ICOS-B7 Homologous Protein Interactions Are Necessary for Mercury-Induced Autoimmunity
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免疫学杂志 2005年第5期
Abstract
After exposure to subtoxic doses of heavy metals such as mercury, H-2s mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of one of the members of the CD28-B7 costimulation families, ICOS-B7 homologous protein (B7h), in the regulation of mercury-induced autoimmunity. The expression of ICOS on T cells was more enhanced in susceptible A.SW mice than in non-responsive C57BL/6 and DBA/2 mice after HgCl2 treatment. Furthermore, in A.SW mice treated with HgCl2, administration of a blocking anti-ICOS Ab effectively inhibited anti-nucleolar autoantibodies and total serum IgE production. Taken together, these results indicate that the ICOS-B7h costimulation pathway is required for this autoimmune syndrome and suggest that targeting this pathway might have therapeutic benefits for human autoimmune diseases.
Introduction
Mouse or rat strains expressing certain MHC Ags are susceptible to the heavy metal induction of a complex autoimmune syndrome (1, 2, 3). In susceptible H-2s mice, subtoxic doses of HgCl2 induce an autoimmune dysfunction characterized by the production of anti-nucleolar Abs (ANoA),3 lymphoproliferation, and hyperglobulinemia (especially pronounced for IgG1 and IgE). The increase in serum Ig peaks 2–3 wk after the beginning of the HgCl2 injections, whereas ANoA can persist for several months after the induction phase.
A primary costimulation signal is delivered through the CD28:B7-1(CD80)/B7-2(CD86) interactions during the initial immune response (4). Engagement of CTLA-4 (CD152) by the same B7-1/B7-2 ligands results in attenuation of T cells responses (5). The CD28/CTLA-4:B7-1/B7-2 pathways are crucial for HgCl2-induced autoimmunity in mice (6, 7). In addition to these pathways, other B7 family ligand-receptor pairs including ICOS-B7 homologous protein (B7h) (8, 9), PD-1:PD-L1/L2 (10, 11, 12) are involved in cognate interactions and may be critical for the pathogenesis of systemic autoimmune diseases.
ICOS is a costimulatory receptor homologous to CD28 and CTLA-4 (13). The ICOS gene is closely linked to the genes for CD28 and CTLA-4 on human chromosome 2q33 and mouse chromosome 1 (14, 15). Expression of ICOS occurs on activated, but not resting, T cells (13) and is dependent upon TCR and CD28 signals (16). However, ICOS expression is not absolutely dependent upon CD28 signals because activated human CD8+ T cells that do not express CD28 can express ICOS (13), and some T cell responses in CD28-deficient mice can be modulated with ICOS-Ig (17), suggesting a distinct costimulatory effect from CD28. The ligand for ICOS, B7h (18), also known as B7-related protein-1 (B7RP-1) (19) or GL50 (20), is constitutively expressed on unstimulated B cells and is inducible on macrophages and peripheral blood-derived dendritic cells (18, 20, 21). ICOS-deficient mice show defective T cell activation and proliferation and greatly enhanced susceptibility to experimental autoimmune encephalomyelitis (22). A lack of ICOS in these mice also results in severely defective T cell-dependent B cell responses, germinal center formation and Ig class switching, including IgE (23, 24). However, class switching can be restored in ICOS-deficient mice by CD40 stimulation, indicating that ICOS promotes T-B cells collaboration through the CD40/CD40L (CD154) pathway (23).
We have investigated the role of ICOS in regulating HgCl2-induced autoimmunity by using a blocking anti-ICOS mAb. Our data show that ICOS expression on effector T cells is up-regulated in susceptible mice after HgCl2 treatment. The blockade of the ICOS-B7h pathway significantly inhibits autoantibody and serum total IgE production. Taken together, our studies indicate that ICOS may represent a therapeutic target during systemic autoimmune diseases.
Materials and Methods
Flow cytometry
Splenocytes isolated from HgCl2- or PBS-treated mice were stained with PE-labeled anti-mouse ICOS (7E.17G9), FITC-labeled anti-mouse CD4 (L3T4, GK1.5), and FITC-labeled anti-mouse CD8a (Ly-2, 53-6.7). All conjugated Abs and control Abs were obtained from BD Biosciences. Flow cytometry was performed using a FACSort flow cytometer (BD Biosciences). The software used for data acquisition is Lysis II (BD Biosciences), and the software used for analysis is WinMDI version 2.8 (J. Trotter, The Scripps Research Institute, La Jolla, CA, available online from http://facs.scripps.edu/software.html).
