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The Journal of Immunology, 2006, 177: 5708-5717.
Copyright © 2006 by The American Association of Immunologists, Inc.

Amelioration of Mercury-Induced Autoimmunity by 4-1BB1

Dass S. Vinay*, Jung D. Kim*,{dagger} and Byoung S. Kwon2,*,{dagger}

* Louisiana State University Eye Center, Louisiana State University Health Science Center School of Medicine, New Orleans, LA 70112; and {dagger} Immunomodulation Research Center and Department of Biological Sciences, University of Ulsan, Ulsan, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In certain strains of mice, subtoxic doses of HgCl2 (mercuric chloride; mercury) induce a complex autoimmune condition characterized by the production of antinucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1/IgE Abs, and deposition of renal immune complexes. 4-1BB is an important T cell costimulatory molecule that has been implicated in T cell proliferation and cytokine production, especially production of IFN-{gamma}. To elucidate T cell control mediated by the 4-1BB signaling pathway in this syndrome, we assessed the effect of administering agonistic anti-4-1BB mAb on mercury-induced autoimmunity. Groups of A.SW mice (H-2s) received mercury/control Ig or mercury/anti-4-1BB or PBS alone. Anti-4-1BB mAb treatment resulted in a dramatic reduction of mercury-induced antinucleolar Ab titers, serum IgG1/IgE induction, and renal Ig deposition. These effects may be related to the present finding that anti-4-1BB mAb decreases B cell numbers and function. The anti-4-1BB mAb-treated mercury group also showed a marked reduction in Th2-type cytokines but an increase in Th1-type cytokines and chemokines. Increased IFN-{gamma} production due to anti-4-1BB mAb treatment appears to be responsible for the observed B cell defects because neutralization of IFN-{gamma} in vivo substantially restored B cell numbers and partly restored IgG1/IgE. Collectively, our results indicate that 4-1BB mAb can down-regulate mercury-induced autoimmunity by affecting B cell function in an IFN-{gamma}-dependent manner and thus, preventing the development of autoantibody production and tissue Ig deposition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mercury and its compounds are ubiquitous in nature, primarily due to their natural release into the environment and because of their widespread use in dental amalgams, cosmetics, preservatives, fumigants, fertilizers, and vaccine preparations (1, 2). Despite their known deleterious effects, mercury and its compounds continue to be major constituents of products used daily (3, 4).

Druet and colleagues (5, 6) were the first to observe that mercury can cause immune dysfunction in Brown Norway rats. The exact mechanism by which mercury activates the immune system leading to the development of a systemic autoimmune disorder is not well understood. Not all strains develop characteristic mercury related autoimmune lesions. Strains such as DBA/2 (H-2d) and CBA (H-2k) are resistant to mercury-mediated effects (reviewed in Ref. 7). Genetic analysis showed that at least three to four genes are involved in mercury-mediated autoimmunity and one of them lies within the MHC class II region (4, 8, 9). The H-2s mice such as SJL, B10.S, and AS.W regardless of their genetic background, are highly susceptible to mercury-induced autoimmunity (10, 11). Conversely, A/J mice with H-2a genotype develop partial immunological alterations in that these mice generate some autoantibodies but no kidney deposits were noted (12). In susceptible mouse strains, the autoimmune lesions caused by exposure to mercury involve the production of autoantibodies, elevation of serum Igs (including IgE), polyclonal activation of B and T cells, and deposition of renal immune complexes resulting in glomerulonephritis (13, 14, 15, 16). The autoantibodies are directed inter alia against dsDNA, phospholipids, glomerular basement membrane, and fibrilarin (17, 18, 19, 20, 21, 22). Most of these symptoms resolve within 4–5 wk even when mercury treatment is continued (23, 24).

Mercury-induced autoimmunity in H-2s mice provides a useful model for chemically related autoimmunity in humans (23, 24). Knowledge derived from the human and mouse models of these mercury effects suggested that Th2-type cytokines, especially IL-4, are critical for the switch to IgG1/IgE (23, 24, 25, 26, 27). However, blockade of IL-4 with either neutralizing Abs or use of IL-4-null mice prevented some but not all components of the autoimmunity (28, 29), suggesting that immune modulators other than IL-4 may be involved in the development of mercury-induced autoimmunity. There is compelling evidence implicating the role of IFN-{gamma} in the regulation of mercury-induced autoimmunity especially at early induction phase of mercury exposure (30). Thus, when mercury-intoxicated mice were treated with IL-12 or anti-IFN-{gamma} mAbs to boost Th1 immunity, the production of autoantibody against some components was significantly reduced (31, 32). Additionally, deletion of IFN-{gamma} regulating genes such as IL-12p35, IL-12p40, STAT-4, and IL-1-converting enzyme, while falling short of completely reversing mercury-induced autoimmune lesions, could significantly influence the outcome (30). In contrast, the IFN-{gamma}–/– or IFN-{gamma}R–/– or IFN regulatory factor-1–/– mice showed significant suppression of nearly all components of autoimmune lesions (reviewed in Refs. 30 , 33). However, in susceptible mice when treated with mercury and IFN-{gamma}, only a few parameters of autoimmunity were resolved (34) despite reports that early IFN-{gamma} is a key regulator of mercury-induced autoimmunity (reviewed in Ref. 30). Taken together the knowledge that both IL-4–/– mice and anti-IL-4 treatment of susceptible mice (28, 29) did not fully recover the autoimmune lesions. And given the protective effects seen in IFN-{gamma}–/– and IFN-{gamma}R–/– mice (30, 33), the current dogma strongly proposes that IFN-{gamma} is an integral component of mercury autoimmunity.

