Neonatal induction of tolerance to Th2-mediated autoimmunity in rats
Anne-Christine Field,
Laure Caccavelli,
Jacqueline Fillion,
Joëlle Kuhn,
Chantal Mandet,
Philippe Druet1 and
Blanche Bellon
INSERM U430 Hôpital Broussais, Pavillon Leriche, 96 rue Didot, 75674 Paris Cedex 14, France
1 INSERM U28 Hôpital Purpan, Pavillon Lefebvre, Place du Dr Baylac, 31059 Toulouse Cedex, France
Correspondence to:
B. Bellon
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Abstract
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Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl2) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4+ Th2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of Th2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl2- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl2: (i) do not develop the mercury disease, (ii) remain resistant to HgCl2-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl2, (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl2-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for Th2-mediated autoimmunity.
Keywords: heavy metal salts, neonatal tolerance, rodents, Th2 cells
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Introduction
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For a long time the neonatal period has been thought to be a privileged period to manipulate the immune system, and particularly to tolerize against self and non-self antigens (1). Revisiting neonatal tolerance, recent papers have demonstrated that regarding the tolerance induction, the neonatal period is rather quantitatively different from the later stages of development (24).
Three non-mutually exclusive mechanisms have been posited to account for acquired unresponsiveness of T cells: clonal deletion, clonal anergy and active suppression (5). In this latter case, tolerance was shown to be restored or broken by passive transfer or depletion of regulatory T cells respectively (6).
Most autoimmune disorders, either organ-specific, such as experimental allergic encephalomyelitis (EAE), or systemic, such as the MRL/lpr lupus model, depend upon Th1 cells (79). Induction of tolerance has been well established in these autoimmune conditions by either intra-thymic injection (1012) or by oral administration of the corresponding autoantigen with regulatory T cells being generated (13) or by passive transfer of regulatory T cells (14). By contrast, Th2 cells appear to be much less frequently involved in autoimmune diseases. They play a role in EAE in immunocompromised mice (15), in lupus syndrome developed in B/W mice (16,17) and in allogeneic reactions (18). Moreover, Th2 cells were first accepted as not susceptible to tolerization (19,20) and even though induction of peripheral tolerance of Th2 cells is no longer controversial (21), a tolerant state appears more difficult to achieve in Th2 than in Th1 cells (22). For example, injections of parental spleen cells in F1 neonates lead to tolerance of Th1 cells, whereas Th2 cells escape this phenomenon and induce autoimmunity (23,24).
Mercury- or gold-induced autoimmune disorders in the Brown-Norway (BN) strain of rats represent another example of Th2-mediated autoimmunity. Indeed BN rats injected with mercuric chloride (HgCl2) or sodium aurothiopropanolsulfonate (ATPS) develop a similar lupus-like syndrome with lymphoproliferation (2527), hypergammaglobulinemia affecting mainly IgE and IgGl (28), production of numerous autoantibodies (against laminin, DNA, type II and IV collagen, and thyroglobulin) (26,2931), and an autoimmune glomerulonephritis due to the deposition of anti-laminin antibodies (3234). All the immune disorders autoregulate and thereafter animals are relatively resistant to rechallenge with HgCl2 (35,36). In humans, mercury and gold salts are also associated with the occurrence of various immune-mediated manifestations (37) and thus make the animal models valuable to study the role of environmental factors in the development of systemic autoimmunity.
For both mercury- and gold-induced immune disorders, previous studies pointed to the pivotal role of autoreactive CD4+ T cells that proliferate in the presence of naive syngeneic MHC class II+ cells (34,38,39) and induce a polyclonal B cell activation (26,34). These CD4+ T cells belong to the Th2 subset (18) and Th2 cell lines derived from gold salt-injected rats were demonstrated to transfer the whole autoimmune syndrome in naive, CD8-depleted recipients (40). Taking advantage of this strong mediation by the Th2 subset (34,4043), we investigated, in this study, the conditions to induce a solid tolerance to Th2-mediated autoimmunity. We show that neonatal injections of HgCl2 in BN rats establish a solid metal-specific tolerance to HgCl2-induced immune manifestations. Moreover, transfer of spleen cells from animals neonatally exposed to HgCl2 is shown to protect syngeneic naive rats against mercury disease, therefore suggesting a role for an active suppressive mechanism.
