Autoimmunity TNF Family in Chemically Induced a Role for B

A Role for B Cell-Activating Factor of the TNF Family in Chemically Induced Autoimmunity

This information is current as Yan Zheng, Stefania Gallucci, John P. Gaughan, Jane A. of October 1, 2021. Gross and Marc Monestier J Immunol 2005; 175:6163-6168; ; doi: 10.4049/jimmunol.175.9.6163 http://www.jimmunol.org/content/175/9/6163 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Role for B Cell-Activating Factor of the TNF Family in Chemically Induced Autoimmunity1

Yan Zheng,* Stefania Gallucci,‡ John P. Gaughan,† Jane A. Gross,§ and Marc Monestier2*

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 observe that HgCl2 administration in susceptible mice results in the elevated production of B cell- activating factor of the TNF family ((BAFF) also known as BLyS, TALL-1, zTNF-4, THANK, and TNSF13B), a B cell growth factor belonging to the TNF family. A transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI)-Ig fusion protein (which neutralizes both BAFF and a proliferation-inducing ligand (APRIL), another TNF family member) inhibited Hg-induced autoantibody or serum IgE production. These results are discussed in the context of the inhibitory effect of TACI-Ig on B cell maturation at the transitional stage. The Journal of Immunology, 2005, 175: 6163–6168. Downloaded from ollowing productive Ig gene rearrangements in the bone TACI fusion protein) and TACI-Ig transgenic mice have fewer marrow, newly emerging B cells in the periphery go transitional T2 and MB. Furthermore, TACI-Ig treatment inhibits through transitional stages associated with negative selec- disease progression of collagen-induced arthritis (16). In BLySϪ/Ϫ F 3 tion steps before becoming fully mature B cells (MB). Early re- mice, B cell maturation arrests at the transitional T1 stage (16, 17). ports described two transitional stages, transitional type 1 B cells However, APRILϪ/Ϫ mice do not show abnormalities in T and B

