ToxSci Advance Access originally published online on February 22, 2006
Toxicological Sciences 2006 91(1):113-122; doi:10.1093/toxsci/kfj142
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Immune Modulation by Cadmium and Lead in the Acute Reporter Antigen–Popliteal Lymph Node Assay
,1,2
* Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland; and Environmental Research Institute, University College Cork, Cork, Ireland
2 To whom correspondence should be addressed. Fax: + 353 21 4343211. E-mail: f.vanpelt{at}ucc.ie.
Received November 25, 2005; accepted February 20, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immune modulation by heavy metals may cause serious adverse health effects in humans, although the mechanisms involved are not well understood. Both cadmium and lead are important environmental and occupational toxins. Therefore, in the current study, the costimulatory/adjuvant effects and the T-cell–activating potential of these metals (i.e., CdCl2 and PbCl2), are examined. These immune-modulating properties are critical in the development of conditions such as allergy, hypersensitivity, and autoimmunity. Using the direct popliteal lymph node assay (PLNA) and reporter antigen–popliteal lymph node assay (RA-PLNA) both metals were examined individually for immunotoxicity. Mercury (i.e., HgCl2) was included for comparative purposes as its effects in the RA-PLNA are well documented. Seven days following a single footpad injection containing metal and/or RA (trinitrophenyl-ovalbumin [TNP-OVA] or TNP-Ficoll), BALB/c mice were sacrificed and the popliteal lymph nodes (PLNs) removed. PLN cellularity, TNP-specific antibody-secreting cells (ASCs), and lymphocyte subsets were assessed. All three metals strongly stimulated T- and B-cell proliferation and ASC production following coinjection with the RA TNP-OVA. In each case, ASC production was skewed towards the IgG1 isotype. In addition, all three metals induced IgG production to TNP-Ficoll (although relatively weakly in the case of Cd). These results show that each of these metals can provide adjuvant signals to promote lymphocyte proliferation and enhance adaptive immune responses to unrelated antigens. Skewing of immune responses towards T helper type 2 responses suggests that each of these metals can enhance allergic and hypersensitivity reactions to environmental antigens. Furthermore, the induction of IgG responses to TNP-Ficoll, a T-cell–independent antigen, indicates that each of these metals can activate neoantigen-specific T cells. T-cell activation by metals can lead to metal hypersensitivity and has been implicated in the development of autoimmunity. This is the first report of immune modulation by CdCl2 and PbCl2 in the RA-PLNA.
Key Words: heavy metals; cadmium; lead; mercury; popliteal lymph node assay; immunotoxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heavy metals are a prominent class of immunotoxins associated with both specific and nonspecific immunoenhancement and immunosuppression. As a result of this ability to alter immune system homeostasis, some metals have been implicated as causative agents or aggravating factors in the development of chemical hypersensitivity, allergy, and autoimmune disease or in increased susceptibility to infections (Bigazzi, 1999
; Dayan, 1990; Druet, 1995; Lawrence and McCabe, 2002; Peden, 2002). However, the immunotoxic properties of most heavy metals are not well documented, and in all cases the underlying mechanisms are only poorly understood. Therefore, the purpose of the present study was to examine immune-modulating properties of the important environmental and occupational toxins cadmium and lead (i.e., CdCl2 and PbCl2) in the reporter antigen–popliteal lymph node assay (RA-PLNA) and to compare these properties with those of the well-known autoimmunogen mercury (i.e., HgCl2).
Mercury can cause hypersensitivity and autoimmunity in susceptible rodents, and its hypersensitizing effects in man are well documented (Cederbrant and Hultman, 2000; Druet, 1995; Fournie et al., 2002; Hu et al., 1999; Hultman and Hansson-Georgiadis, 1999; Nielsen and Hultman, 2002; Pelletier et al., 1988). Indeed, much of the extant knowledge of low–molecular weight chemical (LMWC) involvement in hypersensitivity and autoimmunity stems from the work carried out with mercury (Bigazzi, 1994, 1999; Dayan, 1990; Druet, 1995; Lawrence and McCabe, 2002). Rodents suffering from mercury-induced autoimmunity characteristically display T helper type 2 (Th2) responses, such as immune-complex deposits (IgG) in their glomeruli, raised serum IgE, IgG1, and autoantibodies of varying specificities (Nielsen and Hultman, 2002; Pelletier et al., 1988). Consistent with this, HgCl2 also stimulated B- and T-cell proliferation in the PLNs and enhanced production of IgG1 and IgE antibody–secreting cells (ASCs) specific for the RAs trinitrophenyl-ovalbumin (TNP-OVA) and TNP-Ficoll in the RA-PLNA (Albers et al., 1996, 1997, 1998, 1999).