Costimulation is mandatory for successful T cell activation because in its absence T cells enter the state of anergy, becoming nonresponsive to subsequent signals (35). 4-1BB, a member of the TNFR superfamily, is an activation-dependent molecule expressed by both T cells and non-T cells and is involved in costimulation (36, 37, 38). It binds a high affinity ligand, 4-1BB ligand (4-1BBL),3 present on a variety of APCs (36, 37, 38). The role of the 4-1BB/4-1BBL pathway in immune regulation is well characterized and has been confirmed using 4-1BB-null and 4-1BBL-null mice (39, 40). Although CD4+ and CD8+ T cells express 4-1BB at comparable levels, signaling via 4-1BB markedly enhances CD8+ T cell expansion and has been used to treat several autoimmune and tumor models by boosting Th1-type immunity to target autoreactive T cells (41, 42, 43, 44). An additional but important feature of anti-4-1BB mAb immunotherapy has been its unique ability to affect B cell function, which is thought to occur in an IFN-{gamma}-dependent manner (42).

We hypothesized that administration of agonistic anti-4-1BB mAbs, which induces a potent IFN-{gamma} response and suppresses B cell function (36, 37, 38, 41, 42, 43, 44), would cause rapid induction of regulatory Th1 cells and, therefore, affect B cell function and additionally circumvent the Th2 phase of the mercury disease as the Th1/Th2 choice is reciprocally regulated (45). If this response were true, the mercury-elicited increase in autoantibody production and tissue Ig deposition would be neutralized by the anti-4-1BB-induced elevation of Th1 immunity, in particular by IFN-{gamma}. In the present study we show that this reaction is the case. Administration of agonistic anti-4-1BB mAb strongly inhibited the development of unconstrained as well as established mercury-induced autoimmunity in A.SW (H-2s) mice by a mechanism involving up-regulation of Th1 immunity and down-regulation of Th2 immunity and chiefly targeting B cell numbers and function. This finding suggests a beneficial role for this T cell costimulatory molecule, which is particularly important because of the unprecedented increase in the occurrence of autoimmune diseases. A recent review on the epidemiology and significance of autoimmune diseases in health care suggests that ~20% of the human population suffer from at least one form of autoimmune disease (46).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female A.SW-H2s H2-T18b/SnJ mice (AS.W, stock no. 000471) were obtained from The Jackson Laboratory. All mice were housed in the specific pathogen-free animal facility of the Louisiana State University Health Sciences Center (New Orleans, LA). All experiments were performed using mice matched for strain, age, and sex. The animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of the Louisiana State University Health Sciences Center.

HgCl2 and Ab treatment

Groups of mice at least 2-mo-old were injected with PBS alone or HgCl2 (mercuric chloride, mercury; Sigma-Aldrich) together with control rat IgG (control Ig; Sigma-Aldrich), or mercury together with anti-4-1BB mAb (rat IgG1 clone 3H3), which was a gift from Dr. R. S. Mittler, Emory Vaccine Center (Atlanta, GA). Mice were injected s.c. three times a week with 30 µg of mercury in 100 µl of sterile PBS throughout the duration of the experiment. They were either left without additional treatments or treated with anti-4-1BB mAb (100 µg/day i.p. three times per week) at the time of mercury administration. Mice were killed 1, 2, 3, 4, or 5 wk after the first HgCl2 or anti-4-1BB mAb treatment.

Detection of antinucleolar Abs (ANoA)

ANoA levels in serial dilutions of mouse serum were determined by indirect immunofluorescence, as previously described (22). Sera diluted in PBS were incubated in HEp-2 slides (Antibodies Incorporated) for 45 min, and ANoA were detected with FITC-conjugated goat anti-mouse IgG1 or IgG2a Abs (Southern Biotechnology Associates). The titers were expressed as the reciprocal value of the highest serum dilution that gave a clear positive reaction.

Flow cytometry

Spleens were removed and single-cell suspensions prepared. The cells were then treated with RBC lysing solution (BioWhittaker) to eliminate erythrocytes, washed, and suspended (1 x 106/assay) in ice-cold staining medium (PBS, 5% FBS, and 0.09% NaN3). Monoclonal Abs used were: anti-B220, anti-CD4, anti-CD5, anti-CD8beta, anti-CD23 (eBioscience), and anti-CD21 (BD Biosciences). After staining (30 min at 4°C), cells were washed and analyzed with a BD FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

ELISA for serum IgG1, IgG2a, and IgE

Total serum IgG1, IgG2a, and IgE levels were determined using a sandwich ELISA kit (Bethyl Laboratories), according to the manufacturer’s instructions. Briefly, plates were coated overnight at 4°C with appropriate affinity-purified anti-mouse Igs in carbonate buffer (pH 9.6). After three washes with PT buffer (PBS and 0.05% Tween 20), the wells were blocked with PBTN (PBS containing 1% BSA (Sigma-Aldrich), 0.05% Tween 20) for 60 min. Sera serially diluted in PBTN buffer were then added and the wells left overnight at 4°C. The wells were washed five times with PT, and HRP-conjugated anti-mouse IgG1 or IgG2a or IgE secondary Abs, diluted in PBTN, was added for 60 min, and washed out with five washes of PT. The tetramethylbenzidine substrate (TMB; eBioscience) was added and allowed to develop for 5 min, and absorbance was read at 450 nm. Standard curves were generated with concentrations of IgG1, IgG2a, and IgE from 3.125 to 1000 ng/ml.

ELISA for serum self- and foreign Ags

Levels of serum IgG1 Abs against various self- and foreign Ags were measured by ELISA (25, 39). Briefly, microtiter ELISA plates (MaxiSorp; Nunc) were coated with 50 µl/well ssDNA (catalog no. D-8899, 10 µg/ml in PBS; Sigma-Aldrich), dsDNA (catalog no. D-4522, 10 µg/ml in PBS; Sigma-Aldrich), laminin (catalog no. L-2020, 10 µg/ml in carbonate buffer (pH 9.6); Sigma-Aldrich), 3-sn-phosphatidyl ethanolamine (catalog no. P-7693, 25 µg/ml in ethanol; Sigma-Aldrich), thyroglobulin (catalog no. T-1001, 10 µg/ml in carbonate buffer (pH 9.8); Sigma-Aldrich), or TNP-BSA (catalog no. T-5050, 10 µg/ml in PBS; Biosearch Technologies). The plates were incubated overnight at 4°C, except for the 3-sn-phosphatidyl ethanolamine-coated plates, which were evaporated overnight at room temperature. After washing, unbound sites in each well were blocked by incubation with 100 µl of 1% BSA in PBS at room temperature for 2 h, except for the 3-sn-phosphatidyl ethanolamine-coated plates, which were blocked with 10% FBS in PBS. Serially diluted sera were added to the wells followed by overnight incubation at 4°C. After washing, HRP-labeled goat anti-mouse IgG1 (1/3000; Southern Biotechnology Associates) was added to each well, bound immune complexes were detected with TMB substrate (eBioscience), and the absorbance was read at 450 nm.

