Genetic Control of Resistance to Mercury-Induced Immune/Autoimmune Activation

Genetic Control of Resistance to Mercury-Induced

Immune/Autoimmune Activation

M. ABEDI-VALUGERDI, M. HANSSON, & G. MOÈ LLER

Department of Immunology, the Wenner-Gren Institute, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden

(Received 27 February 2001; Accepted in revised form 30 March 2001)

Abedi-Valugerdi M, Hansson M, MoÈller G. Genetic Control of Resistance to Mercury-Induced Immune/

Autoimmune Activation. Scand J Immunol 2001;54:190±197

Previous studies have shown that genetic factors control the susceptibility to mercury-induced immuno-

globulin (Ig)G1 antibody formation, IgE synthesis, renal IgG deposits and antinucleolar autoantibodies

(ANolA) production in the susceptible mice. In this study, we examined the genetic control of resistance to

these characteristics after HgCl2 injection in F1 hybrid crosses between the highly mercury resistant DBA/2

and mercury susceptible NZB (H-2d), SJL (H-2 s), A.CA (H-2f) and DBA/1 (H-2q) mice and also in

backcross hybrids between (DBA/2 � SJL)F1 and SJL mice. We observed that mercury-induced immune/

autoimmune manifestations were profoundly downregulated in most (if not all) of the F1 hybrids, indicating

that the resistance to mercury was a dominant trait. Analysis of mercury-induced immune/autoimmune

responses in the (DBA/2 � SJL) � SJL backcross hybrids suggested that only one gene or a cluster of genes

determined the resistance to the ANolA production, whereas the resistance to other characteristics was

controlled by two and/or three gene loci. By H-2 genotyping the backcross mice, it was found that H-2d

haplotype per se could confer resistance to ANolA production. However, we did not find any significant

association between the H-2d haplotype and the resistance to increase of IgG1 and IgE synthesis and the

development of renal IgG1 deposits. Thus, while in DBA/2 mice, gene(s) in the H-2 loci strictly contribute to

the inheritance of resistance to ANolA production; non-H-2 genes mainly govern the inheritance of

unresponsiveness regarding other characteristics.

Dr M. Abedi-Valugerdi, Department of Immunology, the Wenner-Gren Institute, Arrhenius Laboratories for

Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden. E-mail: [email protected]

INTRODUCTION

The heavy metal ion mercury is able to induce immune

responses with autoimmune characteristics in susceptible

animals such as mice, rats and rabbits [reviewed in 1±3]. In

highly susceptible mice, mercury-induced immune responses are

characterized by a T-cell-dependent polyclonal B-cell activation

with mainly increased serum levels of IgG1 and IgE antibodies,

the production of ANolA and the formation of IgG deposits

in the kidney [1±4]. The exact mechanisms by which mercury

activates the immune system leading to the development of a

systemic autoimmune disorder are not well understood.

However, several studies have demonstrated that susceptibility

to mercury-induced immune/autoimmune manifestations is

genetically controlled [5±12]. For instance, it has been shown

that genes within H-2 loci control the susceptibility to

mercury-induced ANolA production, i.e. only mouse strains of

H-2 s, H-2q, and H-2f genotypes, irrespective of their back-

ground genes produced ANolA after mercury treatment [5±12].

By using intra-H-2 recombinant mouse strains, susceptibility to

mercury-induced ANolA production could be mapped to the I-A

loci of H-2 class II genes [7]. It has been shown that the other H-

2 class II locus (I-E) either suppressed [6] or did not influence

the mercury-induced ANolA response [7]. Moreover, H-2 genes

in combination with other not yet defined non-H-2 genes could

also confer susceptibility to mercury-induced B-cell activation

and formation of renal IgG deposition [7±12]. It seems that non-

H-2 background genes mainly control the susceptibility to

mercury-induced increase in serum IgE levels [12].

Although genetic analysis of susceptibility to murine

mercury-induced immune/autoimmune activation has been

investigated earlier [5±12], very few studies explored the

Scand. J. Immunol. 54, 190±197, 2001

q 2001 Blackwell Science Ltd

genetics of resistance to mercury [13]. This was partly owing to

the public interest in finding the susceptible genes for a given

disease (here mercury-induced autoimmunity) and partly owing

to the fact that very few studies (if any) attempted to define

mouse strains, which were fully resistant to mercury-induced

immune/autoimmune manifestations.