HgCl2 and Ab treatment
The blocking rat anti-ICOS mAb (12A8) was generated as previously described (25). An irrelevant rat IgG2b mAb was used as control Ab. Mercury-induced autoimmunity was induced in groups of five mice according to a standard protocol by three s.c. injections at days 0, 2, and 4 (each injection consisted of 30 μg of HgCl2 in 100 μl of sterile PBS) (26). In addition to HgCl2, some groups of mice received anti-ICOS or control Abs. Anti-ICOS Ab injections in protocol 1 and protocol 2 were identical to those used by previous investigators (27) and are described in Fig. 1. In all cases, Abs were administered i.p. and mice received 100 μg of each Ab in 500 μl of sterile PBS per injection.
FIGURE 1. Time course of HgCl2 and anti-ICOS Ab injection protocols. In both protocol 1 and 2, mice received s.c. injections of 30 μg of HgCl2 in 100 μl of sterile PBS three times during the first week. In protocol 1, eight injections of anti-ICOS mAb or control rat IgG2b Ab were administered daily from day 0 to day 7. In protocol 2, Abs were administered daily from day 7 to day 14. In all cases, Abs were administered i.p., and mice received 100 μg of each Ab in 500 μl of sterile PBS per injection.
ANoA immunofluorescence
Serum ANoA IgG1 and IgG2a (the main isotypes produced during HgCl2-induced autoimmunity) levels were determined by indirect immunofluorescence, as described previously (26). Sera diluted in PBN (PBS containing 1% BSA and 0.02% sodium azide) were incubated with HEp-2 slides (Antibodies, Inc.) for 30 min, and ANoA were detected with FITC-conjugated goat anti-mouse IgG1 or IgG2a Abs (Southern Biotechnology Associates). The inverse of the highest serum dilution at which nucleolar fluorescence could be detected was defined as the ANoA titer.
ELISA for mouse serum IgG1 and IgE
Total serum IgG1 and IgE levels were determined using a sandwich ELISA as previously described (26).
Statistical methods
Using SAS version 9.0 (SAS Institute, Cary, NC), the dependent variables were treated as continuous variables for all analyses. Mean values, SD values, and number of observations are presented. The experimental unit was each individual mouse.
The experiment used a repeated measures design with each mouse evaluated at five periods. The null hypothesis was that there would be no difference between group and week. Before analysis, all data were tested for normality using the Shapiro-Wilk test. The data were significantly non-normal for all variables. To apply ANOVA methods, a "normalized-rank" transformation was applied to the data. The rank-transformed data was analyzed using a mixed-model ANOVA for repeated measures followed by multiple comparisons to detect significant mean differences between groups at each week. Multiple pair-wise comparisons used the Bonferroni adjustment to maintain an experiment-wise type I error of 0.05 or less. Differences between means (rejection of the null hypothesis) were considered significant if the probability of chance occurrence was 0.05 using two-tailed test. Adjusted p values are presented for each analysis.
Results
Expression of ICOS on effector T cells from HgCl2-treated susceptible A.SW mice
To investigate the effect of HgCl2 treatment on the expression of ICOS, two groups of A.SW mice were treated with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-labeled anti-ICOS, FITC-labeled anti-CD4, and anti-CD8 mAb or with the appropriate fluorochrome-conjugated control Abs. Staining was analyzed by flow cytometry. An electronic gate was set on CD4+ and CD8+ T cells, and the expression of ICOS is shown in Fig. 2. Susceptible A.SW mice receiving 1 wk of HgCl2 treatment showed a 2.9-fold increase in ICOS expression on the surface of activated CD4+ T cells when compared with control mice treated with PBS alone (Fig. 2A). We also observed that CD8+ T cells exhibited low ICOS expression and that there was no up-regulation of ICOS on CD8+ T cells upon HgCl2 administration (Fig. 2B). In addition, there was virtually no ICOS expression and no up-regulation of ICOS on non-CD3+ T cells (data not shown).
FIGURE 2. Expression of ICOS on splenocytes in HgCl2-treated susceptible A.SW mice. Mice were treated either with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-conjugated anti-ICOS, FITC-conjugated anti-CD4 or anti-CD8 mAb, or with appropriate fluorochrome-conjugated control Ig. An electronic gate was set on CD4+ or CD8+ T cells, and the expression of ICOS on T cells is represented by a dotted line with the control Ig staining as a solid line.