Detection of renal Ig deposits

After 3–4 wk of treatment, kidneys were removed from the various groups of mice, and bisected halves were snap-frozen in 1–2 ml of OCT compound (Miles). Cryostat sections (7 µm) were prepared with a microtome and fixed in ice-cold ethanol for 20 min. They were then soaked in PBS for 20 min and blocked with PBS containing 10% rabbit serum (Sigma-Aldrich) in a moist chamber. After rinsing briefly in PBS, they were incubated for 10 min with anti-Fc block (clone 2.4G2, undiluted culture supernatant; raised in-house) followed by incubation with dilutions of FITC-conjugated detection Abs specific for IgG (Southern Biotechnology Associates). The titers were enumerated as the reciprocal value of the highest anti-IgG1-FITC dilution that gave a clear positive reaction.

Immunohistochemistry

Frozen tissues were thaw-mounted onto glass slides, fixed in ice-cold ethanol and rehydrated in TBS (pH 7.6) for 20 min, followed by immersion in 10% rabbit serum for an additional 30 min. Staining for germinal centers was performed with biotinylated peanut agglutinin (PNA, 1/20; Vector Laboratories) and visualized with avidin biotinylated enzyme complex reagents (Vector Laboratories). Diaminobenzidine reagent (Vector Laboratories) was used to visualize the immunostaining followed by methyl green (Vector Laboratories) counterstaining and covering with coverslips. Microphotographs were taken with an Eclipse E600 microscope (Nikon) and FDX35 camera (Nikon). For histological studies, 7-µm sections were cut and stained with H&E by routine methods, and photographed.

T cell purification and RNase protection assays

Single-cell spleen suspensions were prepared, RBCs lysed, and CD4+ cells enriched by incubation with anti-CD4 microbeads followed by purification over MACS columns (Miltenyi Biotec). Column-bound cells were used as the source of CD4+ cells. Total RNA was isolated from the purified CD4+ T cells using TRIzol reagent (Invitrogen Life Technologies). Cytokine and chemokine mRNA levels were quantified by RNase protection assays according to the manufacturer’s instructions (Riboquant; BD Biosciences). Briefly, 10 µg of total RNA was hybridized overnight with [{alpha}-32P] 5'-UTP-labeled riboprobes at 56°C. Unhybridized ssRNA was digested by RNase treatment, and the dsRNA was purified by phenol/chloroform extraction and ethanol precipitation. The samples were fractionated by electrophoresis on a 6% polyacrylamide/7 M urea gel, dried and subjected to autoradiographic analysis.

In vivo cytokine blockade

Mice were injected three times per week s.c. with 30 µg of mercury in 100 µl of sterile PBS for 3 wk. They were either left without additional treatments or treated with anti-IL-4 (clone 11B11, rat IgG1; generated in house), anti-IL-5 (clone TRFK5, rat IgG1; eBioscience), anti-IL-13 (clone 38213.11, rat IgG2b; R&D Systems) or anti-IFN-{gamma} (clone R4-6A2, rat IgG1; generated in house). All anti-cytokine Abs were administered 100 µg/day i.p. for the duration of the experiment. Mice were killed 3 wk after first HgCl2 or anti-cytokine Ab treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
4-1BB signaling reduces mercury-imposed autoimmune lesions

Autoimmune lesions resulting from exposure to mercury in susceptible mice are characterized by elevated levels of serum Igs, especially IgG1/IgE, ANoA directed against self- and foreign Ags, and deposition of immune complexes in the kidneys (23, 24). We and others have shown that 4-1BB enhances Th1 immunity in several animal models (36, 37, 38, 41, 42, 43, 44). To determine whether treatment with agonistic anti-4-1BB mAbs causes Th1 dominance and overrides the Th2 immune response that is often seen in later stages of mercury autoimmunity (23, 24, 25, 26, 27), we injected groups of AS.W (H-2s) mice with mercury/control Ig, or mercury/anti-4-1BB mAb three times per week for the entire duration of the experiment. Control AS.W mice were mock-treated with PBS as controls for the mercury group. Serum IgG1 and IgE levels were greatly elevated in the mice exposed to mercury/control Ig, peaking at after 3 wk (in the case of IgG1) or at 3 wk (in the case of IgE) following the initial treatment (Fig. 1, A and C), and this effect was drastically reduced in the mice receiving mercury/anti-4-1BB mAb (Fig. 1, A and C). Levels of serum IgG2a (Fig. 1B) and IgG2b (data not shown) displayed only an initial spike that was not sustained.


Figure 1
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  		FIGURE 1. Spontaneously arising autoimmune lesions in mercury-treated mice are prevented by anti-4-1BB mAb treatment. Groups of AS.W mice were repeatedly injected s.c. with either mercury or PBS. Where indicated they also received anti-4-1BB mAb (i.p.) as described in Materials and Methods. A–C, At different times after injection, serum (n = 9) was collected by cardiac puncture and tested for IgG1, IgG2a, and IgE by ELISA. Data are the mean ± SD. D, Representative H&E staining of kidney sections showing cropped images of individual glomeruli. E, Infiltrating cells in each glomerulus were visually counted under a microscope. A minimum of at least 10 glomeruli was counted and the mean ± SD calculated. Infiltrating cells within each glomerulus were counted visually under microscope. Cells from a minimum of at least 10 glomeruli were counted and expressed as mean ± SD. F, Sections (7-µm) of kidneys from various animal groups were stained with FITC-conjugated anti-mouse IgG1 3 wk (n = 9) after first injection. G, Staining of kidney sections was achieved as mentioned in Materials and Methods and titers of IgG1 deposits in the glomeruli was assessed as mentioned. H, Results of ELISA showing production of Abs against self- and non-self-Ags 3 wk after control Ig- and anti-4-1BB mAb treatment (n = 9). The immune complexes were detected with HRP-conjugated goat anti-mouse IgG1 conjugate in conjunction with tetramethylbenzidine substrate. Data are mean ± SD. P. Ethanolamine, phosphatidyl ethanolamine. I, Representative HEp-2 staining pattern obtained with serum diluted 1/100 and detected with FITC-conjugated goat anti-mouse IgG1 conjugate. J, Staining of HEp-2-coated slides was achieved as mentioned in I. Serial dilution of test sera (n = 9) was applied to slides, developed as before, and titers of IgG1 ANoA were enumerated as described in Materials and Methods. Significant differences between mercury-treated and anti-4-1BB mAb treated mice were calculated using Student’s t test, *, p < 0.01; **, p < 0.001. Original magnification x20 (I) and x40 (D and F).

