Effects of the Murine Genotype on T Cell Activation and Cytokine Production in Murine Mercury-induced Autoimmunity

Journal of Autoimmunity (1997) 10, 347–355

Effects of the Murine Genotype on T Cell Activation and Cytokine Production in Murine Mercury-induced Autoimmunity Uno Johansson, Birgitta Sander and Per Hultman Departments of Pathology, Linko¨ping University, Linko¨ping, Sweden

Received 10 September 1996 Accepted 23 April 1997 Key words: autoimmunity, cytokines, mercury, mice, Th1/Th2

Mercury induces a systemic autoimmune condition characterized by autoantibodies to the nucleolar protein fibrillarin (AFA) and systemic immunecomplex (IC) deposits in genetically susceptible mouse strains. This study examines T cell activation and cytokine production following mercury exposure in genetically susceptible and resistant strains. Mercury injected s.c., according to the protocol for induction of autoimmunity, caused an early T cell activation, measured as an increase of IL-2-producing cells, and increased expression of the IL-2-receptor proteins CD25 and CD122 and of the proliferation marker CD71 on days 2–4 in the susceptible A.SW and A.TH strains. This was followed by a long-lasting increase in the number of T cells, dominated by CD4 + cells. Mice of the susceptible A.SW strain showed a modest increase of TNF-á-, IFN-ã-, and IL-4-producing cells after 4–6 days, and a very distinct increase of IL-4-producing cells on days 8–10. The susceptible SJL strain (H-2s), severely deficient in Th2-promoting CD4 + , NK1.1 + T cells, showed no increase of IL-4 + cells on days 8–10. Instead, the number of IFN-ã-producing cells was increased. Susceptible mice developed an increase of Ig-producing cells, AFA, and systemic IC-deposits. Genetically mercury-resistant A.TL mice showed a minimal increase of T cells, but no increase in cytokine-producing cells. We conclude that autoimmunogenic doses of HgCl2 induce an activation and proliferation of T cells in genetically susceptible mouse strains, as well as a broad increase of cytokine-producing cells, followed by a late predominance of the Th2-associated IL-4. One strain, severely deficient in Th2-promoting CD4 + , NK1.1 + T cells, lacked the increase in IL-4 + cells, indicating that a predominantly Th2-response is not necessary for induction of autoimmunity by mercury. However, a Th2-dominated response led to a faster and stronger B cell activation. © 1997 Academic Press Limited

Introduction

fibrillarin (reviewed in [1]), or indirectly via activation of proteases in antigen presenting cells (APC) [7]. Therefore, mercury has the potential to create altered peptides of fibrillarin, which may lead to presentation of cryptic epitopes to T cells. In contrast to this, induction of CD4 + cells reacting to normal (not mercury-modified) syngeneic class II molecules on B cells [8], leading to B cell activation and production of autoantibodies against a number of self- and non-selfantigens [6, 9], is the established mechanism in the rat model. As in rats [10], many mouse strains develop T and B cell proliferation after treatment with mercury [11, 12]. This is in accordance with in vitro studies showing that mercury is able to induce proliferation of B and T lymphocytes in a number of species, including mice (reviewed in [13]). Although intact T cell function is critical for the induction of autoimmunity by mercury in mice [14], the relationship between lymphocyte

Mercury exposure induces a systemic autoimmune disease in susceptible rodent strains (reviewed in [1]). Susceptibility is genetically determined with the most important genes residing in MHC class II loci [2, 3]. Despite similarities between mercury-induced autoimmunity in the mouse and the rat, there are important differences; the main autoantibody specificity is the 34 kDa nucleolar protein fibrillarin in mice [4, 5], and the basement membrane protein laminin in rats [6]. Recent studies indicate different autoimmune mechanisms: mercury might interact with the main autoantigen (fibrillarin) in murine autoimmunity, either via direct binding to cysteine residues in Correspondence to: Per Hultman, Department of Molecular and Immunological Pathology, University Hospital, S-581 85 Linko¨ping, Sweden. Fax: +46-13132257; E-mail: [email protected]. 347 0896-8411/97/040347+09 $25.00/0/au970149