Expression of ICOS on effector T cells from HgCl2-treated non-responsive C57BL/6 and DBA/2 mice
To further examine the effect of HgCl2 treatment on ICOS expression, two additional mouse strains were tested. The first one is C57BL/6 mice, which does not produce ANoA but displays a mild increase in serum IgG1 and IgE (lower than the susceptible A.SW strain). The second one is the completely resistant DBA/2 strain, which produces neither autoantibodies nor polyclonal activation after mercury treatment. Mice from these strains were treated with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-labeled anti-ICOS, FITC-labeled anti-CD4, or with the appropriate fluorochrome-conjugated control Abs. Staining was analyzed by flow cytometry. An electronic gate was set on CD4+ T cells, and the expression of ICOS is shown in Fig. 3. These strains displayed a higher ICOS baseline than A.SW mice, but ICOS expression did not increase as much as in A.SW mice after HgCl2 treatment. As shown in Fig. 3A, C57BL/6 displayed only a moderate increase, 1.8-fold vs 2.9-fold in A.SW mice (Fig. 2A). The mercury-resistant strain DBA/2 displayed a fractional increase (around 1.2-fold) in ICOS expression after HgCl2 injection (Fig. 3B). As in A.SW mice, we also observed weak ICOS expression and no up-regulation on non-CD4+ T cells (data not shown). These results led us to further investigate the involvement of ICOS-B7h pathway in HgCl2-induced autoimmunity in mice, using a blocking anti-ICOS mAb.
FIGURE 3. Expression of ICOS on splenocytes in HgCl2-treated non-responsive C57BL/6 and DBA/2 mice. Mice were treated either with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-conjugated anti-ICOS, FITC-conjugated anti-CD4, or with appropriate fluorochrome-conjugated control Ig. An electronic gate was set on CD4+ T cells, and the expression of ICOS on T cells is represented by a dotted line with the control Ig staining as a solid line.
Inhibition of ANoA and serum IgE production by anti-ICOS mAb treatment during disease induction
To assess the roles of ICOS/B7h interaction in HgCl2-induced autoimmunity, we initially conducted a series of experiments (Fig. 1, protocol 1), in which groups of A.SW mice received HgCl2 injection three times at days 0, 2, and 4. During the same period, anti-ICOS mAb or control rat IgG2b were administered at 100 μg/day from days 0 to 7 (see details in Fig. 1). The anti-ICOS mAb treatment significantly inhibited the ANoA-IgG1 (p < 0.001) and ANoA-IgG2a (p < 0.001) production for several weeks (Fig. 4), when compared with the group of mice receiving isotype-matched control Abs. In addition to the ANoA production, the increase of certain serum Ig isotype levels, especially IgG1 and IgE, is another important hallmark of HgCl2-induced autoimmunity. Mice receiving HgCl2 and anti-ICOS mAbs developed temporarily lower (p < 0.01) serum total IgE levels than mice receiving HgCl2 plus isotype-matched control Abs, although both groups later developed a similar peak level of IgE. The total serum IgG1 and IgG2a levels were not significantly different between these two groups (Fig. 4).
FIGURE 4. Blockade of ICOS-B7h pathway during the induction of the disease inhibits ANoA production and reduces serum IgE levels in A.SW mice. Groups of five A.SW mice received HgCl2 and Ab injections as described in protocol 1 (Fig. 1). ANoA were detected by immunofluorescence on HEp-2 cells using isotype-specific FITC conjugates as described in Materials and Methods. Results are expressed as serum titers (inverse of the highest serum dilution that yielded nucleolar fluorescence) ± SD. Serum Ig levels were measured by ELISA as described in Materials and Methods and are expressed in milligrams per milliliter ± SD (IgG1 and IgG2a) or micrograms per milliliter ± SD (IgE). **, p < 0.01; ***, p < 0.001.
Inhibition of ANoA and serum IgE production by anti-ICOS mAb treatment after disease induction
Because ICOS expression occurs later in the course of an immune response, we postponed the Ab administration until after the HgCl2 treatment. In protocol 2 (Fig. 1), groups of A.SW mice received HgCl2 injection three times at days 0, 2, and 4 in the first week. During the following week, anti-ICOS mAbs or control rat IgG2b were administered at 100 μg/day for the entire week (see details in Fig. 1). As was the case with protocol 1, the administration of anti-ICOS mAbs significantly inhibited the ANoA-IgG1 (p < 0.001) and ANoA-IgG2a (p < 0.01) production for several weeks, compared with the control group receiving rat IgG2b Abs (Fig. 5). At the same time, these mice receiving anti-ICOS mAbs developed significantly lower (p < 0.001) peak levels of serum total IgE than mice treated with isotype-matched control Abs (Fig. 5). No significant differences were observed between these two groups in terms of total serum IgG1 and IgG2a (Fig. 5). These observations are consistent with previous findings showing ICOS–/– mice have defective isotype class-switching to IgE (23, 24).