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Methods
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Animals
BN rats, originating from the CSEAL (Orléans, La Source, France), were bred in our own animal facilities. Animals were weaned at 3 weeks of age, and were cared for and handled according to the principles expressed in the Declaration of Helsinki on the use of animals in research. Neonates and 2- to 9-month-old female and male rats were used in the following experiments.
Experimental procedure
BN rats were s.c. injected with HgCl2 3 times a week for 2 weeks at a dose of 100 µg/100 g body wt (33) starting within 24 h after birth. Control rats received the same volume of distilled water adjusted to the same pH (3.8) as the HgCl2 solution, following the same schedule as for HgCl2 injections. At 812 weeks of age, several BN rats received a second set of injections of either HgCl2 or H2O as above, or of ATPS at a dose of 2 mg/100 g body wt 3 times a week for 8 weeks, as already described (34).
Experimental groups
BN rats were neonatally injected with HgCl2 (Hg rats) or H2O rats) (first set of injections). Rats from each of these two groups were either sacrificed at 2 weeks of age or were injected, at 812 weeks of age, with HgCl2 (Hg-Hg or H2O-Hg rats) or H2O (Hg-H2O or H2O-H2O rats) or ATPS (Hg-ATPS or H2O-ATPS rats) (second set of injections). In another set of experiments, BN rats neonatally injected with HgCl2 were re-exposed to HgCl2 either at 2, 4, 6 or 9 months of age.
Transfer experiments
Spleen cells obtained from 2- to 4-month-old BN rats neonatally injected with HgCl2 or H2O were i.v. transferred into 137Cs
-irradiated (200 rad) BN rats of 812 weeks of age. Twenty-four hours after adoptive transfer, irradiated BN rats were exposed to 50 µg/100 g body wt of HgCl2 3 times a week as described (33).
Proteinuria and renal immunofluorescence studies
Proteinuria was assessed once a week using the biuret method and was considered as abnormal when exceeding 20 mg/24 h (33). Open wedge kidney biopsy was performed in 8- to 12-week-old rats on day 15 of the second set of injections. Kidneys were obtained after killing of 2-week-old rats on day 15 of the first set of injections or of 8- to 12-week-old rats on day 30 of the second set of injections. Kidney cryostat sections were stained with a fluoresceinated sheep antibody to rat Ig as previously described (33).
Detection of anti-laminin and anti-DNA antibodies in serum
Individual serum titers of antibodies to laminin and DNA were measured by ELISA as already described (44,45). Results were expressed as percent of maximum binding activity of a standard pool of sera originated from BN rats injected with HgCl2.
Quantification of serum IgE concentration
Individual serum IgE concentrations were determined by a sandwich ELISA as follows. Microtiter plates (Maxi-Sorp; Nunc, Rocksilde, Denmark) were coated with 100 µl of the mouse monoclonal MARE antibody to the rat
chain (Immex, Brussels, Belgium), diluted to 5 µg/ml in PBS containing 0.01% NaN3 for 90 min at 37°C and overnight at 4°C. Rat serum samples were diluted in PBS buffer containing 0.1% gelatine and 0.01% Tween 20 (PBS gel Tw) and incubated for 2 h at 37°C. Mouse monoclonal MARK-1 antibody to the rat
chain labeled with horseradish peroxidase (HRP) (a gift from H. Bazin, Brussels, Belgium) was used as a second antibody, diluted 1:6 000 in PBS gel Tw and incubated for 1 h at 37°C; bound HRP activity was revealed as described (44) and absorbance at 490 nm was determined with a microplate ELISA reader (MR610; Dynatech, Alexandria, VA). Results were expressed by comparison to a standard pool of BN rat sera containing known amounts of rat IgE.