In contrast to mercury, the immune-stimulating and immune-sensitizing properties of cadmium and lead are still unclear. In fact, much of the existing data actually suggest a suppressive role for both these metals on immune function (Cook et al., 1975; Descotes, 1992; Ewers et al., 1982; Exon et al., 1986; Fischbein et al., 1993; Fujimaki et al., 1983; Ritz et al., 1998; Sarasua et al., 2000; Sata et al., 1997; Yucesoy et al., 1997). However, cadmium exposures did correlate with increased levels of self-reactive antibodies in one study of occupationally exposed individuals (Bernard et al., 1987). Lead has also been shown to exacerbate autoimmune conditions such as lupus in susceptible rodents and has been linked with an increased production of self-reactive antibodies directed against neural proteins (El-Fawal et al., 1999; Waterman et al., 1994). Therefore, in order to better characterize the immune-modulating properties of these metals they were tested in the direct PLNA and RA-PLNA and their immune-modulating properties compared with those of mercury.
In the simplest form of the PLNA (i.e., direct PLNA), the test compound is injected into the footpad of naïve mice. The draining PLN is later removed and examined for chemical-induced inflammation. Importantly, many compounds have been tested in the PLNA and responses show good correlation with clinical data in humans (Pieters, 2001). More recently, the well-defined RAs TNP-OVA and TNP-Ficoll have been incorporated into this assay. These are injected with test compounds at subsensitizing doses. By measuring the formation of RA-specific ASCs it is possible to differentiate among immune-sensitizing chemicals, nonsensitizing yet immunostimulatory (irritant) chemicals, and innocent chemicals (Pieters et al., 2002). For instance, the T-cell–dependent antigen TNP-OVA requires cognate T- and B-cell interactions and costimulatory/adjuvant signals for effective immune responses (Nierkens et al., 2002). Therefore, the production of costimulatory inflammatory mediators following chemical exposure can be detected as increased ASC responses to coexposed TNP-OVA. The T-cell–independent antigen TNP-Ficoll can stimulate B cells to produce IgM ASCs. However, isotype switch to IgG production requires T cell help. As TNP-Ficoll is unable to activate T cells, TNP-specific IgG production following coinjection of test compound and TNP-Ficoll indicates that the test compound independently activated "neoantigen-specific" T cells and is therefore capable of immune sensitization. The ability to provide costimulation for unrelated adaptive immune responses and to sensitize the immune system are important immunotoxic properties and would allow chemicals to influence hypersensitivity reactions to environmental antigens (allergy) and to self (autoimmunity). In addition to assessing these critical immune-modulating properties, the PLN responses induced by each metal were further characterized using flow cytometric identification of PLN lymphocyte subsets.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice.
A breeding colony of BALB/c mice was established from mice procured from Harlan, Bicester, U.K. The breeding program was managed by the Biological Services Unit of the National University of Ireland, Cork, Ireland. The animals were housed under conventional conditions, i.e., suspended polycarbonate cages, 12 h light/dark cycle, 20 ± 2°C, and 50 ± 10% humidity. Tap water and commercial laboratory rodent food were freely available at all times. Mice were approximately 8 weeks old at the start of each experiment.
Chemicals and reagents.
Methanol, HgCl2, CdCl2·2
H2O, and PbCl2 were obtained from Alkem, Shannon, Ireland. All other chemicals used were of the highest grade available and were obtained from Sigma-Aldrich, Dublin, Ireland, unless otherwise indicated. 5-Bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt (BCIP) and nitro blue tetrazolium chloride (NBT) were purchased from Promega, Dublin, Ireland. Alkaline phosphatase–conjugated goat anti-mouse IgM, IgG1, and IgG2a were purchased from Southern Biotechnology Associates, Birmingham, AL. All flow cytometry antibodies used were obtained from BD Biosciences, Cowley, U.K. Polyvinylidene difluoride (PVDF) membrane (0.2 µm pore size) was purchased from Bio-Rad, Hemel Hempstead, U.K. All phosphate- buffered saline (PBS) buffers employed in RA-PLNA were adjusted to pH 7.2–7.4 before use.