FIGURE 5. Blockade of ICOS-B7h pathway after the induction of the disease inhibits ANoA production and reduces serum IgE levels in A.SW mice. Groups of five A.SW mice received HgCl2 and Ab injections as described in protocol 2 (Fig. 1). ANoA were detected by immunofluorescence on HEp-2 cells using isotype-specific FITC conjugates as described in Materials and Methods. Results are expressed as serum titers (inverse of the highest serum dilution that yielded nucleolar fluorescence) ± SD. Serum Ig levels were measured by ELISA as described in Materials and Methods and are expressed in milligrams per milliliter ± SD (IgG1 and IgG2a) or micrograms per milliliter ± SD (IgE). **, p < 0.01; ***, p < 0.001.
Discussion
The discovery of the ICOS-B7h costimulatory pathway has focused attention on its functional role and its relationship with the CD28/CTLA-4:B7-1/B7-2 pathway during T cell activation. The costimulatory function of both CD28 and ICOS raises the possibility that they may share overlapping signaling pathways. However, although both CD28 and ICOS signaling can costimulate T cell proliferation, ICOS signaling does not trigger IL-2 production, but is instead critical for the production of several other cytokines including IL-4, IFN-, TNF-, and IL-10 (13, 21). The cytoplasmic tail of ICOS lacks the PXXP site implicated in IL-2 production by CD28 engagement, which may account in part for the distinct functions of CD28 and ICOS (28).
Although several studies have shown a critical role for the CD28/CTLA-4:B7-1/B7-2 pathway in HgCl2-induced autoimmunity in mice (6, 7), the role of the ICOS-B7h pathway had not been explored in this model. In this study, we observed that, upon HgCl2 treatment, susceptible A.SW mice significantly up-regulate ICOS expression on effector CD4+ T cells, whereas the HgCl2-induced increase in ICOS expression was much lower in C57BL/6 mice and even absent in the resistant DBA/2 strain. CD28 is constitutively expressed on T cells and is critical for optimal expression of ICOS (16). In contrast, ICOS is only fully expressed on T cells after activation (13), which suggests a hierarchy of costimulation, with a primary role for CD28 in the initial phase of immune responses, and a significant involvement of ICOS in the effector phase of immune reactions.
Blockade of the ICOS-B7h pathway by an anti-ICOS mAb decreased some of the Hg-induced autoimmune manifestations, as indicated by the almost complete suppression of autoantibodies (ANoA) production and a significant reduction in IgE production. However, the serum IgG1 and IgG2a increases were not affected by this treatment. In contrast, the blockade of CD28-B7 pathway by using CTLA-4-Ig or a combination of anti-B7-1 and anti-B7-2 mAbs completely prevented all the manifestations in HgCl2-induced autoimmunity, including the polyclonal IgG increase (6, 7). These observations suggest that the major contributions of the CD28-B7 and ICOS-B7h costimulation pathways occur at different times during HgCl2-induced autoimmunity, implicating CD28 engagement during the initial stage and ICOS engagement during the effector stage of the disease. The observation that the total serum IgG1 and IgG2a levels were not significantly different between anti-ICOS-treated and control groups also supports the view that different regulatory mechanisms control the various manifestations of the syndrome.
In summary, our data along with recent reports on the collagen-induced arthritis and murine lupus nephritis models (29, 30) demonstrate that blocking ICOS-B7h pathway is effective at preventing autoimmune manifestations. Although blocking the ICOS-B7h pathway is effective against autoantibody production, the effect is not as drastic as the blockade of CD28-B7-1/2 interactions, resulting in a potentially less severe immunosuppression. Therefore, manipulation of this pathway represents a promising strategy for the immunotherapy of human autoimmune diseases.
Disclosures
The authors have no financial conflict of interest.
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 ES-09409 and ES-12464 to M.M.
2 Address correspondence and reprint requests to Dr. Marc Monestier, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail address: marcm{at}temple.edu
3 Abbreviations used in this paper: ANoA, anti-nucleolar Ab; B7h, B7 homologous protein.
Received for publication February 5, 2004. Accepted for publication December 23, 2004.