We recently studied the genetics of susceptibility to mercury-

induced autoimmunity in several inbred mouse strains of

different H-2 genotype regarding most of the above-mentioned

characteristics [12]. We found that among the examined strains,

SJL (H-2 s) and A.SW (H-2 s) mice were highly susceptible

strains with regard to the all tested parameters. The NZB (H-2d)

mice were also highly susceptible, but they did not develop

ANolA. Most of, if not all, other strains were classified as

intermediate responders, because each of them was able to

develop at least one characteristic of mercury-induced auto-

immunity [12]. Among the tested strains, only the DBA/2 (H-2d)

strain was found to be fully resistant to mercury-induced

immune/autoimmune responses. Existence of highly mercury

resistant mice allowed us to investigate further the genetics of

resistance to mercury-induced autoimmunity. In this study we

used F1 hybrids between DBA/2 and susceptible NZB (H-2d),

SJL (H-2 s), A.CA (H-2f) and DBA/1 (H-2q) mice as well as

backcross hybrids between (DBA/2 � SJL)F1 and SJL mice.

After chronic injections with mercury, development of IgG1

antibody formation, IgE synthesis, IgG1 ANolA production and

renal IgG1 deposit formation were studied in these hybrids. Our

results indicate that only one gene or a cluster of genes, which is

located within the H-2 loci controls the resistance to ANolA

production and that more than one gene (at least two or three

genes), which resides outside the H-2 loci confer the resistance

to increase in IgG1 and IgE synthesis as well as formation of

renal IgG1 deposits.

MATERIALS AND METHODS

Mice. Female and/or male SJL (H-2 s), DBA/1 (H-2q), A.CA (H-2f),

NZB (H-2d) and DBA/2 (H-2d) mice were originally purchased from

Charles River (Charles River Sverige AB, Uppsala, Sweden), Harlan

(Harlan UK Limited, Bicester, UK) or Microbiology and Tumour

Biology Centre (Karolinska Institute, Stockholm, Sweden). These strains

and (DBA/2 � SJL)F1 (DBA/2 � NZB)F1 (DBA/2 � DBA/1)F1

(DBA/2 � A.CA)F1 hybrids as well as backcross hybrids between

(DBA/2 � SJL)F1 hybrids and SJL mice [(DBA/2 � SJL)F1 � SJL]BC

were further bred and kept in our animal house at the Department of

Immunology at the University of Stockholm. All mice were 6±8 weeks

old at the beginning of the experiments.

HgCl2 treatment. Mercury treatment was carried out as described

before [14]. Briefly, groups of different F1 hybrids (4±14 mice per

group) and backcross hybrids (40±52 mice per group) were injected s.c.

with either 0.1 ml of HgCl2 solution (1.6 mg/kg body weight) or 0.1 ml

of a sterile 0.9% NaCl solution (control mice) every 3rd day for 4

weeks.

Blood, spleen and kidney collection. At the end of each experiment,

mercury- and saline-treated mice were bled by retro-orbital puncture

under metophane anaesthesia. Thereafter, the same mice were killed by

cervical dislocation and their spleens and kidneys were removed. The

blood of each mouse was allowed to clot at 4 8C and serum was

separated after centrifugation. The sera and kidneys were stored at

220 8C until used.

Protein-A plaque assay. Splenic single cell suspensions were

prepared by teasing spleens gently with forceps in Earle's balanced

Table 1. Association between the H-2 genotype and the resistance to mercury-induced immune/autoimmune manifestations in (DBA/

2 � SJL)F1 � SJL backcross hybrids

Response status to mercury-induced immune responses*

(DBA/2 � SJL)F1 � SJLs/s (DBA/2 � SJL)F1 � SJLs/d

Characteristics

Non/

Low Intermediate High

Non/

Low Intermediate High

IgG1antibody

formation

2 14 16 6 11 3

IgE antibody

synthesis

15 12 5 2 11 7

Renal IgG1

deposits

4 20 8 2 14 4

IgG1 ANolA

production

0 3 29 20 0 0

*Groups of (DBA/2 � SJL)F1 � SJL backcross hybrids were repeatedly injected s.c. with either mercuric chloride (several experiments, 52 mice

in total) or sterile saline (40 mice in total) for 4 weeks. At the end of each experiment, the mice were bled and killed. The spleens were tested for

IgG1 antibody secreting cells. The kidneys were tested for presence of IgG1 deposits. The sera were examined for IgG1 antinucleolar antibodies and

IgE contents and the tails were used for H-2 genotyping. Mercury-injected mice were divided into two groups based on their H-2 genotype (H-2s/s

and/or H-2s/d). Thereafter, they were categorized as low, intermediate and/or high responders.

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 190±197

Genetics of Resistance to Mercury-Induced Autoimmunity 191

salt solution (EBSS). All cell suspensions were washed three times and

re-suspended in EBSS. Antibody-secreting cells of different Ig classes

and subclasses were enumerated in the cell-suspensions by using a

protein-A plaque assay as described by Gronowicz et al. [15]. Rabbit

antimouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC, USA) and

IgG2b (Nordic Immunological Laboratories, Tilburg, the Netherlands)

were used as developing reagents. In this study, the results for IgG1

antibody secreting cells are shown.