In addition, all biological samples and PBS buffers were kept on ice unless otherwise indicated.
Synthesis of TNP antigens.
The RAs TNP-Ficoll and TNP-OVA and the detection antigen TNP-Bovine Serum Albumin ([BSA]; i.e., Enzyme-linked ImmunoSPOT (ELISPOT)-coating antigen) were prepared as described previously (Albers et al., 1996). TNP-carrier ratios were estimated as TNP88-Ficoll, TNP12-Ova, and TNP42-BSA. The conjugate solutions were lyophilized, transferred to appropriate containers, and stored at –20°C.
Direct PLNA and RA-PLNA.
The direct PLNA and RA-PLNA were carried out broadly as described previously (Albers et al., 1996, 1997). Briefly, RAs and metals were each dissolved in 0.9% NaCl. All solutions were sterilized by passage through a 0.2 µm filter (Schleicher & Schuell, London, U.K.) prior to the addition of RA. Eight-week-old female BALB/c mice received injections of RA (5 µg per animal) alone or in combination with metals. The metal concentrations used were 5, 25, and 50 µg HgCl2, 4, 20, and 40 µg CdCl2, and 5, 25, 40, and 50 µg PbCl2 in a 30 µl injection volume. As these doses of HgCl2 have been shown to be stimulatory in the RA-PLNA (Albers et al., 1996), approximately equimolar doses of the other two metals were used (i.e., in 30-µl injection volume: 50 µg HgCl2 = 6.1 mM, 40 µg CdCl2·2H2O = 5.8 mM, 50 µg PbCl2 = 6.0 mM). Injection sites were first sanitized by swabbing with 70% ethanol. All injections were carried out in the right hind footpad in the heel-toe direction, using disposable 30-gauge (30G) needles and insulin syringes (Becton Dickinson, Cowley, U.K.). Seven days after injections, the mice were sacrificed by cervical dislocation. Using scissors and watchmakers' forceps, the right popliteal lymph nodes were removed and freed of excess fatty tissue. Single-cell suspensions were prepared in PBS/1% BSA by gently pressing the lymph nodes between the frosted ends of two microscope slides. Cells were triturated by passage through a 26G needle. Cell suspensions were counted with a Coulter Counter ZM (Beckman Coulter, High Wickham, U.K.) and readjusted to a concentration of 1 x 106 cells/ml.
ELISPOT assay.
The ELISPOT assay was performed using a protocol similar to that described previously (Schielen et al., 1995). PVDF membranes were coated overnight at 4°C with TNP-BSA (10 µg/ml) in PBS/0.05% Tween 20 and blocked for 1 h at room temperature with PBS-Tween 20/1% BSA. Blocked membranes were washed twice with PBS and clamped in a "spot block." The spot block used was a 28-well version of the ELISPOT plate described by Schielen et al. (1995). A total of 5 x 104 cells were centrifuged onto the membrane in each well and incubated at 37°C for 4 h. Following removal from the spot block, membranes were washed vigorously with PBS and PBS/Tween 20 to remove cell debris. Membranes were then incubated overnight at 4°C with alkaline phosphatase–conjugated anti-mouse secondary antibody (anti-IgM, -IgG1, or -IgG2a) diluted 1/2000 in PBS/Tween 20. Membranes were washed and then incubated with substrate solution (BCIP and NBT in alkaline phosphatase buffer, prepared as per manufacturer's instructions [Promega]), to visualize TNP-specific antibody spots. The numbers of spots (i.e., ASCs) formed by each PLN cell sample (5 x 104 cells) were counted under a stereo microscope and expressed in terms of ASCs per 106 PLN cells.
Flow cytometry.