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After exposure to subtoxic doses of heavy metals such as mercury, H-2s mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of one of the members of the CD28-B7 costimulation families, ICOS-B7 homologous protein (B7h), in the regulation of mercury-induced autoimmunity. The expression of ICOS on T cells was more enhanced in susceptible A.SW mice than in non-responsive C57BL/6 and DBA/2 mice after HgCl2 treatment. Furthermore, in A.SW mice treated with HgCl2, administration of a blocking anti-ICOS Ab effectively inhibited anti-nucleolar autoantibodies and total serum IgE production. Taken together, these results indicate that the ICOS-B7h costimulation pathway is required for this autoimmune syndrome and suggest that targeting this pathway might have therapeutic benefits for human autoimmune diseases.
Introduction
Mouse or rat strains expressing certain MHC Ags are susceptible to the heavy metal induction of a complex autoimmune syndrome (1, 2, 3). In susceptible H-2s mice, subtoxic doses of HgCl2 induce an autoimmune dysfunction characterized by the production of anti-nucleolar Abs (ANoA),3 lymphoproliferation, and hyperglobulinemia (especially pronounced for IgG1 and IgE). The increase in serum Ig peaks 2–3 wk after the beginning of the HgCl2 injections, whereas ANoA can persist for several months after the induction phase.
A primary costimulation signal is delivered through the CD28:B7-1(CD80)/B7-2(CD86) interactions during the initial immune response (4). Engagement of CTLA-4 (CD152) by the same B7-1/B7-2 ligands results in attenuation of T cells responses (5). The CD28/CTLA-4:B7-1/B7-2 pathways are crucial for HgCl2-induced autoimmunity in mice (6, 7). In addition to these pathways, other B7 family ligand-receptor pairs including ICOS-B7 homologous protein (B7h) (8, 9), PD-1:PD-L1/L2 (10, 11, 12) are involved in cognate interactions and may be critical for the pathogenesis of systemic autoimmune diseases.
ICOS is a costimulatory receptor homologous to CD28 and CTLA-4 (13). The ICOS gene is closely linked to the genes for CD28 and CTLA-4 on human chromosome 2q33 and mouse chromosome 1 (14, 15). Expression of ICOS occurs on activated, but not resting, T cells (13) and is dependent upon TCR and CD28 signals (16). However, ICOS expression is not absolutely dependent upon CD28 signals because activated human CD8+ T cells that do not express CD28 can express ICOS (13), and some T cell responses in CD28-deficient mice can be modulated with ICOS-Ig (17), suggesting a distinct costimulatory effect from CD28. The ligand for ICOS, B7h (18), also known as B7-related protein-1 (B7RP-1) (19) or GL50 (20), is constitutively expressed on unstimulated B cells and is inducible on macrophages and peripheral blood-derived dendritic cells (18, 20, 21). ICOS-deficient mice show defective T cell activation and proliferation and greatly enhanced susceptibility to experimental autoimmune encephalomyelitis (22). A lack of ICOS in these mice also results in severely defective T cell-dependent B cell responses, germinal center formation and Ig class switching, including IgE (23, 24). However, class switching can be restored in ICOS-deficient mice by CD40 stimulation, indicating that ICOS promotes T-B cells collaboration through the CD40/CD40L (CD154) pathway (23).
We have investigated the role of ICOS in regulating HgCl2-induced autoimmunity by using a blocking anti-ICOS mAb. Our data show that ICOS expression on effector T cells is up-regulated in susceptible mice after HgCl2 treatment. The blockade of the ICOS-B7h pathway significantly inhibits autoantibody and serum total IgE production. Taken together, our studies indicate that ICOS may represent a therapeutic target during systemic autoimmune diseases.
Materials and Methods
Flow cytometry
Splenocytes isolated from HgCl2- or PBS-treated mice were stained with PE-labeled anti-mouse ICOS (7E.17G9), FITC-labeled anti-mouse CD4 (L3T4, GK1.5), and FITC-labeled anti-mouse CD8a (Ly-2, 53-6.7). All conjugated Abs and control Abs were obtained from BD Biosciences. Flow cytometry was performed using a FACSort flow cytometer (BD Biosciences). The software used for data acquisition is Lysis II (BD Biosciences), and the software used for analysis is WinMDI version 2.8 (J. Trotter, The Scripps Research Institute, La Jolla, CA, available online from http://facs.scripps.edu/software.html).