 
Mercury-induced autoimmunity is also characterized by the appearance of immune complexes, mostly in the kidneys but also to some extent in secondary lymphoid organs (23, 24). Both histologic analysis (Fig. 1D) and quantitative enumeration (Fig. 1E) of glomerular infiltrates revealed comparable levels between rat IgG and anti-4-1BB treated mercury groups (Fig. 1E). As with the serum titers of IgG1/IgE and ANoA, anti-4-1BB mAb treatment of mercury-intoxicated mice prevented the accumulation of immune complexes in kidney glomeruli (Fig. 1F). Titration experiments suggested that titers of IgG1 were significantly higher in mercury/rat IgG group compared with mercury/anti-4-1BB group that showed slightly elevated but not significant IgG1 titers (Fig. 1G). Similar trends were noted when complement component 3 (C3) or IgM or IgD were examined (data not shown). These results suggest that treatment with anti-4-1BB mAb inhibits the deposition of kidney Igs responsible for autoimmunity.

Besides elevation of serum Igs and ANoA levels, mercury exposure can result in the generation of Abs to several self- and foreign Ags (23, 24, 31, 47). We next performed experiments to determine whether treatment with anti-4-1BB mAb also attenuated the production of autoantibodies to some of these Ags. Serially diluted sera were subjected to ELISA in microtiter plates coated with individual Ags, as described in Materials and Methods. Mercury treatment alone resulted in the production of autoantibodies to several self- and foreign Ags, and treatment with anti-4-1BB mAb abrogated the development of such autoantibodies (Fig. 1H). Fig. 1H displays the IgG1-specific reactions at the lowest serum dilution (1/500). Other dilutions of the sera, as well as other anti-Ig-specific isotypes, yielded a similar pattern (data not shown).

One of the important features of mercury-induced autoimmunity is the generation of ANoA (23, 24). To further understand the protective role of 4-1BB signaling in the suppression of ANoA production, we treated mice as in Fig. 1, A–C, collected serum at weekly intervals, and measured IgG1-specific ANoA. Fig. 1I shows the kinetics of IgG1-specific ANoA at the lowest (1/100) serum dilution in the treated groups. After 3 wk of treatment, IgG1-specific ANoA was prominent in the mercury-treated mice. The anti-4-1BB mAb treated groups, in contrast, gave no significant ANoA at the dilution shown or at any other dilution (Fig. 1I). Time course analysis also revealed increased IgG1 titers (Fig. 1J) in mercury group over titers seen on other groups. Because anti-4-1BB mAb is a potent IFN-{gamma} inducer (36, 37, 38, 41, 42, 43, 44), we anticipated that ANoA of Th1-representing Ig isotypes (e.g., IgG2a/IgG2b) might develop in this group. However, as in Fig. 1B, no ANoA was detectable upon staining with FITC-conjugated anti-IgG2a/IgG2b (data not shown). This observation was surprising, as both Th1- and Th2-sustaining Ig responses appeared to have been lost in the anti-4-1BB mAb treated group.

Treatment with anti-4-1BB mAb suppresses established autoimmune lesions elicited by mercury

Although our experiments suggest a protective role for anti-4-1BB mAb against unconstrained autoimmune lesions caused by mercury, it was not clear whether such therapy also ameliorated established mercury-provoked pathological events. To see whether this result was the case, we delayed anti-4-1BB mAb therapy for various periods of time after mercury treatment (Fig. 2A). Mice were observed for 4 wk, then killed, and their sera analyzed. The results in Fig. 2, B–E reveal a rapid and striking protective effect of anti-4-1BB mAb. Despite a delay in treatment until the Th2 response was well established, the anti-4-1BB mAb remained effective in down-regulating the production of IgG1 ANoA (Fig. 2, B and C) and IgG1/IgE (Fig. 2, D and E). Taken together, our data strongly suggest that anti-4-1BB mAb therapy is highly effective in protecting mercury-induced autoimmunity.


Figure 2
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  		FIGURE 2. Attenuation of established mercury lesions by anti-4-1BB mAb. A, The mercury and anti-4-1BB mAb injection protocol. Mice (n = 4) received s.c. injections of 30 µg of mercury in 100 µl of sterile PBS three times weekly throughout the experiment. Where indicated they were additionally treated with anti-4-1BB mAb as follows: group a, anti-4-1BB mAb treatment commenced 3 wk after mercury administration; group b, anti-4-1BB mAb treatment commenced 2 wk after mercury administration; group c, anti-4-1BB mAb treatment commenced 1 wk after mercury administration; and group d, anti-4-1BB mAb and mercury administered simultaneously. In all cases, anti-4-1BB mAb was administered i.p., and mice received 100 µg of mAb in 500 µl of sterile PBS per injection. All mice were killed 4 wk after the initial mercury treatment. B, Mice were treated as outlined in A, and representative HEp-2 staining pattern obtained with serum diluted 1/100 and detected with FITC-conjugated goat anti-mouse IgG1 conjugate. C, Sera were collected at various times points after initial treatment and staining and titers of IgG1 ANoA were assessed as described in Materials and Methods. D and E, Four weeks after treatment (n = 4), serum was collected by cardiac puncture and tested for IgG1, IgG2a, and IgE by ELISA. Data are the mean ± SD. Significant differences between mercury and anti-4-1BB mAb-treated mice were calculated using Student’s t test, *, p < 0.01; **, p < 0.001. Original magnification, x20.