ELISA for mouse IgE. Total mouse serum IgE was determined by a

sandwich ELISA assay as described previously [16]. We used a rat

antimouse IgE monoclonal antibodies (MoAb), R35-72 (Pharmingen,

San Diago, CA, USA) as the capture antibody and a biotinylated rat

antimouse IgE MoAb, R35-92 (Pharmingen) as the detection antibody.

Detection of IgG1 antinucleolar antibodies (ANolA). The presence of

IgG1 ANolA in the sera was determined by an indirect

immunofluorescence method. Rat liver sections and/or HEp/2 cells

grown as monolayers on slides were used as substrates and FITC-

conjugated goat antimouse IgG1 (Southern Biotechnology,

Birmingham, AL, USA) as the detecting antibody [16]. The initial

dilution for the sera was 1 : 50. When at this dilution no specific green

fluorescence was detected, the result was recorded as 0 (zero). The

highest serum dilution at which nucleolar fluorescence could be

detected was defined as the titre of IgG1 ANolA.

Detection of renal IgG1 deposits. The presence of glomerular

deposits of IgG1 Igs was detected by a direct immunofluorescence

method, as described previously [16]. Briefly, 5 mm-thick kidney

cryostat sections were fixed in acetone and incubated with serial

dilutions of FITC-conjugated goat antimouse IgG1 antibody (Southern

Biotechnology, Birmingham, AL, USA). The initial dilution for FITC-

conjugated antibody was 1 : 40. When at this dilution no specific green

fluorescence was detected, the result was recorded as 0 (zero). The

highest dilution of the conjugated antibody at which a specific green

fluorescence could be seen was defined as the endpoint titre of the

glomerular deposits.

H-2 genotyping. Genomic DNA samples from offsprings of

SJL � (DBA/2 � SJL)F1 hybrids were prepared from mouse tail

biopsies according to the following procedure: approximately 0.5 cm

of tail biopsies were put in 0.1 ml of DNA digestion buffer (10 mm

Tris-HCl, 5 mm EDTA, 1% SDS, 0.3 m NaAc and proteinase K at

0.1 mg/ml) and incubated overnight at 52 8C. Thereafter, genomic

DNA was first extracted with a phenol/chloroform mixture and then

precipitated by ethanol. The H-2 typing was performed by polymerase

chain reaction (PCR) of genomic DNA, using the D17Mit16 primers

(Invitrogen, Groningen, the Netherlands), which could discriminate

between H-2s and H-2d genotype [17]. The PCR was carried out using

50±100 ng of genomic DNA in the presence of 1.5 mm MgCl2, 1 mm

dNTP, 2 U of Taq DNA polymerase, and 6.6 mM of each primer in a

final volume of 20 ml. After the initial denaturation at 95 8C for 2 min,

the amplification was performed during 30 cycles of 94 8C for 45 s,

55 8C for 45 s, and 72 8C for 1 min, followed by a final extension at

72 8C for 7 min in a thermal cycler (Corbett Research, Mortlake,

Australia). All amplified DNA samples were then electrophorezed

through an agarose gel mix, containing 2% agarose (Gibco BRL, Life

Technologies, Paisley, UK) and 1.5% of low melting point agarose

(Gibco BRL, Life Technologies). After electrophoresis, the gels were

stained with ethidium bromide and photographed.

Grading of susceptibility to mercury-induced immune responses. For

each characteristic, the term `fold increase' was used to define the

difference between mercury-and saline-injected mice. Fold increase in

the number of IgG1 antibody-secreting cells in each mercury-injected

mouse was calculated by dividing the number of IgG1 antibody

secreting cells obtained from the mouse to the mean value of the IgG1

antibody secreting cells obtained from the saline-injected mice.

Mercury-injected backcross mice were considered as non/low,

Fig. 1. Inheritance of resistance to

mercury-induced increase of

immunoglobulin (Ig)G1 and IgE antibody

formation in the F1 hybrid crosses. Groups

of different F1 hybrids were repeatedly

injected subcutaneously with either

mercuric chloride (solid symbols) or

sterile saline (not shown) for 4 weeks. At

the end of each experiment the mice were

bled and killed. The spleens were tested

for IgG1 antibody secreting cells by using

a protein-A plaque assay (A). The sera

were tested for total IgE concentration,

using an ELISA method (B). The data are

shown as the mean values for the fold

increase in the numbers of IgG1 antibody

secreting cells and serum IgE levels in

mercury-injected mice ^ 1 SE (see

Materials and methods for further

description). Significant differences

between the parameters in mercury-injected

F1 hybrid crosses and mercury-injected

susceptible parental strains were calculated

by Wilcoxon±Mann±Whitney test.

*P , 0.05; **P , 0.01; ***P , 0.001.