(T1) and transitional type 2 B cells (T2), in the mouse (1). Certain cell development and functions or in T cell-dependent or -inde- http://www.jimmunol.org/ members of the TNF superfamily and their receptors are critical pendent humoral responses (18). These data indicate that APRIL for the development of the mammalian immune system, being in- may be dispensable and that BAFF is the major player for the volved in the organogenesis of lymphoid organs and the regulation development of the immune system. Research on the BAFF/BLyS of efficient adaptive immune responses (2). In recent years, two system and its receptors has shown its crucial impact on the im- closely related TNF family members, B cell-activating factor of mune response and especially B cell survival (19). the TNF family ((BAFF), also known as BLyS, TALL-1, zTNF-4, Systemic autoimmune diseases result from a complex interac- THANK, and TNSF13B) (3–7) and a proliferation-inducing ligand tion between genetic and environmental factors. Mercury, an en- (APRIL) (8), have been identified that are critical for immune sys- vironmental pollutant, has been used in a wide range of commer- tem function (9). There are three currently identified receptors for cial and medical products, which give rise to accidental and by guest on October 1, 2021 BAFF and APRIL: B cell maturation protein (BCMA) (10), trans- occupational exposures. In humans, chronic exposure to low doses membrane activator and calcium-modulating and cyclophilin li- of elemental mercury or mercuric salt can induce glomerulone- gand interactor (TACI) (11), and BAFF receptor (BAFF-R; BR3) phritis and proteinuria (20, 21), excessive T cell activation, in- (12). Expression of BCMA and BAFF-R is thought to be restricted creased level of serum IgE, and antinuclear Abs in the serum (22, to B cells, whereas TACI is expressed by both B cells and acti- 23). Some of these features are also observed in the HgCl2 model vated T cells (9, 13, 14). Recently, Ng et al. (15) revisited BAFF-R of murine autoimmunity. Mouse or rat strains expressing certain expression and showed its expression on activated/memory subset MHC Ags are susceptible to the heavy metal induction of a com- of T cells. The individual role of these factors or their receptors is plex autoimmune syndrome. In susceptible H-2s mice, subtoxic not fully elucidated, but mice treated with TACI-Ig (a soluble doses of HgCl2 induce an autoimmune syndrome characterized by production of anti-nucleolar Abs (ANoA) and increased serum levels of IgG1 and IgE. The increase in serum Igs peaks 2–3 wk after *Department of Microbiology and Immunology, and †Department of Biostatistics, the beginning of the HgCl injections, whereas ANoA can persist Temple University School of Medicine, Philadelphia, PA 19140; ‡Division of Rheu- 2 matology, Joseph Jr. Strokes Research Institute, The Children’s Hospital of Philadel- for several months after the induction phase (24–26). In the § phia, Philadelphia, PA 19104; and Department of Immunology, ZymoGenetics, Se- present study, we observe that HgCl2 administration induces attle, WA 98102 BAFF production only in susceptible A.SW mice. TACI-Ig (which Received for publication November 10, 2004. Accepted for publication August neutralizes both BAFF and APRIL) administration prevents ANoA 23, 2005. or IgE induction in HgCl2-treated mice. 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. Materials and Methods 1 This work was supported by National Institutes of Health Grants ES-09409 and Mice ES-12646 (to M.M.). A.SW/SnJ (H-2s), C57BL/6 (H-2b), and DBA/2 (H-2d) mice were obtained 2 Address correspondence and reprint requests to Dr. Marc Monestier, Department of from The Jackson Laboratory and maintained in our animal facilities. All Microbiology and Immunology, Temple University School of Medicine, 3400 North of the mice used in our experiments were at least 2 mo old. Broad Street, Philadelphia, PA 19140. E-mail address: [email protected] 3 Abbreviations used in this paper: MB, mature B cells; T1, transitional type 1 B cells; Recombinant TACI-Ig protein T2, transitional type 2 B cells; BAFF, B cell-activating factor of the TNF family; APRIL, a proliferation-inducing ligand; BCMA, B cell maturation protein; TACI, The neutralizing TACI-Ig fusion protein and the control Hu-Ig (the Fc transmembrane activator and calcium-modulating and cyclophilin ligand interactor; portion of human IgG1 produced for TACI-Ig) were prepared as described BAFF-R, BAFF receptor; ANoA, anti-nucleolar Abs; MZ, marginal zone B cells. before (16).

