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Costimulation Requirements of Induced Murine Systemic Autoimmune Disease¹

K. Michael Pollard^2,*, Marc Arnush^*, Per Hultman and Dwight H. Kono

Departments of ^* Molecular and Experimental Medicine and Immunology, W. M. Keck Autoimmune Disease Center, The Scripps Research Institute, La Jolla, CA 92037; and Department of Health and Environment, Division of Molecular and Immunological Pathology, Linköping University, Linköping, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl₂-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L^–/– mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Costimulation is required for productive interaction between T cells and APC, including B cells (1). Major receptor/ligand pairs include CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). Within the context of appropriate engagement of TCR with MHC (signal 1), costimulation mediated by CD28-B7 interaction leads to T cell proliferation and cytokine production (2). CD28-mediated T cell activation is followed by expression of CTLA-4 whose interaction with B7 molecules leads to inhibition of T cell responses (2). CD40L is transiently expressed on activated T cells and is a powerful regulator of T cell-dependent B cell function, including B cell survival (3). Costimulation via CD28/B7 can influence CD40L/CD40 function and vice versa. However CD40L can be induced on T cells from CD28-deficient mice (4), suggesting nondependent expression. It has been argued that CD28-B7 interaction is required for initiating T cell-dependent responses while the role of CD40/CD40L is to amplify such responses (1). However, the requirement for CD40-CD40L interaction for certain B cell responses can be replaced by agonistic anti-CD28 Ab (5), supporting the pivotal role that CD28-B7 interaction plays in T cell-dependent humoral immunity.

Systemic lupus erythematosus is a multigenic autoimmune disease characterized by humoral autoimmunity and multiorgan immunopathology (6). T-B cell costimulation plays a significant role in the development of idiopathic lupus as disease can be blocked by treatment with a chimeric CTLA-4/IgFc construct (7) or anti-CD40L Ab (8), or more effectively by a combination of both reagents (9). Studies using gene knockouts have also demonstrated the importance of costimulation in systemic autoimmunity. CD28 deficiency in lupus prone MRL-Fas^lpr mice results in reductions in serum IgG, IgG autoantibodies, and renal pathology, but serum IgM and IgM rheumatoid factor are unaffected (10). Absence of either CD80 or CD86, ligands for CD28, does not affect autoantibody levels compared with wild-type mice (11, 12). However, CD80-deficient mice have more severe renal disease than wild type, while CD86 knockouts have less severe disease compared with wild-type MRL-Fas^lpr (11, 12). The less active disease of MRL-Fas^lprCD86^–/– mice is associated with a reduction in activated T cells (12). Absence of both CD80 and CD86 in MRL-Fas^lpr mice is associated with reductions in most features of systemic lupus, including serum IgG, autoantibodies, IFN- and IL-12, renal pathology, and proteinuria (13). Like MRL-Fas^lprCD28^–/– mice (10) MRL-Fas^lprCD80/CD86^–/– (13) mice do not have reduced levels of IgM. MRL-Fas^lpr mice lacking CD40L had reduced serum IgG, autoantibodies, and glomerular disease when compared with wild type; however, most of these features are more pronounced than in nonautoimmune micelacking CD40L (14). Serum IgM levels are elevated in MRL-Fas^lprCD40L^–/– mice and low titer anti-small nuclear ribonucleoprotein autoantibodies are also present (14). Another study has confirmed the presence of low titer anti-nuclear Abs (ANA)³ and anti-small nuclear ribonucleoprotein autoantibodies in MRL-Fas^lprCD40L^–/– mice and demonstrated that T cells not only contribute to increased levels of autoantibodies but may also regulate levels of some autoantibody specificities (15).

MRL-Fas^lpr mice have also been used to examine the role of ICAM-1 in systemic autoimmunity. Absence of ICAM-1 does not affect serum Ig or autoantibody levels, but does reduce glomerular disease (16) and pulmonary inflammation (17). Absence of ICAM-1 is also associated with increased life span in MRL-Fas^lpr mice (16, 17).