© 1997 Academic Press Limited

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proliferation and autoimmunity is uncertain. Different requirements regarding dose [15] and genotype [16, 17] indicate that lymphocyte proliferation and autoimmunity need not be linked. In 1991, Goldman et al. [18] suggested that the susceptibility of rodents to develop systemic autoimmune disease in response to metals correlates with the propensity to develop a Th2 response, and that resistance to disease correlates with a predominantly Th1 response. Recently, a number of studies have been published which support this hypothesis in the rat model (reviewed in [19]). In contrast to the experimental evidence for a Th1:Th2 imbalance in the rat model, few studies so far have dealt with the Th1:Th2 imbalance concept in murine mercury-induced autoimmunity. A preferential increase of IL-4 mRNA in B10.S mice susceptible to the induction of autoimmunity, as compared with resistant B10.D2 mice, was shown by van Vliet et al. [12], and increase in the Th2-dependent IgG1 and IgE serum Ig isotypes was suppressed in mercury-treated A.SW mice given antiIL4 mAb [20]. Recently, Biancone et al. [21] described aggravation of autoimmune disease in mercurytreated mice, presumed to be mediated via inhibition of Th1 cells, after in vivo anti-CD2 treatment. We have reported previously that in vivo and in vitro mercury treatment induces IL-1 secretion in both susceptible and resistant mouse strains [22]. Mercury exposure in vitro induces IL-2 and IFN-ã production in mouse lymphocytes [23, 24]. A better knowledge of the cytokine profile in genetically susceptible and resistant mouse strains after in vivo mercury treatment is needed to understand the mechanisms leading to autoimmunity. We report in this study that mercury treatment induces an increase of cells producing Th1- and Th2associated cytokines in susceptible strains. A late predominance of the Th2-associated IL-4 cytokine was seen in most susceptible strains. However, a strain deficient in production of IL-4 developed a systemic autoimmune disease similar to that in strains with intact IL-4 production.

Material and Methods Mice SJL/N, A.SW (H-2s), and BALB/c (H-2d) mice were obtained from Bommice Breeding and Research Centre, Ry, Denmark. A.TH (H-2t2), A.TL (H-2tl), and BALB.B (H-2b) mice were obtained from Harlan Ltd, Oxon, England. All mice were 8–12 weeks old at the onset of the experiments, housed under 12 h dark– 12 h light cycles in a high-barrier unit, kept in steelwire cages, and given sterilized pellets (Type R 36, Lactamin, Vadstena, Sweden) and tap water ad libitum.

Treatment Treatment, in accordance with the standard protocol used for induction of autoimmunity with mercury

Table 1. mAbs used in flow cytometry for phenotyping and activation/proliferation marker expression Antibody CD45R/B220-FITC* CD90/Thy1.2-FITC CD3-FITC CD4-FITC CD8a-PE CD25-biotin CD122-biotin CD69-biotin CD71-biotin

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*All antibodies are from Pharmingen, San Diego, CA, USA.

(multiple injections), consisted of a s.c. injection of 1.0 mg HgCl2/kg body weight in 0.1 ml PBS on the dorsum every third day for up to 48 days. Single injection treatment consisted of giving a total of 1.0 mg HgCl2/kg body weight in 0.1 ml of PBS, injecting 0.05 ml s.c. on the outside of each thigh. Controls received an equal volume of PBS.

Spleen cell preparation Groups of four to five mice were killed at intervals from 12 h to 49 days after starting the injections. Preparation of splenic erythrocyte-free, single-cell suspensions was performed as previously described [25].

Dual colour flow cytometry analysis of T cell phenotypes and activation/proliferation markers The splenic single-cell suspensions were incubated with one or two mAbs (Table 1; Pharmingen Inc., San Diego, CA), and cells acquired using a FACScan (Becton Dickinson, Mountain View, CA) as previously described [25]. Analysis was performed on the lymphocyte population using the Cell Quest program (Becton Dickinson). Tissues from mercury-treated and control mice were always prepared and run simultaneously in the flow cytometer. The mean fluorescence channel (MFC) exhibited by an activation/ proliferation marker in a mercury-treated animal was divided by the mean MFC obtained for this marker in the control animals. In this way, a ratio of fluorescence intensity was obtained for every mercury-treated animal, and the mean fluorescence ratio in each group of mercury-treated mice was expressed as a percentage change compared with the controls.