HgCl2 and TACI-Ig treatment Cell preparation and staining Mercury-induced autoimmunity was induced according to a standard pro- Suspensions of mouse splenocytes were prepared through perfusion of ␮ ␮ tocol by three s.c. injections (30 g of HgCl2 in 100 l of sterile PBS) spleens with FACS buffer (PBS containing 0.5% BSA, 1 mM EDTA, and every other day (27). In addition to HgCl2, some groups of mice received 0.05% sodium azide). Following lysis of RBCs with 0.165 M NH4Cl, cells TACI-Ig or control Hu-Ig. TACI-Ig injection schedules are depicted in Fig. were washed then prestained for 10 min with rat anti-mouse CD16/CD32 1. In all cases, fusion proteins were administered i.p., and mice received (clone 2.4G2) Abs to block Fc␥R, and then incubated with optimal dilu- 100 ␮g of each protein in 500 ␮l of sterile PBS per injection. tions of the indicated Abs in Eppendorf tubes in a final volume of 50 ␮l. After 30 min on ice, cells were washed twice with FACS buffer then fixed ELISA for mouse serum BAFF in 1% formaldehyde in PBS. Serum BAFF levels were determined using an ELISA detection kit for Abs and flow cytometric analyses soluble mouse BAFF (ApoTech). A 96-well ELISA plate was coated over- night at 4°C with 50 ␮l/well of a 5 ␮g/ml solution of mAb to BAFF (5A8) Commercially obtained Abs used in these studies include PE-conjugated diluted in PBS (pH 7.4). After extensive washes and a blocking step with anti-mouse IgM (R6-60.2), FITC-conjugated anti-mouse CD21/CD35 200 ␮l of PBS containing 5% FCS for1hat37°C, a standard of recom- (7G6), PerCP-Cy5.5-conjugated anti-mouse CD19 (ID3), and allophyco- binant mouse soluble BAFF diluted in PBS containing 0.5% FCS and cyanin-conjugated anti-mouse CD45R/B220 (RA3-6B2), all from BD mouse serum samples were added. After incubation at 37°C for 2 h, wells Pharmingen. In parallel tubes, cells were stained with isotype control Abs were extensively washed. Then 50 ␮l/well of a 2 ␮g/ml solution of bio- to subtract nonspecific staining. Analyses were conducted on a dual laser tinylated mAb to BAFF (1C9) diluted in PBS containing 0.5% FCS was flow cytometer (FACSCalibur; BD Immunocytometry Systems). All flow added. After incubation for1hat37°C, wells were extensively washed. A cytometry data were analyzed by CellQuest software (BD Immunocytom- total of 50 ␮l/well of avidin-HRP conjugate (BD Pharmingen) diluted etry Systems). ␮ Ј Ј 1/500 was added for 30 min. After extensive washes, 100 l/well 3,3 ,5,5 - Statistical methods tetramethylbenzidine substrate working solution (BD Pharmingen) was Downloaded from used as a substrate, and 20 min later, 100 ␮l/well stop solution containing Using SAS version 9.0 (SAS Institute), the dependent variables serum 1 M phosphoric acid was added, and absorbance was measured at 450 nm IgG1, IgE, and ANoA-IgG were treated as continuous variables for all subtracted by 570 nm in an ELISA reader. analyses. Means, SDs, and number of observations are presented for each variable. The experimental unit was each individual mouse. ANoA immunofluorescence The experiment used a repeated measures design with each mouse eval- uated at five periods. The null hypothesis was that there would be no Serum ANoA levels were determined by indirect immunofluorescence, as difference between group or week. Before analysis, all data were tested for described previously (27). Sera diluted in PBN (PBS containing 1% BSA normality using the Shapiro-Wilk test. The data were significantly non- http://www.jimmunol.org/ and 0.02% sodium azide) were incubated with HEp-2 slides (Antibodies, normal for all variables. To apply ANOVA methods, a “normalized-rank” Inc.) for 30 min, and ANoA were detected with FITC-conjugated goat transformation was applied to the data. The rank-transformed data was mouse IgG1 (the main subclass in HgCl -induced autoimmunity) Abs 2 analyzed using a mixed-model ANOVA for repeated measures followed by (Southern Biotechnology Associates). The inverse of the highest serum multiple comparisons to detect significant mean differences between dilution at which nucleolar fluorescence could be detected was defined as groups at each week. Multiple pair-wise comparisons used the Bonferroni the ANoA titer. adjustment to maintain an experiment-wise type I error of 0.05 or less. ELISA for mouse serum IgG1 and IgE Differences between means (rejection of the null hypothesis) were consid- ered significant if the probability of chance occurrence was Յ0.05 using Total serum IgG1 and IgE levels were determined using a sandwich ELISA two-tailed test. Adjusted p values are presented for each analysis. as previously described (27). Results by guest on October 1, 2021

3.1 HgCl2 treatment induces BAFF production only in susceptible A.SW mice Because BAFF plays an essential role in B cell maturation and can affect immune tolerance (28), we investigated whether it contrib-

utes to the emergence of autoreactive B cells in HgCl2-induced autoimmunity. To assess this question, we ﬁrst measured BAFF serum levels in susceptible A.SW mice, as well as resistant

C57BL/6 and DBA/2 mice (29), all of which received three HgCl2 injections at days 0, 2, and 4. As expected, susceptible A.SW mice showed signiﬁcant increases in serum IgG1, IgE, and ANoA levels, whereas resistant strains did not show any signiﬁcant response (Fig. 2). Moreover, we observed steadily increasing serum BAFF levels exclusively in susceptible A.SW mice for several weeks

even though HgCl2 administration was only conducted between days 0 and 4 (Fig. 2). BAFF levels remained undetectable in