Murine HgCl₂-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease characterized by lymphadenopathy, hypergammaglobulinemia, autoantibodies, and immune complex deposits (18). Although the importance of mercury to human autoimmunity has yet to be determined, mHgIA has nonetheless proven to be a useful model of systemic autoimmunity that exhibits numerous features of murine lupus (18). Among the most prominent lupus-like features are anti-chromatin autoantibodies and glomerulonephritis (19). mHgIA is also characterized by a MHC class II-restricted autoantibody response against the nucleolus, particularly fibrillarin (20), an autoantibody response characteristic of scleroderma (20). Significantly, exposure of lupus-prone mice to low doses of mercury exacerbates idiopathic disease (21), suggesting that idiopathic lupus and mHgIA may share common pathogenic mechanisms. Analogous with idiopathic lupus mHgIA can be suppressed by anti-CD40 ligand (CD40L) Ab or CTLA-4 Ig (22), or a combination of anti-B7-1 (CD80) and anti-B7.2 (CD86) Abs (23). Individually, anti-CD86 Ab suppressed anti-nucleolar autoantibodies (ANoA) and reduced IgE but anti-CD80 Ab was only able to partially reduce the ANoA response (23). In vitro anti-CD40L, anti-CD80, and to a lesser extent anti-CD86 Ab inhibited mercury-induced T cell proliferation although none of these Abs was as effective as anti-IL-1 Ab (24). The role of ICAM-1 in mHgIA has not been studied.

In this report, we examined the mechanism of costimulation in mHgIA by attempting to induce disease in mice deficient for CD40L, CD28, or ICAM-1. Apart from a reduction of glomerular immune complex deposits, absence of ICAM-1 appeared to have little effect. In contrast deficiencies in either CD40L or CD28 had significant ameliorating effects. This was particularly true for CD40L where heterozygous mice showed reduced autoantibody and immunopathology. Markers of T cell activation such as CD44 and intracellular CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of intracellular CTLA-4 expression in CD40L^–/– mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings support the hypothesis that the severity of mHgIA relies on positive costimulation via both CD28 and CD40L.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Mice with targeted disruption of the gene for CD40L (B6.129S-Tnfsf5^tm1Imx), CD28 (B6.129S2-Cd28^tm1Mak), or ICAM-1 (CD54) (B6.129S7-Icam1^tm1Bay) as well as wild type (B10.S-H2^s/SgMcdJ) were obtained from The Jackson Laboratory (Bar Harbor, ME). Subsequent breeding and maintenance were performed under specific-pathogen-free conditions at The Scripps Research Institute Animal Facility (La Jolla, CA). H-2^s CD40L^–/–, CD28^–/–, or ICAM-1^–/– mice were generated by backcrossing homozygous knockout mice for two generations to B10.S (H-2^s/s) mice. Mice, homozygous for H-2^s and heterozygous for the gene of interest, were then intercrossed to create +/+, +/–, and –/– H-2^s/s littermates. H-2 typing was performed by PCR of genomic DNA using D17 Mit16 primers that are polymorphic for H-2^b and H-2^s haplotypes. Wild-type and knockout genotypes were also determined by PCR of genomic DNA. For CD28 wild type, the primers used were CD28-R (5'-AGTTCCATTGCTCCTCTCGTTGTC-3') and CD28-F (5'-CAGTCGCCCCTGCTTGTGGTAGATAGC-3') as previously described (25). The knockout gene was typed using generic Neo primers (Neo-R2, 5'-ATACTTTCTCGGCAGGAGCA-3'; Neo-L2, 5'-TGAATGAACTGCAGGACGAG-3'). PCR conditions were 35 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s. CD28-R and CD28-F gave a 300-bp fragment for the undisrupted gene and Neo-R2 and Neo-L2 a 171-bp fragment for the disrupted gene. The CD40L wild-type gene and knockout gene primers were CD40L-P3 (5'-CCCAAGTGTATGAGCATGTGTGT-3'), CD40L-P4 (5'-GTTCCTCCACCTAGTCATTCATC-3'), CD40L-P5 (5'-GCCCTGAATGAACTGCCAGGACG-3'), and CD40L-P6 (5'-CACGGGTAGCCAACGCTATGTC-3') as previously described (26). PCR consisted of 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The CD40L-P3 and CD40L-P4 gave a 250-bp fragment for the undisrupted gene and CD40L-P5 and CD40L-P6 a 500-bp fragment for the disrupted gene. ICAM-1 wild-type gene PCR was 35 cycles of 94°C for 20 s, 53°C for 45 s, and 72°C for 90 s. The primers used were ICAM-14 (5'-AGAACCACTGCTAGTCC-3') and ICAM-15 (5'- GTTCTTCTGAGCGGCGT-3') as previously described (27). ICAM-14 and ICAM–15 gave a 1200-bp fragment for the undisrupted gene. Neo-R2 and Neo-L2 were used to detect the disrupted gene (171-bp fragment).