Statistical analysis
Comparisons between the different groups of rats were performed using unpaired Student's t-test or Fisher's test as post-hoc procedure after ANOVA.
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Results
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Neonatal injections of HgCl2 make BN rats tolerant to HgCl2-induced autoimmunity
Neonatally HgCl2-injected BN rats sacrificed at 2 weeks of age, i.e. after six injections of HgCl2, exhibited similar very low levels of circulating IgE and antibodies to laminin as neonatally H2O-injected BN rats sacrificed at 2 weeks of age. Moreover, in both groups renal glomeruli were free of IgG deposits (Table I
).
Neonatally H2O-injected BN rats exposed to HgCl2 at 812 weeks of age (H2O-Hg rats) exhibited the typical HgCl2-induced manifestations including a dramatic increase in serum IgE concentration (Fig. 1A
) associated with the production of antibodies to DNA (Fig. 1B
) and to laminin (Fig. 1C
). As previously described, these manifestations peaked on day 15, then declined and were no longer observed in the third month of HgCl2 administration (not shown). Moreover, on day 15 all these H2O-Hg rats displayed typical linear IgG deposits along the glomerular capillary wall (Fig. 2a
), whereas at the time of sacrifice, by the end of the second month of HgCl2 administration, glomerular IgG deposits were distributed in a granular pattern along the capillary walls (Fig. 2b
) and in the arteriolar walls. Finally, all of the H2O-Hg rats developed proteinuria (Fig. 1D
).

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Fig. 1. BN rats neonatally injected with HgCl2 are resistant to mercury disease under HgCl2 exposure at 812 weeks of age. BN rats, when neonates, were injected with H2O or HgCl2 and then, when 812 weeks old (adults), were exposed to H2O or HgCl2 (see Methods). Serum IgE concentration (A), circulating anti-DNA (B) and anti-laminin (C) antibody titers were measured using specific ELISA, and proteinuria (D) was measured using the biuret method. Data represent peak values obtained during the second set of injections, i.e. in adult rats, and are expressed as the mean ± SD from 1116 rats. Statistical analysis for Hg-Hg rats versus H2O-Hg rats: **P<0.01 and ***P<0.001.
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Fig. 2. Immunofluorescence studies. BN rats, when neonates, were injected with H2O or HgCl2 and then, when 812 weeks old (adults), were exposed to H2O or HgCl2 (see Methods). Kidney cryostat sections were stained with FITC-labeled sheep anti-rat IgG antibodies. In H2O-Hg rats (n = 16), on day 15 of HgCl2 exposure (a; original magnification x250), IgG deposits are observed along the glomerular capillary walls in a linear pattern, and on day 60 of HgCl2 exposure (b; original magnification x250), in a granular pattern along the glomerular capillary walls and in the arteriolar walls (arrow). In contrast, in Hg-Hg rats (n = 11), only on day 60 of HgCl2 exposure (c; original magnification x160) very light granular IgG deposits are disseminated within the mesangium and (d; original magnification x250) granular IgG deposits are seen mainly in arteriolar walls. No staining is ever seen either in H2OH2O rats (n = 14) (2; original magnification x160) or in HgH2O rats (n = 12) (f; original magnification x160).