192 M. Abedi-Valugerdi et al.


intermediate, and high responders when they exhibited either a 1±2.5-

fold (non/low responder), or a 3±8-fold (intermediate responder) and/or

a . 8-fold (high responder) increase in the number of IgG1 antibody

secreting cells. In order to calculate the fold increase in the serum levels

of IgE, we first calculated the mean absorbance for a dilution, which

was within the linear part of the titration curve for saline-injected mice.

Then, in each mercury-injected mouse, we considered the serum

dilution at which it reached the mean absorbance for the saline-injected

mice. Finally, fold increase in the serum IgE levels was calculated by

dividing the obtained serum dilution to the serum dilution at which the

mean absorbance was calculated. Mercury-injected backcross mice

were considered as non/low, intermediate, and high responders when

they exhibited either a 1±2-fold (non/low responder), or a 3±7-fold

(intermediate responder) and/or a . 7-fold (high responder) increase in

the levels of serum IgE levels. Fold increase in the titres of renal IgG1

deposits in each mercury-injected mouse was calculated by dividing the

reciprocal titres of renal IgG1 deposits to 40 (the initial reciprocal

dilution for FITC-conjugated antibody). Mercury-injected backcross

mice were considered as non/low, intermediate, and high responders

when they showed either a 0±2-fold (non/low responder), or a 3±8-fold

(intermediate responder) or a . 8-fold (high responder) increase in the

titres if renal IgG1 deposits. Fold increase in the serum titres of IgG1

ANolA in each mercury-injected mouse was calculated by dividing the

reciprocal titres of serum IgG1 ANolA to 50 (the initial reciprocal

dilution for the serum). Mercury-injected backcross mice were

considered as non/low, intermediate, and high responders when they

exhibited either a 0±4-fold (non/low responder), or a 5±8-fold

(intermediate responder) or a . 8-fold (high responder) increase in

the serum titres of IgG1 ANolA.

Statistical analysis. Fold increase in antibody-secreting cells of

different isotypes, serum IgE levels, serum titres of IgG1 ANolA and

titres of renal IgG1 deposits in mercury-injected mice were shown as the

means ^ 1 standard error (SE). We estimated the SE, because it represents

the expected standard deviation of the statistic in the case where a large

number of samples (here animals) had been used. The differences between

these parameters in mercury-injected susceptible parental strains and F1

hybrid crosses were analyzed with the Wilcoxon±Mann Whitney (rank

sum) test. The X2 test was performed to analyze whether the observed

genetic segregation ratios statistically correlated with the expected values.

RESULTS

Resistance to mercury-induced immune/autoimmune

activation is inherited as a dominant trait

In order to study the inheritance of resistance to mercury-

induced autoimmunity, groups of F1 hybrid crosses between the

mercury resistant DBA/2 mice and mercury susceptible NZB,

A.CA, SJL and DBA/1 mice were continuously treated with

mercury or as controls, with saline for 4 weeks. At the end of

each experiment, the mice were tested for the development of

mercury-induced immune/autoimmune characteristics. As a first

characteristic, we studied the mercury-induced IgG1 antibody

formation by enumerating the IgG1 antibody secreting cells in

the spleens. As shown in Fig. 1(A), the numbers of IgG1

antibody secreting cells dramatically decreased in the F1 hybrids

Fig. 2. Inheritance of resistance to mercury-

induced renal IgG1 deposit formation and

IgG1 ANolA production in the F1 hybrid

crosses. The kidneys removed from the

same experimental mice (Fig. 1) were

analyzed for the presence of renal deposits

of IgG1 antibodies by using a direct

immunofluorescence (DIF) method (A).

The sera obtained from the same

experimental mice (Fig. 1) were tested for

the presence of IgG1 antinucleolar

antibodies using an indirect

immunofluorescence (IIF) method (B).

Data are shown as the mean values for the

fold increase in the titres of renal IgG1

deposits and serum IgG1 ANolA in

mercury-injected mice ^ 1 SE (see

Materials and methods section for further

description). Significant differences

between the parameters in mercury-injected

F1 hybrid crosses and mercury-injected

susceptible parental strains were calculated

by Wilcoxon±Mann±Whitney test.

*P , 0.05; **P , 0.01; ***P , 0.001.

The percentage numbers in the parentheses

(Fig. 2B) are the percentage of the mice,

which were positive for ANolA production

and the statistical analysis have been made

on these numbers.



as compared with their mercury-treated susceptible parental

strains (NZB, A.CA, SJL and DBA/1 mice).

We next measured the levels of IgE antibodies in the sera of

F1 hybrid crosses. Significant decreases in the serum IgE levels

were also found in the mercury-treated F1 hybrids when

compared with the serum IgE levels in their mercury-treated

susceptible parental strains (Fig. 1B). However, the decrement

in the serum IgE levels in (DBA/2 � NZB)F1 and (DBA/

2 � SJL)F1 hybrids was not as striking as that in (DBA/

2 � A.CA)F1 and (DBA/2 � DBA/1)F1 hybrids (Fig. 1B).