Mercury treatment

Mice were injected twice per week for 4 wk s.c. with 40 µg HgCl₂ in PBS as previously described (28). Controls received PBS alone. In some experiments (see below), mice were injected for 2 wk using the same injection schedule.

Serology

ANA. ANA were detected as previously described (20) using HEp-2 cells as substrate (Bion Enterprises, Park Ridge, IL). Sera were diluted 100-fold in PBS containing 0.5% BSA, 0.1% bovine -globulin, 0.001% gelatin, and 0.05% Tween 20 before assay. Goat anti-mouse IgG-FITC or anti-mouse IgM-FITC Abs (Caltag Laboratories, South San Francisco, CA), diluted 100-fold in PBS containing 0.5% bovine -globulin, 0.1% BSA, and 0.05% Tween 20, were used as detecting reagents. Intensity of fluorescence for ANoA uses the 0–4 scale routinely used in scoring ANA by immunofluorescence. An intensity of 1 indicates a clearly discernable staining of the nucleolus while 4 is maximal signal intensity. Sera were scored by an experienced observer (K.M.P.) blinded to the identity of the samples.

Anti-chromatin Abs. Anti-chromatin Abs were detected by ELISA (29). Sera were diluted 100-fold before assay, and chromatin-bound Abs were detected with HRP-conjugated goat anti-mouse IgG or anti-mouse IgM Abs (Caltag Laboratories) diluted 2000-fold.

Serum Ig quantitation

Serum IgG and IgM levels were quantified by ELISA (28). ELISA plates were coated with 200 µl of 2 µg/ml goat anti-mouse L chain Ab (Caltag Laboratories) diluted in PBS and incubated overnight at 4°C. Plates were postcoated for 1 h with 0.1% gelatin in PBS followed by three washes with PBS-0.05% Tween 20. Sera were diluted in serum diluent (30). A standard curve was generated by serial dilutions of polyclonal mouse reference serum containing predetermined levels of Ig isotypes (The Binding Site, Birmingham, U.K.). Diluted sera were incubated in duplicate while shaking for 2 h 30 min, followed by three washes with PBS-0.05% Tween 20. HRP-conjugated goat anti-mouse IgG or IgM Abs (Caltag Laboratories) were diluted in anti-Ig diluent (30) and incubated with shaking for 90 min. After three washes with PBS-0.05% Tween 20 and four washes with PBS, ABTS substrate solution was added and the OD read at 405 nm. Serum IgG and IgM concentrations were calculated by extrapolation from the linear portion of standard curves.

Immunopathology

Sections of kidney and spleen were prepared and stained for direct immunofluorescence as previously described (28). Briefly, 4- to 5-µm-thick cryostat sections were fixed in ethanol and incubated with serial dilutions of FITC-conjugated goat Abs to IgG (-chain specific) and C3 (Southern Biotechnology Associates, Birmingham, AL). The end point titer of the immune deposits was defined as the highest dilution of Ab at which specific fluorescence could be detected. Vessel wall deposits were graded on a 0–4+ scale. The slides were examined without knowledge of treatment or other results.

T cell activation

The contribution of CD28, CD40L, and ICAM-1 to T cell activation in mHgIA was examined by determining expression of CD25, CD44, and CTLA-4 after 2-wk exposure to HgCl₂. Mice were injected twice per week with 40 µg HgCl₂ in PBS or PBS only. Forty-eight hours after the last injection, single-cell suspensions were prepared from spleens as previously described (24). Cell surface marker expression was measured by flow cytometry (see below). In some experiments, wild-type or CD40L^–/– mice were treated with anti-CD28 Ab to enhance T cell activation (5). Anti-CD28 Ab (75 µg, clone 37.51; BD Pharmingen, San Diego, CA) or anti-keyhole limpet hemocyanin (KLH) Ab of the same isotype was administered on days 1, 4, and 8 by i.p. injection and HgCl₂ (40 µg) in PBS was given on days 1, 4, 8, and 11 by s.c. injection. Splenocytes were harvested 48 h after the last injection.