HgCl2 and Ab treatment
The blocking rat anti-ICOS mAb (12A8) was generated as previously described (25). An irrelevant rat IgG2b mAb was used as control Ab. Mercury-induced autoimmunity was induced in groups of five mice according to a standard protocol by three s.c. injections at days 0, 2, and 4 (each injection consisted of 30 μg of HgCl2 in 100 μl of sterile PBS) (26). In addition to HgCl2, some groups of mice received anti-ICOS or control Abs. Anti-ICOS Ab injections in protocol 1 and protocol 2 were identical to those used by previous investigators (27) and are described in Fig. 1. In all cases, Abs were administered i.p. and mice received 100 μg of each Ab in 500 μl of sterile PBS per injection.
FIGURE 1. Time course of HgCl2 and anti-ICOS Ab injection protocols. In both protocol 1 and 2, mice received s.c. injections of 30 μg of HgCl2 in 100 μl of sterile PBS three times during the first week. In protocol 1, eight injections of anti-ICOS mAb or control rat IgG2b Ab were administered daily from day 0 to day 7. In protocol 2, Abs were administered daily from day 7 to day 14. In all cases, Abs were administered i.p., and mice received 100 μg of each Ab in 500 μl of sterile PBS per injection.
ANoA immunofluorescence
Serum ANoA IgG1 and IgG2a (the main isotypes produced during HgCl2-induced autoimmunity) levels were determined by indirect immunofluorescence, as described previously (26). Sera diluted in PBN (PBS containing 1% BSA and 0.02% sodium azide) were incubated with HEp-2 slides (Antibodies, Inc.) for 30 min, and ANoA were detected with FITC-conjugated goat anti-mouse IgG1 or IgG2a Abs (Southern Biotechnology Associates). The inverse of the highest serum dilution at which nucleolar fluorescence could be detected was defined as the ANoA titer.
ELISA for mouse serum IgG1 and IgE
Total serum IgG1 and IgE levels were determined using a sandwich ELISA as previously described (26).
Statistical methods
Using SAS version 9.0 (SAS Institute, Cary, NC), the dependent variables were treated as continuous variables for all analyses. Mean values, SD values, and number of observations are presented. The experimental unit was each individual mouse.
The experiment used a repeated measures design with each mouse evaluated at five periods. The null hypothesis was that there would be no difference between group and week. Before analysis, all data were tested for normality using the Shapiro-Wilk test. The data were significantly non-normal for all variables. To apply ANOVA methods, a "normalized-rank" transformation was applied to the data. The rank-transformed data was analyzed using a mixed-model ANOVA for repeated measures followed by multiple comparisons to detect significant mean differences between groups at each week. Multiple pair-wise comparisons used the Bonferroni adjustment to maintain an experiment-wise type I error of 0.05 or less. Differences between means (rejection of the null hypothesis) were considered significant if the probability of chance occurrence was 0.05 using two-tailed test. Adjusted p values are presented for each analysis.
Results
Expression of ICOS on effector T cells from HgCl2-treated susceptible A.SW mice
To investigate the effect of HgCl2 treatment on the expression of ICOS, two groups of A.SW mice were treated with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-labeled anti-ICOS, FITC-labeled anti-CD4, and anti-CD8 mAb or with the appropriate fluorochrome-conjugated control Abs. Staining was analyzed by flow cytometry. An electronic gate was set on CD4+ and CD8+ T cells, and the expression of ICOS is shown in Fig. 2. Susceptible A.SW mice receiving 1 wk of HgCl2 treatment showed a 2.9-fold increase in ICOS expression on the surface of activated CD4+ T cells when compared with control mice treated with PBS alone (Fig. 2A). We also observed that CD8+ T cells exhibited low ICOS expression and that there was no up-regulation of ICOS on CD8+ T cells upon HgCl2 administration (Fig. 2B). In addition, there was virtually no ICOS expression and no up-regulation of ICOS on non-CD3+ T cells (data not shown).
FIGURE 2. Expression of ICOS on splenocytes in HgCl2-treated susceptible A.SW mice. Mice were treated either with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-conjugated anti-ICOS, FITC-conjugated anti-CD4 or anti-CD8 mAb, or with appropriate fluorochrome-conjugated control Ig. An electronic gate was set on CD4+ or CD8+ T cells, and the expression of ICOS on T cells is represented by a dotted line with the control Ig staining as a solid line.