Enumeration of cytoplasmic Ig + cells on slides Single-cell splenocyte suspensions were separated on Lympholyte-M (Cedarlane, Ontario, Canada), and 2×105 mononuclear cells were centrifuged onto glass slides (Shandon Cytospin 2, Shandon, UK) at 80×g for 7 min and allowed to dry at RT. The cytospin slides

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were fixed in absolute ethanol containing 5% glacial acetic acid at −20°C for 20 min and stored in PBS at 4°C until analysed further. The slides were then incubated with biotin-conjugated rat mAb to mouse IgG1, IgG2a, IgG2b, IgGE, IgM (Pharmingen), or FITC-conjugated goat anti-mouse IgG3 Ab (Southern Biotechnology, Birmingham, AL), diluted in HBSS containing 2% FCS for 20 min, washed and, if appropriate, incubated with streptavidin–FITC (Dakopatts) for 20 min. After washing the slides were mounted using buffered glycerol containing 2% diazabicyclo2,2-octane (Sigma, Milwaukee, WI). On each slide, 2×105 cells were examined and the number of cells showing a bright cytoplasmic (i.e. positive) staining was scored.

Assay for cytokine-producing cells Inguinal and mesenterial lymph nodes were examined for the presence of cytokine-producing cells using indirect immunofluorescence as previously described [26]. This method has been compared in vivo with quantitative PCR as well as staining of cytokines in frozen tissues and has been found to give very similar results [27]. Briefly, 10 ìl of a suspension of 5×106 cells/ml was added to a reaction field (BioRad adhesion slides, Munich, Germany), the cells were allowed to adhere, fixed with paraformaldehyde, then dried and stored at −70°C. After thawing, the cells were permeabilized by saponin, unspecific staining blocked by FCS, and a mAb or a pair of mAbs was incubated on the reaction fields. The mAbs used were IFN-ã (clone XMG1.2) [28], TNF-á (clone XT22) [29], IL-2 (clone S4B6) [30], and IL-4 (clones 11B11 and 1D11) [31, 32]. mAbs of the corresponding isotype and concentration, but with irrelevant specificity, were used as a first step control antibody. Hamster anti-rat IgG was used as the detecting antibody. The whole reaction field (slide well) was examined for the presence of cells with an intracellular staining pattern of Golgi type [26]. For all strains except A.TL the number of cells in each of the examined lymph node stations in a single animal was sufficient for analysis of the different cytokines. However, due to the low number of lymph node cells in the A.TL strain, lymph nodes from a specific lymph node station in two mice were pooled and treated as one sample.

Serum antinuclear antibodies The presence, pattern and titer of serum antinuclear antibodies of the IgG class was determined by indirect immunofluorescence using HEp-2 cells as a substrate [14].

Tissue immune deposits Pieces of the left kidney were examined using direct immunofluorescence, performed as previously described [14], using FITC-conjugated goat anti-mouse

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IgG antibodies (Southern Biotechnology, Birmingham, AL) and anti-C3c antibodies (Organon-Technica, West Chester, PA). The titer was determined by serial dilution of the antibodies to 1:5120.

Statistics All differences between mercury-treated animals and controls were assessed by the Mann–Whitney U-test.

Results Number of T cells with different phenotypes after multiple HgCl2 injections Multiple injections of HgCl2 (see Material and Methods) caused an increased number of splenocytes positive for T cell markers in mice of the strains A.SW, SJL (H-2s), and A.TH (H-2t2), which are all H-2KsAsEsSs (Figure 1A–C). The A.SW strain (Figure 1A) showed a significant increase of T helper (CD4 + ) cells, reaching a maximum after 5 days and then showing a slow decline in this increase up to day 28. The number of cells positive for the pan-T cell marker CD90 was maximal after 14 days. The A.TH strain showed an increase of CD90 + cells on days 7–28, and an increase of CD4 + cells on days 14–28 (Figure 1B). The SJL strain showed a significant increase of CD90 + and CD4 + cells only on day 28 (Figure 1C). The A.TL strain (H-2t1:H-2KkAkEkSk), resistant to induction of autoimmunity by mercury [3], developed a slight increase in the number of CD90 + cells on days 7–28 without any significant increase of CD4 + or CD8 + cells (Figure 1D). BALB/c (H-2d) mice which are susceptible, and BALB/B (H-2b) mice which are resistant, to induction of systemic IC-deposits by mercury [3] showed no increase in the number of splenic CD90 + , CD4 + or CD8 + cells on day 10 after starting multiple s.c. injections of mercury (data not shown).