Flow cytometry

To assess T cell subsets and activation status, single-cell suspensions of splenocytes were stained with two sets of Abs (BD Pharmingen): FITC-anti-CD4, PerCP-anti-B220, and allophycocyanin-anti-CD3, or PE-anti-CD44, FITC-anti-CD4, PerCP-anti-CD8, and allophycocyanin-anti-CD62L. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). For detection of intracellular CTLA-4, lymphocytes were first stained with allophycocyanin-conjugated anti-CD25 and FITC-conjugated CD4 (BD Pharmingen), then fixed and permeabilized in 250 µl of Cytofix/Cytoperm solution (BD Pharmingen) for 20 min at 4°C, washed twice with Perm/Wash buffer (BD Pharmingen), stained with PE-conjugated anti-CTLA-4 for 2 h at 37°C in Perm/Wash buffer, and analyzed on a FACSCalibur. Cell surface CTLA-4 expression was determined using unfixed and unpermeabilized cells.

Statistics

Unless otherwise noted, all data are expressed as mean and SD. Groups were compared by Mann-Whitney U test or unpaired t test as appropriate using InStat by GraphPad Software (San Diego, CA). A value of p 0.05 was considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characteristics of genetically deficient mice

Mice used in this study were produced by mating H-2^s/s mice heterozygous for the gene of interest. Mice were genotyped by PCR as described in Materials and Methods, and all offspring appeared to be healthy. Subsequently, CD40L^–/–, CD28^–/–, and ICAM-1^–/– mice have been maintained by brother and sister mating in the vivarium of The Scripp’s Research Institute’s Department of Molecular and Experimental Medicine for the past 2 years. All three strains of genetically deficient mice are fertile and viable. Compared with their wild-type and heterozygous littermates, PBS-treated CD40L^–/– mice had reduced serum IgG (p < 0.005) and IgM (p = 0.002) levels (Table I). CD28^–/– mice had a lower mean level of IgG, but neither IgG nor IgM levels were significantly different from those of wild-type mice. ICAM-1^–/– mice had reduced IgM levels compared with wild-type littermates (p = 0.016). Thus, as expected, the lack of CD40L resulted in a greater humoral immunodeficiency than absence of the other costimulatory molecules.

Exposure of CD40L^–/– mice to HgCl₂ failed to elicit hypergammaglobulinemia or autoantibodies (Table I). Both wild-type (p = 0.0002) and heterozygous (p = 0.0003) littermates showed increased serum IgG, but IgM levels were unchanged by mercury treatment (Table I). Mercury increased IgG and IgM levels in wild-type mice compared with knockout mice (p = 0.0002). Mercury induced IgG ANA in six of eight wild-type mice and two of eight heterozygous mice. IgM ANA were found in two wild-type mice. Mercury-induced IgG ANoA autoantibodies were found in all wild-type (eight of eight) and in seven of eight heterozygous mice; however, the intensity of nucleolar staining was significantly different between the two groups (+/+ vs +/–, 1.81 ± 0.96 vs 0.81 ± 0.46, p = 0.02). Mercury exposure failed to induce IgM ANoA in any mice (Table I). With the exception of two PBS-treated heterozygous mice positive for IgG and IgM ANA, none of the PBS controls exhibited ANA or ANoA.

Compared with PBS-treated controls, IgG anti-chromatin Abs were significantly increased in HgCl₂- exposed wild-type (p = 0.005) but not heterozygous or knockout mice (Table I). IgG anti-chromatin Ab levels were elevated in mercury-exposed wild-type mice compared with heterozygous (p = 0.017) and knockout (p = 0.003) mice. Compared with PBS-treated controls, IgM anti-chromatin Abs were significantly increased in HgCl₂- exposed wild-type (p = 0.0002) and heterozygous (p = 0.015) but not knockout mice (Table I). IgM anti-chromatin Ab levels were elevated in mercury-exposed wild-type compared with knockout (p = 0.0006) mice. Although IgM anti-chromatin Ab levels were higher in mercury-exposed wild-type mice than in heterozygous mice (Table I), the difference was not statistically significant. Both IgG and IgM anti-chromatin Ab responses exhibited a gene dosage effect with regard to CD40L; compared with wild-type mice, heterozygous animals had a reduced response which was further depressed in homozygous knockout mice (Table I). IgM anti-ssDNA Abs were not elevated by mercury exposure (data not shown).

Pathological features of autoimmunity in mercury-exposed CD40L-deficient mice

Kidney immune deposits. Mercury exposure did not result in glomerular deposits of IgG or C3 in CD40L^–/– or CD40L^+/– mice (Table II). Mercury-exposed wild-type mice had deposits of IgG (p = 0.005) and C3 (p = 0.002) and such deposits were significantly greater than those of knockout (IgG, p = 0.02; C3, p = 0.0009) or heterozygous mice (IgG, p = 0.016; C3, p = 0.019). Immune deposits were not found in kidney vessels.