HgCl2-treated C57BL/6 and DBA/2 mice. As a control, A.SW mice also received injections with PBS alone, and BAFF level also remain undetectable by ELISA (data not shown). 3.2 TACI-Ig treatment during the induction phase reduces

autoantibody production in HgCl2-induced autoimmunity

To assess the effect of TACI-Ig in HgCl2-induced autoimmunity, we initially conducted a series of experiments (protocol 1; Fig. 1) FIGURE 1. Time course of HgCl and TACI-Ig fusion protein injection 2 in which groups of susceptible A.SW mice received three HgCl protocols. In both Protocols 1 and 2, mice received s.c. injections of 30 ␮g 2 ␮ injections during the ﬁrst week. TACI-Ig or control Hu-Ig was of HgCl2 in 100 l of sterile PBS three times on days 0, 2, and 4. A.SW mice were treated via i.p. injection with 100 ␮g of TACI-Ig fusion protein administered three times a week i.p. for 2 wk (from day 0) during or control Hu-Ig (the Fc portion of human IgG1) three times per week the induction phase of the disease (see details in Fig. 1). Mice starting from day 0 for 2 wk (Protocol 1) or starting from day Ϫ7for3wk treated with both HgCl2 and TACI-Ig fusion protein developed (Protocol 2). signiﬁcantly lower ( p Ͻ 0.01) ANoA IgG1 levels than mice The Journal of Immunology 6165

FIGURE 2. Serum BAFF levels increase only in

HgCl2-treated susceptible A.SW mice. A.SW, C57BL/6, and DBA/2 mice were treated with HgCl2 three times on days 0, 2, and 4 during the first week. Serum BAFF levels were measured by ELISA as described in Mate- rials and Methods and are expressed in ng/ml Ϯ SD. 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 titer (inverse of highest serum dilution that yielded nucleolar fluorescence) Ϯ SD. Serum Ig levels were measured by ELISA as described in Materials and Methods and are expressed in mg/ml Ϯ SD (IgG1) or ␮g/ml Ϯ SD (IgE). Downloaded from http://www.jimmunol.org/

treated with HgCl2 and Hu-Ig (Fig. 3). No ANoA IgM Abs were itself showed a dramatic loss of MB and T2/MZ B cells with detected in either group (data not shown). In contrast, TACI-Ig accumulation of T1 B cells (Fig. 5 and Table I). These results treatment did not prevent the Hg-induced increase in total serum confirmed previous reported data that blocking BAFF induces a IgG1 and IgE levels (Fig. 3). specific arrest in B cell development at the immature transitional T1 stage and has dramatic effect on survival of MB cells (10, 16). 3.3 TACI-Ig treatment before the induction phase reduces IgE Similar results were found when we compared group II and IV, in by guest on October 1, 2021 production in HgCl2-induced autoimmunity which both groups of mice received HgCl2 administration. Con- Because the TACI-Ig regimen in protocol 1 did not affect the poly- sistent with previous study showing that HgCl2 administration re- clonal induction of IgG1 and IgE, we administered TACI-Ig 1 wk sulted in an increase in splenic B cell numbers by week 2 (27, 30), Ϫ (day 7) before the first injection of HgCl2 at day 0 (protocol 2). our HgCl2 administration also increased absolute number of B TACI-Ig or control Hu-Ig was administered 3 times a week i.p. for cells, including MB, T2/MZ, and T1. More importantly, TACI-Ig