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In sharp contrast, neonatally HgGl2-injected BN rats exposed to HgCl2 at 812 weeks of age (Hg-Hg rats) had similar circulating anti-DNA (Fig. 1B
) and anti-laminin (Fig. 1C
) antibody titers as Hg-H2O or H2O-H2O control rats on day 15 (Fig. 1B and C
) or at any time thereafter (not shown). In Hg-Hg rats serum IgE concentration significantly increased (P<0.05) during the second week of HgCl2 exposure as compared to H2O-H2O rats and then plateaued (48 ± 24 versus 11 ± 4 µg/ml), but this increase was much lower (P<0.001) as compared to H2O-Hg rats (48 ± 24 versus 5420 ± 3050 µg/ml). Moreover, none of the Hg-Hg rats developed proteinuria (Fig. 1D
); they only displayed scarce IgG deposits in the mesangial areas (Fig. 2c
) and granular IgG deposits in the arteriolar walls (Fig. 2d
) at the time of sacrifice (8 weeks after starting the second set of HgCl2 injections). No renal staining was ever observed either in H2O-H2O or Hg-H2O rats (Fig. 2e and f
).
Neonatally HgCl2-induced tolerance is transient and dependent upon the presence of HgCl2
BN rats neonatally injected with HgCl2 received a second set of HgCl2 injections at 2, 4, 6 or 9 months of age (Hg-Hg rats). As shown in Fig. 3
, as compared to control BN rats that received only one set of HgCl2 injections at 2 months of age, in Hg-Hg rats, serum IgE concentration (Fig. 3A
) and circulating anti-DNA (Fig. 3B
) antibody titers were lower but gradually increased with time. Moreover, in Hg-Hg rats, circulating anti-laminin antibody titers were significantly lower in rats of 2 months of age, but no significant difference was observed in rats of 4, 6 or 9 months of age as compared to control rats (Fig. 3C
). These data indicate that the tolerant state to the mercury disease is transient. However, in rats neonatally injected with HgCl2 and then receiving a second set of HgCl2 injections every 2 months (Hg-nHg rats), the tolerant state to the mercury disease was sustained (Fig. 3A
C).

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Fig. 3. Neonatally induced tolerance to mercury disease is transient and depends upon the presence of HgCl2. BN rats received a first set of HgCl2 injections when neonates and a second set either at 2, 4, 6 or 9 months of age (Hg-Hg rats, n = 39) or every 2 months starting at 2 months of age (Hg-nHg rats, n = 5). Serum IgE concentration (A) circulating anti-DNA (B) and anti-laminin (C) antibody titers were determined by specific ELISA. Data represent peak values obtained during the last set of HgCl2 injections as compared to data obtained from rats exposed once to HgCl2 at 2 months of age (control rats, n = 3). Statistical analysis for Hg-Hg rats versus control rats: *P<0.05, **P<0.01 and ***P<0.001. Statistical analysis for Hg-nHg rats versus Hg-Hg rats exposed again to HgCl2 at 2 months of age: non significant.
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BN rats neonatally injected with HgCl2 are still susceptible to gold salt-induced immune manifestations
In susceptible BN rats, HgCl2 and gold salts induce similar immune manifestations characterized by polyclonal B cell activation depending upon autoreactive Th2 cells specific for MHC class II molecules (18,40,,42). To address the specificity of the heavy metal-induced effects, gold salts were administered in BN rats neonatally exposed to HgCl2. As shown in Fig. 4
, BN rats neonatally injected with H2O and exposed at 812 weeks of age to ATPS (34) (H2O-ATPS rats) behaved like unmanipulated BN rats and exhibited an increase in serum IgE level (Fig. 4A
), and in circulating antibodies to DNA (Fig. 4B
) and to laminin (Fig. 4C
). They also demonstrated glomerular linear IgG deposits (Fig. 5a
) associated with proteinuria (Fig. 4D
). Interestingly, BN rats neonatally injected with HgCl2 and administered with ATPS at 812 weeks of age (Hg-ATPS rats) demonstrated ATPS-induced immune manifestations characterized by a closely similar increase in serum IgE concentration (Fig. 4A
) and the same titer of anti-laminin antibodies (Fig. 4C
) as those of H2O-ATPS rats. The titers of circulating anti-DNA antibodies were even significantly (P<0.01) higher in Hg-ATPS than in H2O-ATPS rats (Fig. 4B
). In the Hg-ATPS rats, glomerular IgG deposits were distributed in the same linear pattern as in H2O-ATPS rats and associated with granular IgG deposits in arteriolar walls (Fig. 5b
); moreover, Hg-ATPS rats developed a proteinuria significantly (P<0.05) higher than H2O-ATPS rats (Fig. 4D
).