Humoral autoimmunity in mercury-exposed CD28-deficient mice

Exposure of CD28^–/– mice to HgCl₂ also failed to produce hypergammaglobulinemia or autoantibodies (Table I). In wild-type mice, HgCl₂ exposure led to increases in serum IgG (p = 0.0002) and IgM (p = 0.03), while only IgG was increased (p = 0.015) in heterozygous mice. Mercury increased IgG levels in wild-type mice compared with heterozygous (p = 0.005) and knockout mice (p = 0.0002). Mercury increased IgM levels in wild-type mice compared with heterozygous (p = 0.016) and knockout mice (p = 0.027). HgCl₂ induced IgG ANA in seven of eight wild-type mice, and IgM ANA in six of eight, with four mice positive for both IgG and IgM ANA. Six mice were positive for IgG ANoA but none for IgM ANoA. HgCl₂ induced IgG ANA in 9 of 12 heterozygous mice and IgM ANA in 8 of 12, with 7 mice positive for both IgG and IgM ANA. Eight of 12 mice were positive for IgG ANoA but none for IgM ANoA. None of the HgCl₂-treated knockout mice were positive for ANA or ANoA. None of the PBS-treated controls were ANA or ANoA positive.

In wild-type mice, HgCl₂ exposure led to increases in serum IgG and IgM anti-chromatin Abs (p = 0.0002). The increase in IgG anti-chromatin Abs in mercury-treated heterozygous mice barely reached statistical significance (p = 0.05), but the increase in IgM anti-chromatin Abs was significant (p = 0.015). Mercury increased IgG (p = 0.0006) and IgM (p = 0.0002) anti-chromatin Abs in wild-type mice compared with those of knockouts. Unlike the CD40L littermate study, there was no gene dosage effect with CD28; both wild-type and heterozygous mice produced similar levels of autoantibodies. Mercury exposure resulted in an increase in IgM anti-ssDNA Abs in wild-type mice compared with PBS controls (p = 0.02) and this response was greater than in mercury-treated knockout mice (p = 0.002; data not shown).

Pathological features of autoimmunity in mercury-exposed CD28-deficient mice

Kidney immune deposits. Mercury exposure did not result in significant glomerular deposits of IgG or C3 in CD28^–/– mice (Table II). Mercury-exposed wild-type mice had deposits of IgG (p = 0.01) and C3 (p = 0.015) and such deposits were significantly greater than those in knockout mice (IgG, p = 0.01; C3, p = 0.007). Although mercury exposure did result in IgG deposits in heterozygous mice (p = 0.006), they were not as pronounced as those in wild-type mice (p = 0.04). Immune deposits in kidney vessels were not found.

Splenic immune deposits. Mercury-exposed CD28^–/– mice also failed to develop immune deposits in the spleen, unlike wild-type (IgG, p = 0.01; C3, p = 0.02) and heterozygous mice (IgG, p = 0.03; C3, p = 0.015; Table II). Compared with knockout mice, wild-type animals had significantly more IgG (p = 0.01) and C3 (p = 0.02) deposits.

Humoral autoimmunity in mercury-exposed ICAM-1-deficient mice

In contrast to CD40L- and CD28-deficient mice, exposure of ICAM-1^–/– mice to HgCl₂ led to expression of humoral autoimmunity, although IgG hypergammaglobulinemia was not found (Table I). HgCl₂ exposure led to increases in serum IgG for wild-type mice (p = 0.006) but not heterozygous or knockout mice. Mercury increased IgM in wild-type (p = 0.014) and knockout mice (p = 0.01). Mercury-increased IgG levels in wild-type mice were different from those of knockout (p = 0.01) but not heterozygous mice. Mercury increased IgM levels in wild-type mice compared with those of knockout mice (p = 0.04). HgCl₂ induced IgG ANA in two of eight wild-type mice and IgM ANA in five of eight, including the two IgG ANA-positive mice. All eight mice were positive for IgG ANoA, but none for IgM ANoA. HgCl₂ induced IgG ANA in five of eight heterozygous mice and IgM ANA in four of eight, with two mice positive for both IgG and IgM ANA. All eight mice were positive for IgG ANoA, but none for IgM ANoA. Three of the HgCl₂-treated knockout mice were positive for IgG ANA, two of which had IgM ANA. Seven mice were positive for IgG ANoA, but none had IgM ANoA. PBS treatment did not induce ANA or ANoA.