As the third phenotype, we evaluated the formation of IgG1

deposits in the kidneys of mercury-treated F1 hybrid crosses.

None of the F1 hybrids exhibited any significant increase in the

titres of renal IgG1 deposits as compared with their mercury-

treated susceptible parental strains (Fig. 2A).

Finally, we studied the inheritance of resistance to mercury-

induced ANolA production in the F1 hybrid crosses. As shown in

Fig. 2(B), there was a variation in production of IgG1 ANolA

among the F1 hybrids. For instance (DBA/2 � A.CA)F1 and

(DBA/2 � SJL)F1 hybrids showed either no or very low serum

titres of IgG1 ANolA as compared with their susceptible parental

strains (A.CA and SJL mice). Conversely (DBA/2 � DBA/1)F1

hybrids exhibited either no or high titres of serum IgG1 ANolA,

which were comparable to those in their susceptible, DBA/1

parental strain (Fig. 2B). Taken together, these findings show that

the expression of most, if not all, of the mercury-induced

immune/autoimmune manifestations is downregulated in the F1

hybrid crosses between susceptible and resistant strains.

Inheritance of mercury-induced immune/autoimmune

responses in the backcross hybrids

In the next series of experiment, the formation of IgG1,

synthesis of IgE, development of renal IgG1 deposits and

production of IgG1 ANolA were analyzed in the mercury-

treated backcross hybrids of (DBA/2 � SJL)F1 � SJL mice (52

animals). As shown in Fig. 3(A±D), the backcross hybrids could

be graded as non/low, intermediate and high responders with a

defined phenotypic ratio for each characteristic. For instance, for

the IgG1 antibody formation the backcross hybrids exhibited a

phenotypic ratio of 8 : 25 : 19 (Fig. 3A). This ratio was in

satisfactory agreement with the expected 1 : 2 : 1 ratio

(x2 � 3.8, 0.2 . P . 0.15) [18], which suggests that two loci

determine the resistance to increase of IgG1 antibody formation.

For the increase in the serum levels of IgE, the backcross

hybrids expressed a phenotypic ratio of 17 : 23 : 12 (Fig. 3B).

This ratio was also compatible with the expected ratio 1 : 2 : 1

(x2 � 1.65, 0.6 . P . 0.5) [18], and again implies that two

loci govern the resistance to increase of IgE serum levels. The

backcross hybrids exhibited a phenotypic ratio of 6 : 34 : 12 for

the increase in the titres of renal IgG1 deposits (Fig. 3C). This

ratio was consistent with the expected ratio 1 : 6 : 1 (x2 � 5.3,

P � 0.08) [18], indicating that three loci are involved in

conferring resistance to development of renal IgG1 deposits. In

contrast to other characteristics, the expression of IgG1 ANolA

production phenotype was more distinct in the backcross

hybrids as they showed a phenotypic ratio of 20 : 3 : 29

(Fig. 3D). This ratio correlated with the expected ratio 1 : 1

(x2 � 1.4, P � 0.23) [18], which suggests that only one gene or

a cluster of genes contributes to resistance to IgG1 ANolA

synthesis.

Contribution of H-2 genotype to the resistance to

mercury-induced immune/autoimmune manifestations

It had been shown that the susceptibility to mercury-induced

Fig. 3. Inheritance of resistance to

mercury-induced increase of IgG1 and IgE

synthesis, development of renal IgG1

deposits and synthesis of IgG1 ANolA in

the backcross hybrids. Groups of (DBA/

2 � SJL)F1 � SJL backcross hybrids were

repeatedly injected s.c. with either mercuric

chloride (52 mice in total) or sterile saline

(40 mice in total) for 4 weeks. At the end of

each experiment, the mice were bled and

killed. As described in Figs 1 and 2, the

spleens were tested for IgG1 antibody

secreting cells (A), the kidneys were tested

for presence of IgG1 deposits (C) and the

sera were examined for IgE and IgG1

antinucleolar antibodies (B and D,

respectively). Thereafter, based on the

degree of response for each characteristic,

mercury-injected mice were categorized as

non/low, intermediate and/or high

responders (see Materials and methods for

further description).



autoimmunity was partly controlled by H-2 genes [5±12]. This

led us to test whether H-2 loci inherited from the DBA/2 mice

could participate in conferring resistance to mercury-induced

autoimmunity. To do this, the mercury-injected backcross

hybrids were genotyped for H-2 genes and analyzed for

expression of each characteristic. As shown in Table 1, 32 out

of 52 mice were homozygous (H-2 s/s) and 20 were hetero-

zygous (H-2 s/d) in the H-2 loci (Table 1).