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Fig. 4. The tolerance is drug specific. BN rats, when neonates, were injected with H2O or HgCl2 and then, when 812 weeks old (adult), were exposed to H2O or ATPS (see Methods). Serum IgE concentration (A), circulating anti-DNA (B) and anti-laminin (C) antibody were measured using specific ELISA, and proteinuria (D) was measured using the biuret method. Data represent peak values obtained during the second set of injections, i.e. in adult rats, either on day 14 or 21 of ATPS exposure, and are expressed as the mean ± SD from 514 rats. Statistical analysis for HgCl2-ATPS rats versus H2O-ATPS rats: *P<0.05 and **P<0.01.
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Fig. 5. Immunofluorescence studies. BN rats, when neonates, were injected with H2O or HgCl2 and then, when 812 weeks old (adults), were exposed to H2O or ATPS (see Methods). Kidney cryostat sections were stained with FITC-labeled sheep anti-rat IgG antibodies. In H2O-ATPS rats (n = 8) (a; original magnification x 250) linear staining along the glomerular capillary walls is observed; in HgCl2-ATPS rats (n = 5) (b; original magnification x250). IgG deposits are observed in a linear pattern along the glomerular capillary walls and in a granular pattern in the arteriolar walls (arrow). No staining is ever seen either in H2O-H2O rats (n = 14) (c; original magnification x160) or in Hg-H2O rats (n = 12) (d; original magnification x160).
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Tolerance to mercury-induced autoimmunity can be adoptively transferred
Lightly irradiated BN rats, that have received spleen cells originated from naive BN rats and then have been exposed to HgCl2 (control rats), exhibited an increase in serum IgE concentration that peaked at 3180 ± 2600 µg/ml (Fig. 6A
), and developed anti-laminin and anti-DNA antibodies whose concentration peaked at 59.3 ± 32.8 and 36.5 ± 17.7 AU respectively (Fig. 6C and B
).

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Fig. 6. Tolerance is adoptively transferred by spleen cells. Naive recipient BN rats were 137Cs -irradiated (200 rad) and i.v. injected with 100 x106 spleen cells originating from naive BN rats or from BN rats neonatally exposed to HgCl2 (Hg-tolerant rats); transfer of spleen cells was done the day of irradiation; 24 h later, BN recipients and mercury-tolerant littermates (Hg-Hg rats) received injections of 0.5 mg/kg HgCl2 as described in Methods. Serum IgE concentration (A), circulating anti-DNA (B) and anti-laminin (C) antibody titers were determined by specific ELISA. Data represent peak values and are expressed as the mean ± SD from two to six rats. Statistical analysis for recipients of cells from mercury-tolerant rat versus recipients of cells from naive rats: *P<0.05 and **P<0.01.
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Lightly irradiated BN rats that had received spleen cells originating from BN rats neonatally exposed to HgCl2 and then been exposed to HgCl2 (transfer rats) exhibited, at the peak of production, a significant lower increase in serum IgE concentration as compared to control rats (P<0.001) (Fig. 6A
). Similarly, the peak of production of anti-laminin antibodies was significantly (P<0.02) lowered as compared to control rats (Fig. 6C
). Circulating anti-DNA antibody titers were also decreased as compared to control rats, although not significantly (Fig. 6B
). In transfer rats, maximal circulating anti-autoantibody titers were never significantly different as compared to BN rats neonatally exposed to HgCl2 and exposed again to HgCl2 after 2 months of age (Hg-Hg rats) (Fig. 6B and C
). At the time of sacrifice, i.e. after 8 weeks of HgCl2 exposure, in transfer rats, kidney IgG deposits displayed the same pattern in the mesangial areas as in Hg-Hg rats (not shown); whereas, in control rats, glomerular IgG deposits were distributed in a typical granular pattern along the capillary walls and in the arteriolar walls (not shown).