HgCl₂ exposure led to increases in serum IgG anti-chromatin Abs in wild-type (p = 0.0002), heterozygous mice (p = 0.0002), and knockout mice (p = 0.0006). HgCl₂-induced IgM anti-chromatin Abs were increased in wild-type (p = 0.002), heterozygous (p = 0.0002), and knockout (p = 0.0006) mice. Mercury did not increase IgG or IgM anti-chromatin Abs in wild-type mice compared with heterozygous or knockout mice. Mercury exposure resulted in an increase in IgM anti-ssDNA Abs in wild-type mice (p = 0.005), heterozygous mice (p = 0.02), and knockout mice (p = 0.03) when compared with PBS controls. Levels of HgCl₂-induced IgM anti-ssDNA Abs were not different between the groups (data not shown).

Pathological features of autoimmunity in mercury-exposed ICAM-1-deficient mice

Kidney immune deposits. Mercury exposure did result in significant glomerular deposits of IgG in ICAM-1^–/–(p = 0.02), ICAM-1^+/– (p = 0.0005), and wild-type (0.002) mice (Table II). Complement deposits were also increased by mercury exposure in ICAM-1^+/– mice (p = 0.01) and ICAM-1^–/– mice (p = 0.01). Glomerular deposits were not significantly different among the three groups. Significant immune deposits in kidney vessels were not found in this group of mice.

Splenic immune deposits. Mercury-exposed ICAM-1^–/– mice had splenic deposits of IgG (p = 0.025), but the level of C3 deposits failed to reach statistical significance (p > 0.05; Table II). Both wild-type (IgG, p < 0.0001; C3, p = 0.01) and heterozygous (IgG, p = 0.0002; C3, p = 0.01) mice had mercury-induced immune deposits. The level of mercury-induced IgG (p = 0.013) but not C3 deposits was different between wild-type and ICAM-1^–/– mice.

Costimulation, T cell activation, and CTLA-4 expression

To examine the effect of CD28 or CD40L deficiency on in vivo T cell activation, mice were exposed to HgCl₂ for 2 wk (four injections) and then 2 days later splenocytes were analyzed for lymphocyte subsets. Relative percentages of CD3⁺, CD4⁺, CD8⁺, and B220⁺ lymphocytes were unchanged by HgCl₂ exposure (Table III). In contrast to mHgIA-prone wild-type (p = 0.015) and ICAM-1^–/– (p = 0.03) mice, CD40L^–/– and CD28^–/– mice showed no increase in activated T cells as judged by CD4⁺CD44^high T cells (Table IV). Both the wild-type (p = 0.01) and ICAM-1^–/– (p = 0.01) mice showed increased numbers of CD4⁺ T cells with intracellular CTLA-4 following exposure to HgCl₂, indicating T cell activation via CD28. Failure of T cell activation in CD28^–/– mice was confirmed by the absence of increased intracellular CTLA-4. CD40L^–/– mice also failed to show an increase in intracellular CTLA-4-positive CD4⁺ T cells, suggesting that T cell activation in these mice fails in part because of inefficient signaling via CD28 and/or a requirement of CD40L expression for intracellular CTLA-4 expression. Levels of CD25⁺, CTLA-4⁺CD4⁺ T cells did not change with the presence or absence of mHgIA.

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Intracellular and surface expression of CTLA-4 on CD4+ T cells in mHgIA. Wild-type, CD40L–/–, and CD28–/– mice were exposed to HgCl2 for 2 wk (four injections of 40 µg HgCl2) and then 2 days later splenocytes were harvested and analyzed for intracellular () and surface () CTLA-4 expression by flow cytometry. Results are expressed as mean ± 1 SEM for four mice in each group.

In vivo, anti-CD28 Ab can act as an agonist in CD40L^–/– mice to enhance Th2-mediated humoral immunity (5). To determine whether such treatment might enhance HgCl₂-induced T cell activation, wild-type and CD40L^–/– mice were treated with mercury and either anti-CD28 Ab or anti-KLH Ab as isotype control. Such treatment increased the numbers of CD4⁺ T cells expressing intracellular CTLA-4 in wild-type (p < 0.03) mice confirming that signaling via CD28 up-regulates CTLA-4 expression (Fig. 2). However anti-CD28 Ab did not result in increased CTLA-4 expression in CD40L^–/– mice, arguing that the presence of CD40L contributes to the expression of intracellular CTLA-4, at least within the context of induction of mHgIA (Fig. 2). CD44 expression of CD4⁺ T cells from wild-type and CD40L^–/– mice was unaffected by anti-CD28 Ab, suggesting that CD44 expression is not regulated by CD28 signaling.