 
Anti-4-1BB mAb therapy augments T cell numbers in mercury-administered mice

The production of mercury-directed autoantibody is controlled by T cells especially CD4+ T lymphocytes (48, 49, 50). Because the primary role of 4-1BB is as a T cell activating molecule (36, 37, 38), and because it is able to suppress mercury-induced autoimmune lesions as shown, we examined the possibility that anti-4-1BB mAb alters T cell function. Groups of AS.W mice were treated with mercury/control Ig or mercury/anti-4-1BB for 3–4 wk, spleen suspensions were prepared, and the percentages and numbers of CD4+ and CD8beta+ T cells were compared by flow cytometry. Although percentages (Fig. 3A) and numbers of CD4+ (Fig. 3C, top) and CD8beta+ (Fig. 3C, bottom) T cells were comparable in the mercury-treated and PBS-treated mice, they were elevated in the spleens of the anti-4-1BB mAb treated mice (Fig. 3, A, left, and C, bottom). Because we administered anti-4-1BB mAb via the i.p. route, we checked whether this treatment affected local (peritoneal) immune competent cells. Surprisingly, we found a massive increase in percentages (Fig. 3B) and numbers of both CD4+ (Fig. 3D, top) and CD8beta+ (Fig. 3D, bottom) T cells in the peritoneum of anti-4-1BB mAb-treated mice. Investigation of the kinetics of CD4+ and CD8beta+ T cell expansion in the mercury/anti-4-1BB mAb-administered mice revealed that elevated levels of these T cell subsets were evident within 2 wk of anti-4-1BB mAb administration (Fig. 3, C and D).


Figure 3
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  		FIGURE 3. Anti-4-1BB mAb treatment results in increased T cell numbers. Three weeks after treatment, mice (n = 4) were killed, spleens removed, single-cell suspensions made, and flow cytometry performed. Representative colored contour plots are shown depicting CD4+ and CD8beta+ cells in the spleen (A) and T cells in the peritoneum (B). Numbers in each panel represent percentage of positive cells of the indicated cell type. C and D, Time course of CD4+ and CD8beta+ cell expression in the spleens (C) and peritoneal exudate cells (D) of PBS, control Ig, and anti-4-1BB mAb-treated mercury-administered mice. Mice were treated as in Fig. 1 and at weekly intervals spleen cell suspensions were subjected to two-color flow cytometry. Absolute cell numbers were calculated for each cell type and shown in histograms. Data shown are mean ± SD. *, p < 0.01; **, p < 0.001.

 
Anti-4-1BB mAb-mediated protection of mercury-induced autoimmunity involves B cell deletion

Although T cells are central to the formation of mercury-induced immunopathological lesions, B lymphocytes, by their cognate interactions with CD4+ T cells, also play a major role by virtue of their ability to produce autoantibodies (23, 24). Because our results suggested that anti-4-1BB mAb treatment suppresses Th2-type immunity with a concomitant rise in CD4+ T cell numbers in peritoneum and spleen, and given our evidence that autoantibody production as well as serum Ig titers were strongly inhibited in the anti-4-1BB mAb-treated mercury groups, we examined B cell numbers and function in the various experimental groups. There was no difference in the percentages (Fig. 4A) and numbers (Fig. 4B) of B cells (B220+) between PBS-treated and mercury/control Ig-treated mice at any point, whereas there was a profound decline in the percentages of B cells in the spleens of the mercury-treated mice receiving anti-4-1BB mAb (Fig. 4, A, top panels, and B). B cells have diverse roles in immune regulation, and B cells with particular phenotypes performing particular functions (51). Conventional B cells (CD5–B220+:B1b cells), which produce Ab and switch Ig class, have been extensively studied in the mercury model (23, 24). However, the role of B1a (CD5+B220+) cells in the autoimmune disease-prone AS.W mouse is still not clear, although these cells have been shown to play a critical role in other autoimmune models in which they produce low-affinity Abs and reduce negative regulation and recruitment to germinal centers and/or the production of IL-10 (52). We found that anti-4-1BB mAb treatment of mercury-administered mice not only led to a reduction in B1a cells (Fig. 4, A; bottom panels, and C) but also severely diminished the number of B1b (Fig. 4, A bottom panels, and D) cells in the spleen. We next evaluated follicular B cells (CD21+CD23+) and marginal zone (MZ) B cells (CD21+CD23–) in the spleen. The percentages (Fig. 4A, bottom panels) and numbers (Fig. 4E) of follicular B cells (CD21+CD23–) was significantly reduced in the anti-4-1BB mAb treated group (Fig. 4A, bottom panels), as were MZ B cell percentages (Fig. 4A, bottom panels) and numbers (Fig. 4F). We also evaluated the splenic B cell compartment using AA4.1 Ab, which recognizes the complement 1q receptor (C1qRp) that is abundantly expressed on splenic transitional B cells; as B cells mature, expression of C1qRp decreases (45, 46, 53, 54). We detected a mercury-induced decrease in C1qRp+ cells (9.6% in the PBS-treated group vs 3.7% in the mercury-treated group) suggestive of an activated B cell phenotype in these mice (data not shown). The reduced ability of the anti-4-1BB mAb-treated mice to produce Ig isotypes (including IgE), as well as to generate ANoA, coupled with the reduction in B1a and B1b cell numbers, is also reflected in the diminished or absent germinal centers (as judged by PNA reactivity) in their spleens (Fig. 4G) and by reduced CD38–PNA+ cell numbers (as revealed by flow cytometry; data not shown). We conclude that the protection against mercury-induced autoimmune lesions afforded by anti-4-1BB mAb involves a reduction in B cell numbers and activity. This reduction was most evident in the peritoneum (where B1a cells are abundant) (Fig. 4H). Analysis of absolute numbers of different B cell subsets in the peritoneum following treatment with anti-4-1BB mAb demonstrated time-dependent reduction in all B cell subsets studied when compared with rat IgG-treated mercury group (Fig. 4, I–K). Because CD4+ and CD8beta+ T cell numbers were also elevated in the peritoneum of the anti-4-1BB mAb groups (Fig. 3, B and D), it is possible, although not yet tested directly, that intense T cell activity and the resultant production of immune modulators suppress B cell numbers. In support of this possibility, evaluation of intracellular IFN-{gamma} expression in peritoneal exudates cells was strikingly higher compared with those revealed by splenocytes or lymph node (inguinal) cells (data not shown) suggesting that the peritoneum is site of intense immune reaction.