3 wk, whereas HgCl2 injections were given at days 0, 2, and 4 (Fig. still was able to reverse this HgCl2-induced phenomenon, showing 1). This TACI-Ig treatment regimen significantly inhibited IgE in- a normal absolute number of B cells (compared with group I) and duction in Hg-treated mice ( p Ͻ 0.05 at week 2), although there a reduction in their transitional populations with a particularly pro- were no significant differences between control and TACI-Ig nounced decrease at the T1 to T2 transition (Fig. 5 and Table I). groups with respect to serum IgG1 levels (Fig. 4). TACI-Ig pretreatment somewhat inhibited ANoA production, but statistical Discussion significance ( p Ͻ 0.001) was only observed at week 2 where The complexity of the TNF family of proteins underlies the intri- ANoA levels had yet to appear in mice that received TACI-Ig cacy of their biological effects on the immune system. An example (Fig. 4). of this complexity is the association of BAFF (BLyS) and APRIL with three known receptors: TACI, BCMA, and BAFF-R (7, 12, 3.4 Effect of HgCl2 and TACI-Ig on splenic B cell development 31). BAFF expression is particularly associated with macrophages, Because TACI-Ig blocks B cell maturation at the transitional stage, monocytes, and dendritic cells (4, 5, 32), whereas APRIL is ex- we also examined splenic B cell subpopulations to determine pressed at low levels by lymphoid cells and at higher levels by which subsets were affected by treatment with TACI-Ig and/or some tumor cells (8). In contrast to TACI and BCMA, BAFF-R

HgCl2. Peripheral B cell maturation occurs in multiple discrete selectively binds BAFF, but not APRIL (12). BAFF and APRIL steps. In the model described by Loder et al. (1), transitional B inﬂuence B cell maturation and survival at several levels. BAFF cells are divided into two subsets, T1 (sIgMhighCD21/35low) and can stimulate B cells in vivo (5, 6, 33) and in vitro (7, 28), whereas T2 (sIgMhighCD21/35high); T2 is envisioned to yield marginal APRIL acts as a costimulator for T cells (33) and enhances the zone B cells (MZ). We have examined the splenic B cell devel- survival of some tumors (8). BAFF transgenic mice produce au- opment according to this model following HgCl2 and/or TACI-Ig toantibodies and develop autoimmune disease manifestations rem- treatment. Splenocytes from four groups of mice treated for 2 wks iniscent of human systemic lupus erythematosus and Sjo¨gren’s with PBS plus Hu-Ig (group I), HgCl2 plus Hu-Ig (group II), syndrome (16, 28, 34–36). Several studies also have revealed the TACI-Ig plus PBS (group III), or HgCl2 plus TACI-Ig (group IV) presence of increased BAFF levels in sera from patients with au- were collected, stained with appropriate Abs, and examined by toimmune diseases, such as systemic lupus erythematosus, rheu- ﬂow cytometry. By comparing group I and III, TACI-Ig treatment matoid arthritis, and Sjo¨gren’s syndrome (35, 37, 38). Consistent 6166 BAFF AND CHEMICALLY INDUCED AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. TACI-Ig treatment during the induction phase prevents the FIGURE 4. TACI-Ig pretreatment prevents IgE induction in Hg-treated production of ANoA in Hg-treated A.SW mice. Groups of five A.SW mice A.SW mice. Groups of five A.SW mice received HgCl and TACI-Ig in- received HgCl and TACI-Ig injections as described in Protocol 1 (Fig. 1). 2 2 jections as described in Protocol 2 (Fig. 1). ANoA were detected by im- ANoA were detected by immunofluorescence on HEp-2 cells using iso- munofluorescence on Hep-2 cells using isotype-specific FITC conjugates type-specific FITC conjugates as described in Materials and Methods. Re- as described in Materials and Methods. Results are expressed as serum titer sults are expressed as serum titer (inversed of highest serum dilution that (inversed of highest serum dilution that yielded nucleolar fluorescence) Ϯ yielded nucleolar fluorescence) Ϯ SD. Serum Ig levels were measured by SD. Serum Ig levels were measured by ELISA as described in Materials ELISA as described in Materials and Methods and are expressed in mg/ and Methods and are expressed in mg/ml Ϯ SD (IgG1) or ␮g/ml Ϯ SD .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء .(ml Ϯ SD (IgG1) or ␮g/ml Ϯ SD (IgE .p Ͻ 0.001 ,ءءء ;p Ͻ 0.05 ,ء .(IgE)