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Discussion
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Susceptible BN rats exposed to HgCl2 or ATPS develop a similar Th2-mediated, systemic autoimmune disease, due to the emergence of autoreactive Th2 cells that recognize MHC class II molecules. In the present study we demonstrate that: (i) neonatal injections of HgCl2 do not induce the mercury disease in 2-week-old BN neonates and induce immunological tolerance since mercury-induced immunopathological manifestations are abrogated, or profoundly reduced, when these rats are challenged with HgCl2 at 8 weeks of age; (ii) this tolerance is mercury specific because gold-induced immunopathological manifestations are still observed in HgCl2-tolerant rats; (iii) this tolerance is transient but can be sustained providing regular exposure to HgCl2; and (iv) this tolerance is dominant since it is adoptively transferable into syngeneic animals by spleen cells from tolerant rats.
To the best of our knowledge, induction of tolerance to a Th2-mediated autoimmune model, following neonatal injection of a chemical, has not been previously reported. In mice as well as in rats, we and others demonstrated that injection of F1 spleen cells into neonates of one parental strain results in transplantation tolerance due to the tolerance of Th1 cells. In contrast, neonate Th2 cells that recognize allogeneic MHC class II molecules are present and responsible for B cell polyclonal activation and systemic autoimmunity (23,24,4648). These results and those from other experimental systems indicate that tolerance is much more difficult to achieve in Th2 than in Th1 cells (19,22). In that respect, it is noteworthy that HgCl2 exposure through oral or respiratory routes not only does not induce tolerance, in contrast to what has been observed in several Th1-mediated autoimmune diseases (13), but leads to systemic autoimmunity (49). However, it has been shown that individuals allergic to bee venom can be desensitized following exposure to tolerogenic amounts of the relevant antigen phospholipase A2 (50) and the tolerance thus obtained is mediated by IL-10 producing cells that are reminiscent of Tr1 cells (51).
The fine specificity of T cells involved in the HgCl2- and ATPS-induced models of systemic autoimmunity is still unsolved. Our previous data, in both models, indicate that T cells are generated that recognize self-MHC class II molecules or, more likely, a ubiquitous peptide presented in the context of MHC class II molecules (52). Those T cells have a Th2 phenotype in BN rats, and induce polyclonal B cell activation both in vivo and in vitro (40,52). In mice exposed to mercury or gold salts, other authors have shown metal-specific T cells but did not evidence their pathogenic role (53). The fact that neonatal injections of HgCl2 induce, in adults, tolerance to HgCl2 but not to ATPS, advocates the existence of such metal-specific T cells. This view is strengthened by our observation that neonatal injections of ATPS induce, in adults, tolerance to ATPS but not to HgCl2 (not shown). Higher anti-laminin antibody titers and proteinuria levels in Hg-ATPS rats than in H2O-ATPS rats than in H2O-ATPS rats may be due to a bystander activation of the previously suppressed autoreactive T cells specific of the mercury-modified (MHC class IIpeptide) complex following the activation of autoreactive T cells primed by the ATPS-modified (MHC class IIpeptide) complex. Whether HgCl2 or ATPS is involved in Th2-mediated autoimmunity, autoreactive T cells that are induced may recognize, in the context of MHC class II molecules, either different ubiquitous peptides or self-peptides specifically altered by the heavy metal (40,54). At this point, one may speculate that whether HgCl2 is administered in neonate or in adult BN rats, T cells of the same specificity are generated but they may differ by their pattern of cytokine production. Those cells, that need to be regularly exposed to HgCl2 in order to maintain their telerogenic potential, are likely to be regulatory cells as demonstrated in other models of tolerance and may produce inhibitory cytokines such as IL-10 or transforming growth factor-ß (5557). This hypothesis is emphasized by the ability of spleen cells from tolerant rats to transfer tolerance in naive syngeneic recipients. The precise phenotype of these cells, their fine specificity and their profile of cytokine production remain to be investigated. Clonal deletion is another mechanism posited to explain peripheral tolerance (5). In previous experiments, we demonstrated that in adult BN rats, HgCl2 induces a polyclonal T cell expansion (58) and in BN rats neonatally injected with HgCl2 no changes occur in the T cell repertoire (not shown). Considering these findings clonal deletion might be ruled out as an associated mechanism of tolerance to the mercury disease. Taken together our data favor specific dominant tolerance due to regulatory cells rather than to clonal deletion; however, anergy as an associated mechanism of tolerance cannot be ruled out.