Expression of ICOS on effector T cells from HgCl2-treated non-responsive C57BL/6 and DBA/2 mice
To further examine the effect of HgCl2 treatment on ICOS expression, two additional mouse strains were tested. The first one is C57BL/6 mice, which does not produce ANoA but displays a mild increase in serum IgG1 and IgE (lower than the susceptible A.SW strain). The second one is the completely resistant DBA/2 strain, which produces neither autoantibodies nor polyclonal activation after mercury treatment. Mice from these strains were treated with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-labeled anti-ICOS, FITC-labeled anti-CD4, or with the appropriate fluorochrome-conjugated control Abs. Staining was analyzed by flow cytometry. An electronic gate was set on CD4+ T cells, and the expression of ICOS is shown in Fig. 3. These strains displayed a higher ICOS baseline than A.SW mice, but ICOS expression did not increase as much as in A.SW mice after HgCl2 treatment. As shown in Fig. 3A, C57BL/6 displayed only a moderate increase, 1.8-fold vs 2.9-fold in A.SW mice (Fig. 2A). The mercury-resistant strain DBA/2 displayed a fractional increase (around 1.2-fold) in ICOS expression after HgCl2 injection (Fig. 3B). As in A.SW mice, we also observed weak ICOS expression and no up-regulation on non-CD4+ T cells (data not shown). These results led us to further investigate the involvement of ICOS-B7h pathway in HgCl2-induced autoimmunity in mice, using a blocking anti-ICOS mAb.
FIGURE 3. Expression of ICOS on splenocytes in HgCl2-treated non-responsive C57BL/6 and DBA/2 mice. Mice were treated either with PBS or HgCl2 three times at days 0, 2, and 4. At day 7, splenocytes from these mice were stained with PE-conjugated anti-ICOS, FITC-conjugated anti-CD4, or with appropriate fluorochrome-conjugated control Ig. An electronic gate was set on CD4+ T cells, and the expression of ICOS on T cells is represented by a dotted line with the control Ig staining as a solid line.
Inhibition of ANoA and serum IgE production by anti-ICOS mAb treatment during disease induction
To assess the roles of ICOS/B7h interaction in HgCl2-induced autoimmunity, we initially conducted a series of experiments (Fig. 1, protocol 1), in which groups of A.SW mice received HgCl2 injection three times at days 0, 2, and 4. During the same period, anti-ICOS mAb or control rat IgG2b were administered at 100 μg/day from days 0 to 7 (see details in Fig. 1). The anti-ICOS mAb treatment significantly inhibited the ANoA-IgG1 (p < 0.001) and ANoA-IgG2a (p < 0.001) production for several weeks (Fig. 4), when compared with the group of mice receiving isotype-matched control Abs. In addition to the ANoA production, the increase of certain serum Ig isotype levels, especially IgG1 and IgE, is another important hallmark of HgCl2-induced autoimmunity. Mice receiving HgCl2 and anti-ICOS mAbs developed temporarily lower (p < 0.01) serum total IgE levels than mice receiving HgCl2 plus isotype-matched control Abs, although both groups later developed a similar peak level of IgE. The total serum IgG1 and IgG2a levels were not significantly different between these two groups (Fig. 4).
FIGURE 4. Blockade of ICOS-B7h pathway during the induction of the disease inhibits ANoA production and reduces serum IgE levels in A.SW mice. Groups of five A.SW mice received HgCl2 and Ab injections as described in protocol 1 (Fig. 1). ANoA were detected by immunofluorescence on HEp-2 cells using isotype-specific FITC conjugates as described in Materials and Methods. Results are expressed as serum titers (inverse of the highest serum dilution that yielded nucleolar fluorescence) ± SD. Serum Ig levels were measured by ELISA as described in Materials and Methods and are expressed in milligrams per milliliter ± SD (IgG1 and IgG2a) or micrograms per milliliter ± SD (IgE). **, p < 0.01; ***, p < 0.001.
Inhibition of ANoA and serum IgE production by anti-ICOS mAb treatment after disease induction
Because ICOS expression occurs later in the course of an immune response, we postponed the Ab administration until after the HgCl2 treatment. In protocol 2 (Fig. 1), groups of A.SW mice received HgCl2 injection three times at days 0, 2, and 4 in the first week. During the following week, anti-ICOS mAbs or control rat IgG2b were administered at 100 μg/day for the entire week (see details in Fig. 1). As was the case with protocol 1, the administration of anti-ICOS mAbs significantly inhibited the ANoA-IgG1 (p < 0.001) and ANoA-IgG2a (p < 0.01) production for several weeks, compared with the control group receiving rat IgG2b Abs (Fig. 5). At the same time, these mice receiving anti-ICOS mAbs developed significantly lower (p < 0.001) peak levels of serum total IgE than mice treated with isotype-matched control Abs (Fig. 5). No significant differences were observed between these two groups in terms of total serum IgG1 and IgG2a (Fig. 5). These observations are consistent with previous findings showing ICOS–/– mice have defective isotype class-switching to IgE (23, 24).