View larger version (10K): [in this window] [in a new window]   FIGURE 2. Anti-CD28 Ab does not enhance HgCl2-induced T cell activation. Wild-type (left) and CD40L–/– (right) mice received anti-CD28 Ab (75 µg, clone 37.51; ) or anti-KLH Ab (75 µg; ) of the same isotype on days 1, 4, and 8 by i.p. injection and HgCl2 (40 µg) in PBS on days 1, 4, 8, and 11 by s.c. injection. Single-cell suspensions were examined by flow cytometry for expression of CD4, CD44, CD25, and intracellular CTLA-4. Results are expressed as mean ± 1 SEM for four mice in each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Mercury exposure increases expression of cell surface markers of T cell activation and proliferation including CD25 (IL-2R), CD71 (34), and CD44 (21), as well as MHC class II on APC (35). Mercury exposure can also perturb signal transduction in T (31, 32, 33) and B (36, 37) cells. In vitro T cell proliferation induced by mercury appears to be Ag driven because it requires an adherent cell population and IL-1 (24) and is inhibited by anti-MHC class II Abs (38). Unlike mitogen stimulation, only a proportion of T cells proliferate in response to HgCl₂ (24). The requirement for T cells and APC suggests an important role for costimulatory molecules in mHgIA, which is supported by studies showing that disease expression can be inhibited by anti-CD40L Abs and CTLA-4-Ig (22). The role of B7 molecules in mHgIA has been examined using anti-B7-1 and anti-B7-2 Abs. Treatment with both Abs combined suppressed both short- and long-term mercury-induced hypergammaglobulinemia and ANoA (23). If mercury exposure was of brief duration anti-B7-1 Abs inhibited, while anti-B7-2 delayed ANoA production; however, these effects could be overcome by prolonged mercury exposure (23). It is unclear why individual B7 molecules differentially influence development of ANoA in mHgIA. Subtly different effects of B7 molecules on development of idiopathic autoimmunity (11, 12) and induced autoimmunity (39) have been reported, suggesting that partial suppression of CD28-B7 interaction can influence multiple facets of autoimmunity.

Absence of CD28 resulted in complete lack of all features of mHgIA. In contrast, lack of CD28 does not lead to a total absence of disease in lupus-prone MRL-Fas^lpr mice (10). In experimentally induced experimental autoimmune encephalomyelitis, absence of CD28 does not impede disease (40, 41), suggesting that autoimmunity can arise by a CD28-independent mechanism. CD28 dependence can be overcome by repeated autoantigen stimulation (40), supporting the notion that a strong or sustained antigenic challenge overcomes the requirement of T cell activation and expansion via CD28/B7 (2). The failure to induce mHgIA in CD28-deficient mice suggests that repeated exposure to HgCl₂, used to induce mHgIA does not lead to costimulation signals strong enough to overcome absence of CD28.

CD40L also was found to play a significant role in the expression of mHgIA with complete inhibition of disease in homozygous knockout mice and partial inhibition in heterozygous mice. Whether CD40L heterozygosity reduces expression of idiopathic autoimmunity has not been examined, but appears unlikely since MRL-Fas^lpr mice deficient in CD40L still exhibit some features of autoimmunity, including autoantibodies to ribonucleoproteins (14, 15). Reduced expression of CD40L has also been associated with suppression of arthritis in IL-1^–/– mice (42). However, effects on autoantibody expression were mixed with those induced by immunization of type II collagen in a powerful adjuvant, CFA, being unaffected while those in the spontaneous human T cell leukemia virus 1-transgenic model were significantly reduced (42). Defective CD40L expression has also been observed following neonatal Ag exposure (43). The resulting anergy was overcome by IL-12 or IFN- and required expression of CD25 (43). Defective CD40L expression in anergic T cells has been known for some time (44). Anergic T cells cannot be activated by CD40-deficient B cells, showing that CD40-CD40L interaction is important for productive T cell responses including prolonged expression of CD25 and CD69 (45). These studies suggest that the lack of expression of mHgIA in CD40L^–/– and CD40L^+/– mice stems from a failure of mercury exposure to elicit sufficiently strong T cell activation signals.