We have previously shown that, besides the induction of autoreactive T cells, HgCl2 induces in vitro IL-4 gene expression in normal T cells following a protein kinase C-dependent pathway (59,60). This latter effect is still observed in tolerant animals (not shown) and likely to explain IgE production that, if deeply reduced, is not completely abrogated and furthermore plateaued after 2 weeks of HgCl2 administration. Thus our data indicate that production of IL-4 associated with the direct effect of HgCl2 on the encoding gene is not affected by neonatal exposure to HgCl2 and nevertheless not sufficient to induce the mercury disease. This suggests a very efficient regulatory process, even more potent than towards the production of autoantibodies. CD8+ T cells that have been involved in IgE tolerance (61) may be at play. Moreover, previous studies have shown that HgCl2 exposure not only induces autoimmune manifestations that spontaneously resolve even if HgCl2 injections are continued, but also makes the animals resistant to rechallenge with full-dose HgCl2 (36). The exact mechanisms involved in regulation and resistance are still ill-defined. There is some evidence that Th1/Th2 balance may be involved (42,43,62,63). Indeed the importance of Th1 cells has been suggested since anti-IL-2 receptor antibody treatment, that preferentially blocks the effect of Th1 cells, delays the regulatory phase (42). It has been shown that depletion of CD8+ T cells partially reverses the resistant state without affecting the regulatory phase (36). Furthermore, adoptive transfer of CD8+ T cells from resistant rats can transfer resistance in naive syngeneic rats (35,36). In the present study, we show that neonatal administration of HgCl2 is not pathogenic and induces the development of disease resistance in adult animals. This neonatally induced resistant state is specific and can be transferred to naive recipients with spleen cells, indicating a role of dominant tolerance. Co-transfer of spleen cells from naive and mercury-tolerant animals remains to be investigated to emphasize this phenomenon of active suppression. Whether this state of neonatally-induced resistance is similar to that described in adult rats treated with HgCl2 remains to be determined; particularly, whether CD4+ or CD8+ T cells are involved in the transfer and maintenance of neonatal tolerance remains to be determined.
In summary, our findings of a solid tolerant state induced by neonatal injections of HgCl2 indicate that neonatal tolerance can be induced to a systemic autoimmune disease mediated by Th2 cells. Further investigation of this dominant tolerance will be of major interest because it might be instrumental in lupus autoimmunity and allergy.
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Acknowledgments
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This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the French Ministère de l'Environnement and the Association pour la Recherche contre le Cancer. J. K. and B. B. are supported by the Centre National de la Recherche Scientifique. We wish to gratefully thank Abdelhadi Saoudi for very fruitful discussions and reading this manuscript. The skillful assistance of M. Paing and P. Kitmacher for photographic work is greatly acknowledged.
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Abbreviations
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ATPS sodium aurothiopropanolsulfonate |
BN Brown-Norway |
EAE experimental autoimmune encephalomyelitis |
HRP horseradish peroxidase |
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Notes
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Transmitting editor: H. Bazin
Received 9 February 2000,
accepted 10 July 2000.
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