If the resistant H-2 haplotype (H-2d) was one of the

responsible genes for the resistance to increase of IgG1 and

IgE and development of renal IgG1 deposits, we expected to

observe that among the H-2 heterozygous (H-2 s/d) backcross

hybrids at least half behave as non/low responders for the

increase to IgG1 and/or IgE (as two genes were involved for

resistance to these phenotypes). We also expected to see that at

least 1/3 of these backcross hybrids would act as non/low

responders for the development of renal IgG1 deposits (as three

genes were involved for resistance to this phenotype). In

addition, none of the H-2 heterozygous backcross mice was

expected to behave as high responder for either of the

characteristics. In spite of our expectation, we observed that

only a small fraction of the H-2 heterozygous (H-2 s/d)

backcross hybrids, behaved as non/low responders for IgG1

antibody formation (6 of 20 mice), increase in IgE synthesis

(two out of 20 mice) and development of renal IgG1 deposits

(two out of 20 mice) (Table 1). Moreover, a substantial number

of the H-2 heterozygous backcross mice acted as high

responders for these characteristics (Table 1). These findings

suggest that resistance to mercury-induced increase in IgG1, IgE

and formation of renal IgG1 deposits is not associated with the

resistant H-2d haplotype.

If H-2d haplotype was responsible for the resistance to IgG1

ANolA production, we expected to observe that most, if not all,

of the H-2 heterozygous (H-2 s/d) backcross hybrids would

behave as non/low responders for this phenotype (as one gene

was involved for resistance to this characteristic). Consistent

with our expectation, we found that all (20 out of 20 mice) of the

heterozygous (H-2 s/d) backcross hybrids exhibited either no or

low titres of IgG1 ANolA in their sera (Table 1). On the other

hand, we found that 29 out of the 32 (90%) homozygous (H-2 s/

s) backcross hybrids behaved as high responders and none were

non/low responders (Table 1). This supports our suggestion that

one gene or a cluster of genes determines the resistance to IgG1

ANolA production and implies that the resistance is associated

with the H-2d haplotype.

DISCUSSION

In the present study, we investigated the inheritance of

resistance to mercury-induced autoimmunity regarding its four

major characteristics, formation of IgG1 antibodies, increase in

serum IgE levels, production of ANolA, and development of

renal IgG1 deposits. The F1 hybrid crosses between mercury

resistant and mercury susceptible strains and backcross hybrids

between mercury susceptible, SJL mice and (DBA/2 � SJL)F1

hybrids were used. Our first observation was that that

development of most, if not all, of the above mentioned

characteristics were downregulated in the F1 hybrids. This

implies that the DBA/2 strain possesses profound dampening

genetic factors that can be transmitted as dominant traits to the

F1 generation. This is in contrast to mercury-induced auto-

immune disease in rats, where it has been shown that the

susceptibility to IgE increase and development of immune

complex-type glomerulonephritis is inherited as a dominant trait

[19±21]. This indicates that the path of inheritance of

susceptibility/resistance to mercury-induced autoimmunity var-

ies among the species.

Several conclusions can be drawn from the results observed

with backcross hybrids. Firstly, the presence of three alter-

natives (low, intermediate and high) in characteristic distribu-

tion for increase in IgG1, IgE and development of renal IgG1

deposits, suggest that resistance to each of these phenotypes is

controlled by more than one gene. Based on this suggestion and

the finding that the resistance was a dominant trait in the F1

hybrids, we can conclude that to fulfil a complete resistance for

each phenotype, heterozygosity in all responsible genes is

required.

Secondly, for the resistance to increase of IgG1, the

phenotypic ratio obtained in the backcross hybrids (before H-2

genotyping) was compatible with a two-gene model [18].

Analysis of IgG1 response in the H-2 genotyped backcross

hybrids showed that the gene(s) within the H-2d loci was not one

of them. Thus, two non-H-2 background loci might be

associated with the resistance to this phenotype in DBA/2 mice.

This is further supported by the observation that among the

mercury-treated mouse strains of H-2d genotype (NZB, BALB/c

and DBA/2 mice), only DBA/2 mice were resistant to IgG1

production [12] and that low magnitudes of IgG1 responses

were found in mercury-treated (NZB � DBA/2)F1 hybrids

which are possibly homozygous for H-2d genotype (H-2d/d)

(this study). In this study, we did not address the location of the

resistant genes. However, because in vivo induction of IgG1

response is known to be T-cell dependent [22], and because it

has been suggested that Fc receptors for IgG (FcgR) play a

pivotal role in the regulation of the B-cell activation [23], it is

likely that the polymorphism in T-cell receptor and Fcg receptor

genes are associated with the resistance to increase in IgG1

response.

