/–) mice in the present study.

The BALB/c wt strain shows elevated serum IgG1 concentration (present study and [32]) and an increase of IL-4-secreting cells 8–10 days after onset of Hg treatment [30]. These observations accord with the fact that IgG1 belongs, together with IgE, to the Th2-associated Ig isotypes (see below). In the present study, lack of the FcγRIIB enhanced the Hg-induced IgG1 response observed in the wt mice. This indicates a down-regulating effect of FcγRIIB on serum IgG1, which accords with observations in pristane-induced murine lupus [33]. Lack of the γ-chain was associated with a slight decrease of mean serum IgG1 in BALB/c mice, and the IgG1 concentration was reduced significantly compared with controls after 5 weeks. However, the differences in IgG1 concentration between Hg-treated and control FcRγ(–/–) mice was small, and due to inter- and intragroup variation the biological significance might be questioned. However, in genetically HgIA susceptible DBA/1 mice (H-2s), the increase in IgG1 was stronger in Hg-treated wt mice than in BALB/c wt mice, and the increase was abolished in FcRγ(–/–) mice (K. Martinsson 2005, unpublished data). This indicates clearly that the effect of the Fcγ-chain on the Hg-induced IgG1 increase observed in BALB/c mice, albeit small, might represent a general effect of the Fcγ-chain for regulation of Ig isotypes.

In contrast, the IgE response was unchanged in the FcRγ(–/–) strain compared with the wt mice, showing that IgE production is not dependent on the FcɛRI in the HgIA model. Furthermore, the IgE response was exaggerated in the FcγRIIB(–/–) mice compared with wt mice, showing a down-regulating effect of the FcγRIIB on IgE production. Therefore, the IgG1 response is dependent on activating FcγRs, whereas the IgE response is not, indicating differences in signalling mechanisms between these two responses. This is also supported by the observation that treatment with blocking anti-inducible co-stimulator (ICOS) mAbs inhibits the IgE but not the IgG1 response during Hg-treatment [34], indicating direct activation of T cells for IgE production by Hg. In contrast, the T cells may need to be primed by antigen-presenting cells expressing activating FcγR to induce B cells to secret serum IgG1 [4], which could explain why the FcRγ-deficient mice in our study showed an IgE but not an IgG1 response.

In addition, Hg-treated FcγRIIB-deficient mice developed an increase in serum IgG2a and IgG2b, which is in agreement with previous results showing that lack of the FcγRIIB causes a general up-regulation of Igs in the BALB/c strain [35].

BALB/c mice do not develop ANoA/AFA in response to Hg, and loss of the inhibitory receptor FcγRIIB could not override this restriction. This is in agreement with a study where B6 mice with the H-2b haplotype did not develop ANoA following Hg treatment despite a lack of FcγRIIB [36]. The development of ANoA/AFA is critically dependent on certain specific H-2 haplotypes, H-2s, H-2q and H-2f [16–18], due probably to binding of Hg-modified fibrillarin peptides to their class II molecules. In contrast, the H-2d and the H-2b class II molecules in BALB/c and B6 mice, respectively, do not bind fibrillarin peptides, and this binding is unlikely to be influenced by the presence or absence of the FcγRIIB. However, all three strains developed ANA during Hg-treatment. Although the specificity of the ANA could not be identified by immunoblotting they consisted of IgG1 and IgG2a, indicating an antigen-specific induction mechanism.

We conclude that in HgIA the activating FcγRs are important for the formation of tissue IC deposits, and are also likely to be important for regulation of the IgG1 concentration, while FcγRIIB is of importance for the regulation of IgG1, IgG2a, IgG2b and IgE responses. Therefore, genetic polymorphism for the Fc receptors in different mouse strains that alter IgG binding capacity could account for the susceptibility to IC-mediated autoimmunity. In humans, certain allelic variants of FcγR genes have been linked to susceptibility for the development of SLE and RA [3,14]. This suggests that a future possibility for treating systemic autoimmunity might be to inhibit signalling specifically via the FcR γ-chain.

Acknowledgments

The technical assistance of Marie-Louise Eskilsson and Christer Bergman is gratefully acknowledged. This study was supported by the Swedish Research Council, Branch of Medicine (project no. 09453) and the Swedish Foundation of Strategic Research.

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