Induction of mHgIA in wild-type and ICAM-1^–/– mice was associated with increased numbers of CD4⁺ T cells containing increased levels of CTLA-4. It is possible that the inability to detect increased CTLA-4 expression in CD40L^–/– and CD28^–/– mice reflects differences in kinetics of expression compared with wild-type mice. However, the absence of other markers of T cell activation in these mice argues against this. Although CTLA-4 expression accompanies T cell activation (46), CLTA-4 has been shown to be a negative regulator of T cell function (2), including T cell proliferation to endogenous self-Ag (47). Although blocking of CD28/B7 with CTLA-4-Ig, or in B7-1/B7-2 knockouts, has been used to inhibit autoimmunity, blockade of CTLA-4 by anti-CTLA-4 allows induction of mHgIA (48) and experimental autoimmune encephalomyelitis (49), and low level expression of CTLA-4 has been argued to contribute to insulin-dependent diabetes mellitus in NOD mice (50). CTLA-4 is therefore thought to control peripheral tolerance and to suppress productive responses of autoreactive T cells. Although increased intracellular CTLA-4 expression in mHgIA is consistent with T cell activation, we were unable to demonstrate a concomitant increase in cell surface expression as expected from a productive T response (51).

Apart from the requirement for costimulation, our experiments do not reveal the mechanism of CTLA-4 expression in mHgIA. The lack of CTLA-4 expression in the absence of either CD28 or CD40L suggests that TCR/MHC signaling in mHgIA is of low affinity, requiring the full complement of costimulation to achieve T cell activation. An examination of the effect of mercury exposure on T cell activation and CTLA-4 expression following stimulation using ligands of differing affinity for the TCR should help address this possibility.

Costimulation requirements in mHgIA are quite specific and cannot be overcome by exogenous stimuli such as anti-CD28 Ab. Although a number of reagents have been used to examine costimulation requirements, agonistic anti-CD28 Abs were used in this study to specifically address the relationship among direct CD28 signaling, CTLA-4 expression, and the presence and absence of CD40L. Augmenting CD28 signaling via the use of agonistic anti-CD28 induces CD40L expression and splenomegaly in BALB/c mice (3), Th2-dependent humoral responses in CD40L^–/– mice (5), and T cell proliferation and CD25 expression in response to peptide presented by CD40^–/– B cells (45). Thus, aggressive signaling via CD28 can render CD40L dispensable not only for T cell activation but also for B cell responses. This appears not to be the case for mHgIA since anti-CD28 Ab was unable to influence CTLA-4 expression in CD40L^–/– mice, even though it increased expression in wild-type mice. Anti-CD28 enhancement of T cell function depends upon the affinity of MHC/TCR for antigenic peptide because supplementing weak agonist peptides with anti-CD28 Ab enhances some of the features of T cell activation, but not others (52). The failure of anti-CD28 Ab to augment HgCl₂-induced T cell activation in CD40L^–/– mice may therefore be due to an inability to augment low-affinity peptide-MHC-TCR interactions. In view of the studies described above and the results of this study, it seems reasonable to hypothesize that mHgIA involves modulation of the activation threshold of low-affinity self-peptide-MHC-TCR interactions resulting in T cell activation via signaling through both CD28 and CD40L. Development of a model of mercury-induced T cell activation using transgenic T cells is being pursued to address the contributions of T cell affinity, costimulatory molecule expression, and cytokine production in T cell activation in xenobiotic-induced autoimmunity.

	Acknowledgments

 
The technical assistance of Deborah Pearson, Jessica Ceisla and Mackenzie Dismore is gratefully appreciated.

	Footnotes

 
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 supported by National Institutes of Health Grants ES07511, ES08080, ES09802, and ES08666 and is publication 16569-MEM from the Department of Molecular and Experimental Medicine.

² Address correspondence and reprint requests to Dr. K. Michael Pollard, Department of Molecular and Experimental Medicine, MEM131, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92017. E-mail address: mpollard{at}scripps.edu

³ Abbreviations used in this paper: ANA, anti-nuclear Ab; mHgIA, murine HgCl₂-induced autoimmunity; CD40L, CD40 ligand; ANoA, anti-nucleolar autoantibody.

Received for publication April 29, 2004. Accepted for publication August 30, 2004.

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