Thirdly, for the resistance of the IgE to increase, the

phenotypic ratio obtained in the backcross hybrids (before H-2

genotyping) was also in agreement with a two-gene model [18].

Analysis of IgE serum levels in the H-2 genotyped backcross

hybrids demonstrated that again the gene(s) within the H-2d loci

were not involved and suggests that two not yet characterized

non-H-2 background loci are linked to the resistance to this

characteristic. It has been suggested that for undergoing isotype

switching to IgE, B cells require the secretion of lymphokines,

especially interleukin (IL)-4 and/or IL-13 and the expression of

CD40 ligand (CD40L) by the T cells [24]. Therefore, further

analysis of expression of cytokines and costimulatory genes



would help to characterize the genes, which are associated with

the resistance to mercury-induced IgE increase.

Fourthly, in the backcross hybrids, the phenotypic ratio

obtained for the resistance to mercury-induced development of

renal IgG1 deposits was consistent with a three-gene model [18].

Evaluation of these backcross hybrids for the formation of renal

IgG1 deposits after being genotyped for H-2 revealed that H-2d

haplotype was not one of these genes. This indicates that three

non-H-2 background genes control the resistance to this

phenotype. This statement gains further support from the

finding that among the mercury-treated mouse strains of H-2d

genotype (NZB, BALB/c and DBA/2 mice), only DBA/2 mice

were resistant to the development of renal IgG1 deposits [12]

and that mercury-treated (NZB � DBA/2)F1 hybrids (H-2d/d)

did not exhibit any significant increase in the titres of renal IgG1

deposits (this study). Further studies on the backcross hybrids

between DBA/2 and (NZB � DBA/2)F1 hybrids would be

useful to characterize the genes, which are linked to resistance to

mercury-induced renal IgG1 deposits.

Fifthly, the finding that there was a variation in IgG1 ANolA

production among the mercury-treated F1 hybrids implies that

IgG1 ANolA phenotype is inherited as an incomplete penetrating

trait and that depending on the susceptible mouse strains used in

the F1 hybrid crosses, different levels of penetration exist.

Sixthly, for the resistance to the induction of IgG1 ANolA

production, the phenotypic ratio obtained in the backcross

hybrids was in agreement with a one-gene model [18]. The

finding that all of the H-2 heterozygous (H-2 s/d) backcross

hybrids, regardless of their background were non/low respon-

ders suggests that resistance to ANolA production is mainly, if

not absolutely, conferred by H-2d haplotype. This statement

confirms and supports the earlier conclusion that the expression

of the resistant H-2 genotype (here class II of H-2 genes) per se

is able to downregulate the ANolA response in otherwise

susceptible mice [10]. The mechanism(s) by which the resistant

H-2 genotype could downregulate (despite the fact that it is

codominantly expressed with the susceptible, H-2s, genotype)

the mercury-induced ANolA response is not well understood.

However, in an adoptive transfer study, Hanley et al. [13] have

demonstrated that all of the lethally irradiated

(B6.SJL � B6.TC)F1(H-2s/b) hybrids, reconstituted with T-cell

depleted bone marrow (BM) from the susceptible parental strain

(B6.SJL (H-2s) produced ANolA after mercury treatment. In

contrast, only a small fraction of the same F1 hybrids

reconstituted with T-cell depleted BM from either identical F1

hybrids or a combination of parental strains [B6.SJL (H-2 s) and

B6.TC (H-2b)] developed ANolA by injection with mercury

[13]. In addition, treatment with mercury in B6.SJL (H-2s) mice

reconstituted with T-cell depleted BM from B6.SJL �B6.TC)F1s/b did not result in production of ANolA [13]. These

findings led to the suggestion that an intrinsic property present

in haplotype-heterozygous B cells is responsible for the

resistance to ANolA synthesis observed in the F1 hybrids

[13]. Thus, this suggestion might be applied for (DBA/

2 � SJL)F1 (DBA/2 � A.CA)F1 hybrids in which the degree

of ANolA penetration was low, but not for (DBA/2 � DBA/1)F1

hybrid, which exhibited a high degree of ANolA penetration (this

study). Therefore, additional studies are required to elucidate how

resistant H-2 haplotypes downregulate the mercury-induced

ANolA production in the H-2 heterozygous hybrids.

ACKNOWLEDGMENTS

The excellent technical assistance of Mrs Lena Israelsson is

gratefully acknowledged. This study was supported by grants

from the Swedish Medical Research Council and the Swedish

Foundation for Health Care Sciences and Allergy Research.

REFERENCES

1 Goldman M, Druet P, Gleichmann E. TH2 cells in systemic

autoimmunity: insights from allogeneic disease and chemically-

induced autoimmunity. Immunol Today 1991;12:223±7.

2 Griem P, Gleichmann E. Metal ion induced autoimmunity. Curr

Opin Immunol 1995;7:831±2.