with these findings, we also observed that HgCl2 treatment resulted in elevated levels of BAFF only in susceptible A.SW mice. The increase in serum BAFF continued for several weeks even though tion of BAFF in the synovial fluid is greater than in the blood (38). mice received only three HgCl2 injections. Lesley et al. (39) have It is therefore likely that inflamed tissue represents a major site of shown that, compared with naive B cells, anergic B cells have a BAFF production. In susceptible animals, administration of HgCl2 greater dependence on BAFF for survival. Moreover, studies have leads to immune-mediated tissue pathology including glomerulo- shown that overexpression of BAFF attenuated apoptosis by nephritis and vasculitis. In our studies, even though HgCl2 was changing the ratio between Bcl-2 family proteins in favor of cell injected only from days 0 to 4, BAFF production increased for survival, predominantly by reducing the proapoptotic Bak and in- several weeks, suggesting that the inflammatory lesions contrib- creasing its antiapoptotic partners, Bcl-2 and Bcl-xL (40, 41). By uted to BAFF production. The absence of BAFF in mice that are rescuing anergic B cells from competitive elimination and accu- resistant to mercury and do not develop tissue lesions support this mulation of autoreactive lymphocytes from attenuation of apopto- view. This production of BAFF during chemically mediated auto- sis, the increase in BAFF serum levels in A.SW mice may thus immunity will result in increased systemic levels, further affecting play a critical role in the susceptibility to HgCl2 observed in this B cells in secondary lymphoid organs and leading to increased strain. autoantibody production. This view is supported by earlier studies In Sjo¨gren’s syndrome, the presence of BAFF can be detected in showing that IFN-␥ is required for autoantibody production in tissue lesions (35). In human rheumatoid arthritis, the concentra- mercury-induced autoimmunity (42) and by observations showing The Journal of Immunology 6167

(7 days before HgCl2 injection), it signiﬁcantly blocked IgE induction although its effect on ANoA was more transient than when given only during the induction phase. The limited effect of TACI-Ig on ANoA production in protocol 2 may be due to a neutralizing response of the mouse immune system toward the human TACI-Ig fusion protein as was reported in a recent study in murine lupus (43). Under both treatment protocols, the TACI-Ig treatment did not substantially change the serum IgG1 level. This suggests

that this feature of HgCl2-induced autoimmunity is BAFF-independent and is consistent with previous findings that mice either transgenic for BAFF or injected with BAFF showed significantly increased levels of serum IgM and IgA, but not IgG (6, 34, 40). Previous studies of this model have also shown that different features such as ANoA production and polyclonal activation can be individually manipulated (44, 45). The present findings confirm that the various manifestations of Hg-induced autoimmunity are separately regulated. After maturation in the bone marrow, newly formed B cells

migrate to the secondary lymphoid organs (spleen and lymph Downloaded from nodes). These B cells do not possess all the characteristics of fully MBs, and they are referred to as transitional B cells. These transitional B cells are immature because they do not proliferate after surface IgM cross-linking. Turnover studies also indicate signiﬁ- cant cell losses at the T1–T2 transition, suggesting that this step is

selection checkpoint where self-reactive B cells may be elimi- http://www.jimmunol.org/