Figure 4
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  		FIGURE 4. Anti-4-1BB mAb treatment of mercury-administered mice results in profound loss of B cell numbers and function. A, Flow cytometric evaluation of different splenic B cell subsets of mercury treated, and control Ig- and anti-4-1BB-administered mice. Three weeks after treatment, spleen cell suspensions were subjected to flow cytometry. The multicolored contour plots in the upper and lower panels show percentage changes in indicated B cell subsets. B–F, Mice were treated as in Fig. 1, and at weekly intervals, spleen cell suspensions were subjected to flow cytometry. Absolute numbers of individual B cell subsets were calculated from the values obtained with flow cytometry and depicted as histograms. Data are mean ± SD. G, Representative cropped images of spleen cell sections showing germinal center reaction (arrows; PNA reactivity). H, Peritoneal cavity cells (Per C) were obtained 3 wk after first injection of PBS or mercury/rat IgG or mercury/anti-4-1BB and flow cytometry performed as in A. Values in panels denote the percentage of positive cells of indicated cell type. I–K, Mice were treated as in B–F, peritoneal cavity cells were collected and flow cytometry performed. Absolute numbers of individual B cell subsets were determined from flow cytometry data and presented as histograms. Significant differences between mercury-treated and anti-4-1BB mAb treated mice were calculated using Student’s t test, *, p < 0.05; **, p < 0.01. Original magnification x40. PBS group (n = 3) and for rat Ig and anti-4-1BB mercury treated groups (n = 4) are examined.

 
Anti-4-1BB mAb-mediated protection results from a shift from Th2- to Th1-type responses

Although IFN-{gamma} is shown to regulate mercury-induced autoimmunity (32, 33), later stages of disease condition reveal elevated Th2-type immune response, notably by IL-4 up-regulation (23, 24). We sought to determine whether there existed a difference in Th1- vs Th2-type cytokine expression between the control Ig-treated and anti-4-1BB mAb-administered mercury intoxicated mice. Because CD4+ T cells are required to help B cells shape the humoral immune component, we isolated CD4+ T population by immunomagnetic purification and performed RNase protection assay to study the cytokine and chemokine profiles of the groups. Our results revealed hitherto unknown aspects of mercury-mediated cytokine responses. Although levels of IL-4 were high in the mercury group, the results in Fig. 5A reveal an important role for other Th2 cytokines (e.g., IL-5 and IL-13) in mercury-mediated autoimmunity. To the best of our knowledge, this evidence is the first for a marked elevation of IL-5 and IL-13 in mercury-treated mice. Surprisingly, IL-5 and IL-13 were both conspicuously absent from the anti-4-1BB mAb-treated group (Fig. 5A). Levels of Th1 cytokines such as IL-2 and IFN-{gamma} were clearly higher in the anti-4-1BB mAb-treated group than the other groups (Fig. 5B). T lymphocytes represent a rich source of chemokines, and these T cell-secreted chemokines play important roles in the regulation of immune responses. The proinflammatory effects of MIP1{alpha}, MIP1beta, and RANTES are all well documented (55, 56, 57). Of note these chemokines were shown to support Th1 immunity (58). For example MIP1{alpha} and MIP1beta are induced in activated T cells, macrophages, and B cells (59, 60, 61). The RANTES expression has been shown in delayed-type hypersensitivity reactions, where the protein seems responsible for the accumulation of CD4+ T cells and macrophages (57). In addition, induction of RANTES has been shown by the type 1 mediators TNF-{alpha} and IFN-{gamma} (62). Given this, we also checked the chemokine profiles of the control Ig- and anti-4-1BB mAb-treated mercury-intoxicated mice. Surprisingly, chemokine expression was lower in the CD4+ cells derived from control Ig/mercury-treated mice (Fig. 5C) than in the anti-4-1BB mAb-treated mice that had enhanced expression of several chemokines, notably RANTES, MIP-1beta, MIP-1{alpha}, and particularly MIP-2 (Fig. 5C). Collectively, our data suggest that anti-4-1BB mAb treatment of mercury-susceptible mice results in down-regulation of the Th2-type immune response (reduced IL-4, IL-5, and IL-13; that are seen in later stages of autoimmunity) (23, 24, 25, 26, 27) by tilting the balance toward the Th1-type response elevated IL-2, IFN-{gamma}, TNF-{alpha}, MIP1{alpha}, MIP1beta, and RANTES).


Figure 5
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  		FIGURE 5. Anti-4-1BB mAb treatment of mercury-intoxicated mice reveals suppression of Th2 cytokine gene expression. An autoradiograph of a representative RNase protection assay showing cytokine (A and B) and chemokine (C) gene expression in purified CD4+ T cell populations 3 wk after treatment (n = 5). Pooled samples from three independent experiments were used for the experiment.