3 EnestroÈm S, Hultman P. Does amalgam affect the immune system? A

controversial issue. Int Arch Allergy Immunol 1995;106:180±203.

4 Hultman P, EnestroÈm E. The induction of immune complex deposits

in mice by peroral and parenteral administration of mercuric

chloride: strain dependent susceptibility. Clin Exp Immunol

1987;67:283±92.

5 Robinson CJC, Balazs T, Egorov IK. Mercuric chloride-, gold

sodium thiomalate-, and d-penicillamine-induced antinuclear anti-

bodies in mice. Toxicol Appl Pharmacol 1986;86:159±69.

6 Mirtcheva J, Pfeiffer C, De Bruijn JA, Jacquesmart F, Gleichmann

E. Immunological alterations inducible by mercury compounds. III.

H-2A acts as an immune response and H-2E as an immune

`suppression' locus for HgCl2-induced antinucleolar autoantibodies.

Eur J Immunol 1989;19:2257±61.

7 Hultman P, Bell LJ, EnestroÈm S, Pollard KM. Murine susceptibility

to mercury. I. Autoantibody profiles and systemic immune deposits

in inbred, congenic and intra-H-2 recombinant strains. Clin

Immunol Immunopathol 1992;65:98±109.

8 Hultman P, Bell LJ, EnestroÈm S, Pollard KM. Murine susceptibility

to mercury. II. Autoantibody profiles and renal immune deposits in

hybrid, backcross, and H-2d congenic mice. Clin Immunol

Immunopathol 1993;68:9±20.

9 Hultman P, Turley SJ, EnestroÈm S, Lindh U, Pollard KM. Murine

genotype influences the specificity, magnitude and persistence of

murine mercury-induced autoimmunity. J Autoimm 1996;9:139±49.

10 Hanley GA, Schiffenbauer J, Sobel ES. Class II haplotype

differentially regulates immune response in HgCl2-treated mice.

Clin Immunol Immunopathol 1997;84:328±37.

11 Johansson U, Hansson-Georgiadis H, Hultman P. The genotype

determines the B cell response in mercury-treated mice. Int Arch

Allergy Immunol 1998;116:295±305.

12 Abedi-Valugerdi M, MoÈller G. Contribution of H-2 and non-H-2

genes in the control of mercury-induced autoimmunity. Int Immunol

2000;12:1425±30.

13 Hanley GA, Schiffenbauer J, Sobel ES. Resistance to HgCl2-

induced autoimmunity in haplotype-heterozygous mice is an

intrinsic property of B cells. J Immunol 1998;161:1778±85.

14 Hu H, MoÈller G, Abedi-Valugerdi M. Mechanism of mercury-



induced autoimmunity: both T helper 1-and T helper 2-type

responses are involved. Immunol 1999;96:348±57.

15 Gronowicz E, Coutinho A, Melchers F. A plaque assay for all cell

secreting Ig of a given type or class. Eur J Immunol 1976;6:588±

90.

16 Al-Balaghi S, MoÈller E, MoÈller G, Abedi-Valugerdi M. Mercury

induces polyclonal B cell activation, autoantibody production and

renal immune complex deposits in young (NZB x NZW) F1 hybrids.

Eur J Immunol 1996;26:1519±26.

17 Dietrich W, Katz H, Lincoln SE et al. A genetic map of mouse

suitable for typing intraspecific crosses. Genetics 1992;131:423±47.

18 Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC, eds. Modern

Genetic Analysis. New York: WH Freeman, 1999:133±7.

19 Sapin C, Hirsch F, Delaporte J-P, Bazin H, Druet P. Polyclonal IgE

increase after HgCl2 injections in BN and LEW rats: a genetic

control. Immunogenetics 1984;20:227±36.

20 Druet E, Sapin C, Gunther E, Feingold N, Druet P. Mercuric

chloride-induced anti-glomerular basement membrane antibodies in

the rats: genetic control. Eur J Immunol 1977;7:348±51.

21 Sapin C, Mandet C, Druet E, Gunther E, Druet E. Immune complex

type disease induced by HgCl2 in Brown-Norway rats: genetic

control of susceptibility. Clin Exp Immunol 1982;48:700±4.

22 Finkelman FD, Holmes J, Katona IM et al. Lymphokine control of

in vivo immunoglobulin isotype selection. Annu Rev Immunol

1990;8:303±35.

23 Fridman WH. Regulation of B-cell activation and antigen

presentation by Fc receptors. Curr Opin Immunol 1993;5:355±60.

24 Bacharier LB, Geha RS. Molecular mechanisms of IgE regulation. J

Allergy Clin Immunol 2000;105:S547±58.



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Genetic Control of Resistance to Mercury-Induced Immune/Autoimmune Activation