FIGURE 5. Effect of TACI-Ig and/or HgCl2 treatment on B cell devel- nated. Therefore, the transitional B cell stages represent important opment. Splenocytes from normal 8-wk-old A.SW mice with or without 2 regulatory milestones in B cell development (46). The character- wk of TACI-Ig and/or HgCl2 treatment (on days 0, 2, 4, 7, 9, 11) were ization of splenic B cells in both TACI-Ig-treated and TACI-trans- stained with PE anti-IgM, FITC anti-CD21/CD35, PerCP-Cy5.5 anti- genic mice revealed a specific arrest in B cell development at the CD19, and allophycocyanin anti-CD45R/B220 Abs for analysis on a dual- immature transitional T1 stage (16). Indeed, we also observed a laser FACSCalibur flow cytometer. Events (200,000) were analyzed. Left, Splenocytes were gated for forward light scatter-side scatter of live cells, similar result, in which there is a dramatic decrease in cell numbers and the percentage of B cells was calculated as percentage of live cells at the T1–T2 transition in mice treated with TACI-Ig. Right As mentioned above, the role of BAFF in B cell activation has positive for CD19 and B220. , The different stages of B cell devel- by guest on October 1, 2021 opment were calculated as percentages of live cells as follows: CD21/ suggested a therapeutic use for TACI-Ig in the treatment of au- CD35 positive-IgM intermediate (M), CD21/CD35 low-IgM high (T1), toantibody-associated autoimmune diseases. Indeed, an earlier and CD21 high-IgM high (T2/MZ). IgM-positive cells are shown. study has shown that TACI-Ig suppresses the production of anti- collagen Abs and disease in a model of collagen-induced arthritis that IFN-␥ up-regulates BAFF production by macrophages and (16). Our results in the Hg-induced autoimmunity model show that monocytes (32). TACI-Ig can be effective at inhibiting autoimmune manifestations, We used a TACI-Ig fusion protein, which neutralizes both BLyS but the specifics of this inhibition (IgE increase vs autoantibody and APRIL, to evaluate the role of these ligands and their receptors production) vary with the schedule of administration of TACI-Ig. Similar considerations may apply in the clinical use of TACI-Ig in in our HgCl2-induced autoimmunity mouse model. The adminis- human patients with autoimmune diseases. tration of TACI-Ig during the induction phase of HgCl2-treated susceptible A.SW mice led to a disruption of autoimmunity, as evidenced by significant lower ANoA levels, although there were no significant differences in terms of serum total IgG1 and IgE Disclosures levels. When TACI-Ig was administered in a prevention regimen The authors have no financial conflict of interest.

Table I. Percentage and numbers of B cell subsets in HgCl2 and/or TACI-Ig-treated mice

I II III IV

ϩ ϩ ϩ ϩ Groups PBS Hu-Ig HgCl2 Hu-Ig PBS TACI-Ig TACI-Ig HgCl2 B cells (%a) 41.61 Ϯ 0.94 42.68 Ϯ 6.48 17.60 Ϯ 0.10 29.44 Ϯ 0.05 B cells absolute numbers (ϫ106) 20.53 Ϯ 0.02 37.01 Ϯ 1.54 8.10 Ϯ 1.34 21.96 Ϯ 2.88 MBb (%) 32.41 Ϯ 1.27 34.44 Ϯ 4.84 14.28 Ϯ 0.25 22.52 Ϯ 0.02 MB absolute numbers (ϫ106) 15.61 Ϯ 0.37 30.12 Ϯ 3.57 6.16 Ϯ 1.18 15.09 Ϯ 2.42 T2/MZ (%) 4.18 Ϯ 0.40 4.56 Ϯ 0.76 1.22 Ϯ 0.11 1.84 Ϯ 0.33 T2/MZ absolute numbers (ϫ106) 2.06 Ϯ 0.15 3.95 Ϯ 0.10 0.56 Ϯ 0.05 1.36 Ϯ 0.06 T1 (%) 0.90 Ϯ 0.34 1.24 Ϯ 0.81 2.02 Ϯ 0.06 3.28 Ϯ 0.60 T1 absolute numbers (ϫ106) 0.44 Ϯ 0.17 1.02 Ϯ 0.50 0.93 Ϯ 0.18 2.42 Ϯ 0.12

a The mean percentage Ϯ SD of cells as a fraction of total splenocytes derived from two mice in each category are shown. b The indicated splenic B cell subsets were gated as illustrated in Fig. 5. 6168 BAFF AND CHEMICALLY INDUCED AUTOIMMUNITY

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