Expression of T cell activation markers after multiple HgCl2 injections Analysis of cell surface activation markers on splenic T (CD90 + ) cells revealed in the A.SW strain a substantial and significant increase in expression of the IL-2R subunit proteins á (p55; CD25) and â (p75; CD122) on days 2–4, which was accompanied by a significantly increased expression of the proliferation marker CD71 (transferrin receptor) on day 4 (Figure 2A). The early activation marker CD69 showed an increased mean expression at 12 h in the A.SW and A.TH mice (Figure 2A & B). The A.TH strain showed a significantly increased expression of the CD25, CD122, and CD71 markers which was maximal after 4 days (Figure 2B). In analogy with the late increase in T cells, the SJL strain did not show a significantly increased expression of the IL-2R proteins or CD71 until days 14–28; all these markers had returned to the baseline

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IL-4 + cells on days 8–10. The cytokine pattern in the mesenterial lymph nodes was similar to that in the draining lymph nodes (Figure 3B). In SJL mice, mercury caused a sustained increase in IL-2 + and IFN-ã + cells on days 6–10 without any increase in IL-4 + cells (Figure 3C). A similar cytokine pattern was seen in the mesenterial lymph nodes (data not shown). A.TL mice given multiple injections showed no increase in cytokine-producing cells, neither in the draining inguinal lymph nodes (Figure 3D), nor in the mesenterial lymph nodes (data not shown). In BALB/c mice multiple Hg injections caused an increase in the number of IFN-ã + and IL-4 + cells on days 8–10 (Figure 3E). A similar pattern was found in the mesenterial lymph nodes (data not shown). When the cytokine pattern was examined on day 10 after starting mercury treatment in BALB.B mice, no increase was seen in any of the cytokines (data not shown). To study further a possible influence of the injection site on the number of cytokine-producing cells, A.SW mice were injected s.c. either on the anterior part of the dorsum or on the outside of the thigh, and the number of cytokine-producing cells assessed in the inguinal, brachial and mesenterial lymph nodes. The pattern of cytokine-producing cells was similar in the different lymph node stations irrespective of the injection site, although the absolute number of cells varied slightly (data not shown).

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Figure 1. The number of splenocytes positive for the T cell phenotype markers CD90, CD4, and CD8 after multiple HgCl2 injections. (A) A.SW mice; (B) A.TH mice; (C) SJL mice; (D) A.TL mice. Four to five mice were used at each point. Bars denote mean±1 SD. *significantly different from controls (P<0.05, Mann–Whitney U-test).

level on day 49 (Figure 2C). The A.TL strain showed no significant increase in expression of T cell activation markers (Figure 2D).

Number of cytokine-producing cells after multiple HgCl2 injections Multiple HgCl2 injections caused a significant increase of IL-2 + cells in the lymph nodes draining the injection site in A.SW mice on days 4–5 (Figure 3A), and the number of IFN-ã + cells was significantly increased on days 3–8 (Figure 3A). There was a large increase of

Number of cytokine-producing cells after a single HgCl2 injection A single s.c. injection of HgCl2 in A.SW mice caused a significantly increased number of IL-2 + cells in the draining lymph nodes on days 3–4 (Figure 4A), and of IFN-ã + and TNF-á + cells on days 4–6. IL-4 + cells first appeared after 3 days and maintained a modest level on days 5–10. A similar increase of cytokineproducing cells was also seen in the mesenterial lymph nodes (not shown). In SJL mice given a single s.c. HgCl2 injection, the draining lymph nodes on day 4 showed a modest increase in the number of IFN-ãand IL-4-producing cells (Figure 4B). A.TL mice given a single injection of HgCl2 did not show any increase of cytokine-producing cells (Figure 4C). A single injection of HgCl2 in BALB/c mice caused a significant increase of IFN-ã + and IL-4 + cells on days 4–5 (Figure 4D). The BALB.B mice showed no significant increase in any cytokine-producing cells (data not shown).