 
Blockade of Th2 cytokines during mercury autoimmunity has complex effects

The cytokine data described suggested that the only difference between the mercury/control Ig and mercury/anti-4-1BB is the presence vs absence of IL-5 and IL-13. Prompted by these findings, we explored whether these cytokines control the fine line between susceptibility and resistance to mercury-induced autoimmunity. We treated groups of AS.W mice with mercury (three times per week) and neutralizing anti-cytokine Abs (once daily) for 3 wk. A mercury/anti-4-1BB mAb treated group was included as a control. As expected, mercury treatment alone increased IgG1-specific ANoA and IgG deposition in the kidneys (Fig. 6A). Treatment with neutralizing Abs to IL-4, IL-5, and IL-13 in each case only reduced IgG1-specific ANoA but fell short of restricting the production of IgG2a-specific ANoA (Fig. 6A). Importantly, treatment with anti-IL-4 and to an extent IL-13 reduced IgG1-specific ANoA much more effectively than IL-5 blockade (Fig. 6A). Interestingly, none of the anti-cytokine Ab treatments completely suppressed immune complex deposition in the kidneys, although the results of anti-IL-4 and anti-IL-13 therapy were slightly better than the others (Fig. 6A). The same is also evidenced by titration experiments (Fig. 6B). We next determined whether serum Ig levels were in any way affected by anti-cytokine therapy. Neutralization of IL-4, IL-5, and IL-13 all suppressed IgG1 and IgE to a significant degree (Fig. 6, C and E), whereas IgG2a was unaffected (Fig. 6D). Taken together, these data suggest that neutralizing Th2 cytokines (such as IL-4, IL-5, and IL-13) during mercury intoxication affects only IgG1/IgE production and IgG1-specifc ANoA but fails to completely protect other components of the disease, such as renal Ig deposition.


Figure 6
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  		FIGURE 6. Neutralization of Th2 cytokines results in only moderate protection against mercury-induced autoimmune lesions. Mice (n = 3) were treated with mercury and Abs as described in Materials and Methods for 3 wk. A, Representative photomicrographs showing serum ANoA and immune complex (IgG) deposition in kidneys. B, Staining of HEp-2-coated slides with various anti-Igs was performed as in A. Titers of individual Ig isotypes was enumerated as outlined in Materials and Methods. C–E, Serum was collected by cardiac puncture and tested for IgG1, IgG2a, and IgE by ELISA. Data are the mean ± SD. Significant differences between mercury-treated and anti-cytokine Ab-treated mice were calculated using Student’s t test, *, p < 0.001. ns, Not significant. Experiment was repeated two times and representative data is shown.

 
Anti-4-1BB mAb-suppressed B cell function is partially restored by neutralizing IFN-{gamma}

We and others have shown that in vivo administration of anti-4-1BB mAb results in a marked increase in IFN-{gamma} expression (36, 37, 38, 41, 42, 43, 44). It has also been shown in an autoimmune lupus model that treatment with anti-4-1BB mAb results in increased IFN-{gamma} production, and that administration of anti-IFN-{gamma} mAb restores B cell numbers and function (42). Our current findings point in the same direction, in that anti-4-1BB mAb treatment of the mercury-intoxicated mice resulted in marked elevation of IFN-{gamma} and caused a significant drop in B cell numbers and function. To see whether these factors were interrelated, we treated AS.W mice with mercury/control Ig, mercury/anti-IFN-{gamma}, mercury/anti-4-1BB, or mercury/anti-IFN-{gamma}/anti-4-1BB for 3 wk. Exposure of mercury-intoxicated mice that received anti-4-1BB mAb to neutralizing anti-IFN-{gamma} mAb significantly reduced the decline in B cell percentages and numbers (Fig. 7, A and B). Interestingly, restoration of IgG2a-specific ANoA was highly significant (Fig. 7, C, lower panels, and E), whereas the IgG1-specific ANoA (Fig. 7, C, top panels, and D), while restored markedly, results were not as striking as those seen with IgG2a. The serum IgE levels (Fig. 7F) were not moderately affected. The reduction in IgG2a-specific ANoA in the mercury/anti-IFN-{gamma} group demonstrates that the anti-IFN-{gamma} mAb regimen was fully effective, and thus supports the conclusion that the anti-4-1BB mAb-mediated deletion of B cells in mercury-induced autoimmunity depends substantially, although not completely, on IFN-{gamma}.


Figure 7
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  		FIGURE 7. Anti-4-1BB mAb-induced B cell loss is partially restored after neutralization of IFN-{gamma}. A and B, Mice (n = 4) were treated with mercury and indicated Abs as described in Materials and Methods. Single-cell suspensions of spleens were prepared and analyzed by flow cytometry. Representative histograms of percentage (A) B220+, CD21+CD23+, and CD21–CD23+ cells and absolute numbers (B) are depicted. C, Serum was diluted 1/100 and applied to HEp-2 slides for the enumeration of ANoA. The reactions were revealed with FITC-conjugated goat anti-mouse IgG1 or IgG2a conjugates. D and E, Various dilutions of test sera were applied to HEp-2-coated slides, and IgG1 and IgG2a reactivity was assessed as mentioned in Materials and Methods. The titers of IgG1 (D) and IgG2a (E) are depicted. F, Serum IgE analysis was performed on samples obtained from mice treated for 3 wk. Data are mean ± SD. Original magnification x20. *, p < 0.01; **, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown that signaling via 4-1BB attenuates both unconstrained as well as established autoimmune lesions evoked by mercury. Several of the anti-4-1BB mAb-directed protective effects were found to stem from elevated Th1 responses and the transition from susceptibility to resistance to mercury in the anti-4-1BB mAb-treated mice was dependent on IFN-{gamma}. The majority of studies agree that 4-1BB signaling supports Th1 immunity, especially involving IFN-{gamma} (36, 37, 38, 41, 42, 43, 44), although some have reported in favor of Th2 (63). One surprising aspect of the present results is that in vivo 4-1BB signaling lowers the mercury-associated autoimmune processes, not by deleting CD4+ Th cells (as reported in Refs. 41, 42, 43, 44) but by targeting B cell function.

Although IL-4, which supports the production of IgG1/IgE at later stages of disease, has been shown to be important in mercury autoimmunity (23, 24), our observations uncovered an important role for IL-5 and IL-13 (in addition to IL-4) in this syndrome. To the best of our knowledge this finding has not been previously reported; in vivo neutralization of IL-13 in particular, but also IL-5 to some extent, markedly prevented several, but not all, of the mercury-imposed autoimmune lesions and in most cases produced effects comparable to those observed with neutralization of IL-4. This observation suggests that IL-4, IL-5, and IL-13 appear to play only restricted roles in the events leading to autoimmune mercury syndrome.