FIGURE 5. Blockade of ICOS-B7h pathway after the induction of the disease inhibits ANoA production and reduces serum IgE levels in A.SW mice. Groups of five A.SW mice received HgCl2 and Ab injections as described in protocol 2 (Fig. 1). ANoA were detected by immunofluorescence on HEp-2 cells using isotype-specific FITC conjugates as described in Materials and Methods. Results are expressed as serum titers (inverse of the highest serum dilution that yielded nucleolar fluorescence) ± SD. Serum Ig levels were measured by ELISA as described in Materials and Methods and are expressed in milligrams per milliliter ± SD (IgG1 and IgG2a) or micrograms per milliliter ± SD (IgE). **, p < 0.01; ***, p < 0.001.
Discussion
The discovery of the ICOS-B7h costimulatory pathway has focused attention on its functional role and its relationship with the CD28/CTLA-4:B7-1/B7-2 pathway during T cell activation. The costimulatory function of both CD28 and ICOS raises the possibility that they may share overlapping signaling pathways. However, although both CD28 and ICOS signaling can costimulate T cell proliferation, ICOS signaling does not trigger IL-2 production, but is instead critical for the production of several other cytokines including IL-4, IFN-, TNF-, and IL-10 (13, 21). The cytoplasmic tail of ICOS lacks the PXXP site implicated in IL-2 production by CD28 engagement, which may account in part for the distinct functions of CD28 and ICOS (28).
Although several studies have shown a critical role for the CD28/CTLA-4:B7-1/B7-2 pathway in HgCl2-induced autoimmunity in mice (6, 7), the role of the ICOS-B7h pathway had not been explored in this model. In this study, we observed that, upon HgCl2 treatment, susceptible A.SW mice significantly up-regulate ICOS expression on effector CD4+ T cells, whereas the HgCl2-induced increase in ICOS expression was much lower in C57BL/6 mice and even absent in the resistant DBA/2 strain. CD28 is constitutively expressed on T cells and is critical for optimal expression of ICOS (16). In contrast, ICOS is only fully expressed on T cells after activation (13), which suggests a hierarchy of costimulation, with a primary role for CD28 in the initial phase of immune responses, and a significant involvement of ICOS in the effector phase of immune reactions.
Blockade of the ICOS-B7h pathway by an anti-ICOS mAb decreased some of the Hg-induced autoimmune manifestations, as indicated by the almost complete suppression of autoantibodies (ANoA) production and a significant reduction in IgE production. However, the serum IgG1 and IgG2a increases were not affected by this treatment. In contrast, the blockade of CD28-B7 pathway by using CTLA-4-Ig or a combination of anti-B7-1 and anti-B7-2 mAbs completely prevented all the manifestations in HgCl2-induced autoimmunity, including the polyclonal IgG increase (6, 7). These observations suggest that the major contributions of the CD28-B7 and ICOS-B7h costimulation pathways occur at different times during HgCl2-induced autoimmunity, implicating CD28 engagement during the initial stage and ICOS engagement during the effector stage of the disease. The observation that the total serum IgG1 and IgG2a levels were not significantly different between anti-ICOS-treated and control groups also supports the view that different regulatory mechanisms control the various manifestations of the syndrome.
In summary, our data along with recent reports on the collagen-induced arthritis and murine lupus nephritis models (29, 30) demonstrate that blocking ICOS-B7h pathway is effective at preventing autoimmune manifestations. Although blocking the ICOS-B7h pathway is effective against autoantibody production, the effect is not as drastic as the blockade of CD28-B7-1/2 interactions, resulting in a potentially less severe immunosuppression. Therefore, manipulation of this pathway represents a promising strategy for the immunotherapy of human autoimmune diseases.
Disclosures
The authors have no financial conflict of interest.
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 ES-09409 and ES-12464 to M.M.
2 Address correspondence and reprint requests to Dr. Marc Monestier, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail address: marcm{at}temple.edu
3 Abbreviations used in this paper: ANoA, anti-nucleolar Ab; B7h, B7 homologous protein.
Received for publication February 5, 2004. Accepted for publication December 23, 2004.
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