Pathogenicity of mercury injections We investigated the number of Ig-secreting cells, serum ANA and systemic IC-deposits in mice treated with single vs. multiple injections of HgCl2. In A.SW and SJL mice, a single injection of mercury caused a slight and a moderate increase, respectively, in the number of IgG2b-producing cells (data not shown). Specifically, neither cells of the Th2-dependent IgG1 and IgE isotypes, nor the Th1-dependent IFN-ãinduced IgG2a isotype were increased. SJL, A.SW,

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Figure 2. The expression of activation and proliferation markers on T (CD90 + ) cells after multiple HgCl2 injections. (A) A.SW mice; (B) A.TH mice; (C) SJL mice; (D) A.TL mice. Four to five mice were used at each point. CD69: early activation marker; CD71: transferrin receptor; CD25: á- and CD122: â-subunit protein of IL-2R. The change in expression of the markers was calculated by comparing the expression in mercury-treated and control mice. Values denote mean change (expressed as a percentage) compared with the controls.

BALB/c and BALB.B mice given a single injection of mercury developed neither serum ANoA, nor systemic immune-complex deposits (data not shown). Multiple mercury injections in A.SW mice induced a large increase in the number of cIg + cells of all isotypes on day 14, whereas SJL mice showed an increase of cIg + cells of the IgG1, IgG2a, or IgG2b isotypes, with a maximum level on day 28 (Johansson et al., pers. obs.). The A.SW and SJL mice started to develop ANoA on day 14, which was followed by systemic immune-complex deposits (Johansson et al., pers. obs.). No increase of Ig-secreting cells, ANoA, or systemic immune-complex deposits was detected in A.TL mice. BALB/c mice given multiple injections showed a significant increase of cIg + cells of the IgG1, IgG2a, IgG2b, and IgG3 isotypes on days 10–14 (data not shown). BALB.B mice (H-2b) treated with multiple injections showed a small but significant increase of cIg + cells of the IgG1 isotype after 10 days (data not shown).

Discussion Injection of HgCl2 caused a transiently increased expression of the CD69 marker on T cells after 12 h in the susceptible A.SW and A.TH strains. CD69, one of

the first cell surface glycoproteins to appear after lymphocyte activation [33], is expressed following engagement of the TCR/CD3 receptor complex in vitro [34] and exposure to mitogens such as LPS and p43 in vivo [35]. The common denominator for induction of CD69 expression in T cells is activation of p21ras [36]. Interestingly, treatment of T cells with mercury in vitro leads to nuclear signalling activating the NF-êB transcription factor, probably mediated by p21ras [37]. The expression of CD69 on T cells after injection of mercury was followed by an increased expression of the á-(CD25) and â-(CD122) chains of the IL-2R on T cells in A.SW and A.TH mice. The presence of both the á- and â-chains is essential for formation of a functional, signal-transducing IL-2R in the mouse [38]. Expression of the IL-2R proteins coincided with an increased number of IL-2 + cells and increased expression of the proliferation marker CD71 on T cells. Taken together, these factors provide evidence for in vivo activation and proliferation of peripheral T cells 2–4 days after starting HgCl2 injections. This is in agreement with the proliferation seen after in vitro stimulation of lymphocytes from A.SW and BALB/c mice with mercury [17, 23]. In our study, strains susceptible to mercury-induced autoimmune disease responded to a single injection of

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Figure 3. Fraction of lymph node cells showing intracellular cytokine staining after multiple HgCl2 injections. (A) A.SW-inguinal lymph nodes; (B) A.SW-mesenterial lymph nodes; (C) SJL-inguinal lymph nodes; (D) A.TL-inguinal lymph nodes; (E) BALB/c-inguinal lymph nodes. Bars on the lines indicate 1 SD. Unfilled bars to the left in each figure denote the mean number of positive cells (+1 SD) found in the controls. An unfilled sign on top of the line indicates a significantly increased (P<0.05) number of cytokine-positive cells in the mercury-treated mice. Four to five mice were used at each point, except for A.TL mice where eight to 10 mice were used. ■, IL-2; m, IFN-ã; ●, IL-4; ◆, TNF-á.

mercury with an early increase of IL-2 + cells, which was followed by a transient increase of IL-4- and IFN-ã-producing cells on days 4–6. This might be interpreted as an early activation of precursor Th cells (pTh) producing IL-2, followed by an activation of Th0 cells producing cytokines of both the Th1 and the Th2 type [39]. The number of IL-4 + cells remained increased on days 5–10, indicating a sustained response dominated by the Th2-associated IL-4 cytokine. However, this increase of IL-4 + cells was not sufficient to induce the strong B cell activation, autoantibody formation, and systemic immunecomplex disease which is characteristically seen in murine mercury-induced autoimmune disease [11]. Multiple injections of mercury caused a different cytokine pattern. The second injection on day 3 was critical in this respect, since it suppressed the increase of cytokine-producing cells seen on days 4–5 after a