We noted that anti-4-1BB mAb therapy not only attenuated the development of unconstrained mercury effects but also showed a remarkable ability to suppress established mercury-induced lesions. Our results suggest that cytokines such as IFN-{gamma} are probably critical for this suppression, as expression of this cytokine was enhanced in the 4-1BB mAb-treated group. Previous studies have suggested that anti-4-1BB mAb suppresses/deletes CD4+ T in an IFN-{gamma}-dependent manner (41, 42, 43, 44). The protective pathway observed in the current study was somewhat different from that previously proposed (41, 42, 43, 44) because the anti-4-1BB mAb did not delete CD4+ T cells despite enhanced IFN-{gamma} production. Importantly, the massive up-regulation of IFN-{gamma} by CD8beta+ T cells reported previously (44) was not evident in the present study because only CD4+ (Fig. 5, A and B) and not CD8+ T cells produced high levels of IFN-{gamma} (CD4– cell fraction; data not shown). Thus these data suggest that anti-4-1BB mAb-mediated protection against certain autoimmune diseases, e.g., mercury syndrome, can occur without deleting CD4+ cells but by altering their activity.

The main protective mechanism operating in the present study involved massive deletion of B cells, including conventional B cells (B2 cells; CD5–B220+) and several other B cell subtypes such as B1 (CD5+B220+), MZ B cells (CD21+CD23–), and follicular B cells (CD21+CD23+) cells. We attempted to establish the mechanism by which anti-4-1BB mAb deletes these B cell subsets. The simple explanation that the increased IFN-{gamma} is responsible for deleting the B cells is supported by the substantial restoration of B cells following neutralization by anti-IFN-{gamma} mAb and is consistent with previous findings that in vivo neutralization of IFN-{gamma} by anti-IFN-{gamma} mAb reversed T/B cell function in a lupus model (42).

Others have suggested that B cell deletion by 4-1BB signaling is mediated by monocytes, and probably results from components secreted by them (64). One of the main agents of monocyte/macrophage activation is NO, which acts on immune cells to modify their effector functions (65, 66). However, when the mercury/anti-4-1BB mAb group of mice was administered aminoguanidine, a known NO synthase inhibitor (relatively selective for inducible NO synthase) (67), the lost B cell function was not restored (data not shown). These results suggest that the absence of Ab production against self- and foreign Ags, as well as the inability to produce Ig isotypes in the anti-4-1BB group, is primarily due to reduced B cell numbers presumably by the actions of IFN-{gamma}+CD4+ cells.

Although anti-4-1BB-mediated IFN-{gamma} appeared to be responsible for the protection seen against mercury-induced lesions (reviewed in Ref. 30), the distinct mechanism underlying such actions is not completely clear at this time especially in the light of the observation that IFN-{gamma} (albeit low) is required for the regulation of this syndrome (30). Moreover, the precise levels and longevity of IFN-{gamma} in the body required to regulate mercury autoimmunity is not clearly understood as when mercury exposed mice treated additionally with IFN-{gamma}, only a few components of autoimmunity were seem to be affected (reviewed in Ref. 30). However, deletion of genes that control IFN-{gamma} expression or mice lacking IFN-{gamma} or IFN-{gamma}R or IFN regulatory factor-1, which showed profound to significant protection against disease, strongly tilt the balance in favor of IFN-{gamma} in the regulation of mercury autoimmunity (30). It is, however, not clear from the reports we mentioned whether B cell number/function was affected when IFN-{gamma} pathway is altered as the experiments described in this study clearly suggest that both loss of B cell number/function in anti-4-1BB group is due to very high levels of IFN-{gamma}. It may also be mentioned that increased IFN-{gamma} production in anti-4-1BB-treated mice although suppressed Th2 immune responses but did not heighten Th1-associated IgG2a isotype (Fig. 1, A–C) merely due to lack of available B cells to carry out this function. Thus restoration observed by anti-4-1BB therapy was not merely due to a tilt of balance between Th2 to Th1. The results described in the current study therefore proposes a unique condition in which anti-4-1BB mAb by its actions (presumably due to high IFN-{gamma} expression and other unknown factors) overrides the regulatory capacity of IFN-{gamma}, which is believed to orchestrate the induction phase of mercury autoimmunity (30, 33, 34), Based on the results described in the present study, we propose that the levels of IFN-{gamma} following exposure to mercury (mercury/rat IgG group) appear to be insufficient to cause disturbances in humoral immunity as revealed by comparable B cell numbers among PBS- and rat IgG-treated mice. Alterations in B cell function was only seen when IFN-{gamma} levels were elevated as seen in the anti-4-1BB treated group that prevented among others the development of autoantibody production, tissue Ig deposition leading to protection against the disease. In summary, our results indicate that high levels of IFN-{gamma} in anti-4-1BB group appear to override the regulatory role of IFN-{gamma}, which is believed to operate in the early induction phase of mercury autoimmunity (30, 33, 34) to target B cell function and form the chief basis of anti-4-1BB mAb-mediated immunotherapy. This conclusion further draws its support from the data that in vivo neutralization of IFN-{gamma} profoundly restored anti-4-1BB-mediated loss of B cell numbers.

Overall, our results suggest that anti-4-1BB mAbs are strong candidates for therapeutic use against autoimmune mercury syndrome. Manipulation of this costimulatory pathway may have important therapeutic benefits in the immunotherapy of autoimmunity.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 in part by U.S. Public Health Service Grants R01EY013325 (to B.S.K.), KRF-2005-201-E00008, KRF-2005-084-E00001, Ulsan Technopark Fund, Science Research Center funds to the Immunomodulation Research Center, University of Ulsan, and grants from Korea Science and Engineering Fund. Back

2 Address correspondence and reprint requests to Dr. Byoung S. Kwon, Immunomodulation Research Center, University of Ulsan, 29 Mukeo Dong Nam-Ku, Ulsan 680-749, Korea. E-mail address: bkwon{at}lsuhsc.edu Back

3 Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; ANoA, antinucleolar Ab; PNA, peanut agglutinin; MZ, marginal zone. Back

Received for publication July 29, 2005. Accepted for publication July 27, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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