single mercury injection. In the A.SW and BALB/c strains, repeated mercury injections caused a distinct increase of IL-4-producing cells on days 8–10, outnumbering the IFN-ã-producing cells. The dominance of IL-4 + cells on days 8–10 indicates an early burst of IL-4 production from specialized T cells, basophils, or mast cells directing the differentiation of CD4 + cells towards the Th2 phenotype [40]. In susceptible rats, mercury induces an increased IL-4 mRNA expression in mast cells [41]. In response to a broad T cell activation following injection with anti-CD3 or superantigen, the highly specialized CD4 + , NK1.1 + T cells produce the early burst of IL-4 in mice [42]. However, injection of Leishmania major causes an equally rapid burst of IL-4 from CD4 + , NK1.1 − T cells in susceptible strains [43]. The relative importance of the different early IL-4-producing cells in promoting a subsequent Th2

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Figure 4. Fraction of inguinal lymph node cells showing intracellular cytokine staining after a single HgCl2 injection. (A) A.SW; (B) SJL; (C) A.TL; (D) BALB/c. Bars on the lines indicate 1 SD. Unfilled bars to the left in each figure denote the mean number of positive cells (+1 SD) in the controls. An unfilled sign over the line indicates a significantly increased (P<0.05) number of cytokine-positive cells in the mercury-treated mice. Four to five mice were used at each point, except for A.TL mice where eight to 10 mice were used. Key as in Figure 3.

response in mercury-treated mice has not been examined. However, SJL mice are markedly deficient in Th2-promoting CD4 + , NK1.1 + T cells [44], which is likely to explain the lack of an increase of IL-4 + cells in these mice (found in the present study). Thus, CD4 + , NK1.1 − T cells, basophils and mast cells apparently did not produce sufficient amounts of IL-4 to compensate for the deficient supply of IL-4 from CD4 + , NK1.1 + cells, since the T cells in SJL mice can be primed to become IL-4-producing Th2 cells if an appropriate amount of IL-4 is provided at an early stage [44, 45]. The predominance of IL-4 + cells on days 8–10 in mercury-treated A.SW and BALB/c mice is likely to be important in the proliferation and maturation of B cells in these strains after mercury treatment [3, 11]. Since SJL mice develop a systemic autoimmune condition very similar to that in A.SW mice after mercury treatment [3, 11], but showed few IL-4 + cells and instead a predominance of IFN-ã + cells on days 8–10, a predominantly Th2-type response seems not to be necessary for the induction of autoimmunity by mercury in mice. However, the B cell response, measured as the number of B cells and levels of serum Ig, was weaker and developed later in SJL mice than in A.SW mice (Johansson et al., pers. obs.), indicating that an IL-4-dominated, Th2-type response augments mercury-induced B cell activation. This is supported by observations in A.SW mice treated with mercury

and anti-IL-4 mAb [20], which showed a reduced increase in serum concentration of Th2-dependent Ig isotypes compared with A.SW mice treated with mercury only. The A.TL mice showed no increase in Ig-producing cells (Johansson et al., pers. obs.), and no systemic autoimmunity [3] in response to mercury. However, there was a small but significant increase of CD90 + (Thy-1.2 + cells), which was not accompanied by a significant increase of CD4 + or CD8 + cells. CD90 + CD4 − CD8 − make up 1–5% of the splenic T cells in normal mice [46]. The increase of such immature peripheral T cells in mercury-treated mice might have been induced by an increased release of immature T cells from the thymus. Thymic atrophy and disordered thymic T cell populations have recently been reported in mercury-treated rats [47]. In contrast to the slight increase in immature peripheral T cells, the A.TL strain showed no increase in either Th1- or Th2-associated cytokines. Specifically, there was no increase in Th1-associated IFN-ãproducing cells. We were also unable to detect any increase of IFN-ã-producing cells in the mercuryresistant [16] BALB.B mice. These observations argue strongly against an activation of predominantly Th1 cells as an explanation of resistance in mercurytreated mouse strains. Instead, we hypothesize that the pTh cells in resistant strains are unable to respond to mercury.

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Acknowledgements This work was supported by a grant from the Swedish Medical Research Council (project no. 09453). The technical assistance of Karin Fredriksson and Hele´n Hansson is gratefully acknowledged.

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