Cell subtypes were assessed using flow cytometry. A total of 2 x 105 PLN cells were centrifuged in round bottom 96-well tissue culture plates (Nunc, Bio-Sciences, Dun Laoghaire, Ireland). Cells were resuspended in the appropriate FITC- and PE-conjugated detection antibodies (BD Pharmingen) or isotype controls at a concentration of 1/200 (vol/vol) in PBS/1% BSA and incubated in the dark for 30 min at 4°C. Following centrifugation, supernatant was discarded, and cells were resuspended in 0.1% (vol/vol) formalin in PBS/1% BSA and stored at 4°C in the dark until measurement. All samples were analyzed within 24 h of staining. Cells were characterized based on the following monoclonal antibodies: CD3-FITC (17A2), CD4-FITC (RM4-4), CD8-PE (53-6.7), CD19-PE (1D3). Flow cytometry was carried out on a FACSCalibur instrument (Becton Dickinson). A minimum of 5000 cells were analyzed for each sample. Data were acquired and analyzed using CellQuest Pro software (Becton Dickinson).
Statistics.
All statistical analyses were carried out using SPSS software (version 11.0.1). Data were tested for normality using the Kolmogorov-Smirnov test. Where necessary, a cube root transformation was carried out to normalize data. Differences between group means were analyzed using a one-way ANOVA with the Tukey post hoc test for multiple comparisons. Significance levels were defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PLN Cell Count
A direct PLNA (without RAs) was carried out to assess the capacities of a single footpad injection of HgCl2, CdCl2, or PbCl2 to induce PLN cell proliferation. Both HgCl2 and PbCl2 significantly increased PLN cellularity, whereas CdCl2 did not (Fig. 1). Because of their comparable atomic weights, the doses used correspond to approximately equimolar levels. However, at this similar dose PbCl2 exerted a much stronger cell-stimulating effect than HgCl2. A cursory examination also showed greater footpad swelling in PbCl2-treated mice. Furthermore, in contrast with HgCl2-treated mice, animals injected with PbCl2 at the highest two doses of PbCl2 (i.e., 40 or 50 µg) had what appeared to be chemical deposits at the sites of injection after 7 days.
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Following coinjection of metals with RA, all three metals stimulated PLN cellularity (Fig. 1). HgCl2, CdCl2, and PbCl2 each exhibited an approximately linear dose-dependent stimulation of PLN cellularity in mice coexposed to TNP-OVA. The order of potency was Pb > Hg > Cd. Similarly, both HgCl2 and PbCl2 significantly stimulated PLN cellularity in a dose-dependent fashion following coexposure to TNP-Ficoll. Although coinjection of CdCl2 with TNP-Ficoll almost doubled the PLN cellularity with respect to controls, this was not statistically significant.
ASC Responses to RAs
The induction and enhancement of RA-specific ASCs in PLNs of treated mice were assessed using the ELISPOT assay. HgCl2 (25 and 50 µg) increased the mean IgM production to both RAs, though not significantly (Fig. 2A). In both cases, the maximal response was evident at the intermediate 25 µg dose. PbCl2 produced a similar pattern of IgM production to TNP-OVA, but the magnitude of the response was greater and was statistically significant (Fig. 2C). CdCl2 provided the most significant enhancement of IgM responses to TNP-OVA (Fig. 2B). The mean IgM response to TNP-Ficoll was also increased by 40 µg CdCl2, but this was not significant.
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All three metals predominantly induced IgG1 production to TNP-OVA challenge (Figs. 2D–2F). For HgCl2 and CdCl2 these effects were detectable at the lowest dose of metal used, while for PbCl2 significant effects were first detected at the 25 µg dose. The effects in each case were dose dependent and for PbCl2 reached maximal at 40 µg. The strong IgG1 response induced by CdCl2 is particularly interesting considering that CdCl2 incurred the least effects on PLN cellularity. Both HgCl2 and PbCl2 also strongly stimulated IgG1 ASC responses to TNP-Ficoll in a dose-dependent fashion. In contrast, CdCl2 similarly induced low responses at all three doses, and only the lowest dose achieved statistical significance.
The induction of IgG2a ASC responses by HgCl2 and PbCl2, to both RAs, exhibited a broadly similar pattern to that seen for IgG1 (Figs. 2G and 2I). Both metals induced significant and dose-dependent IgG2a responses to both RAs. In contrast, CdCl2 had a significant but comparatively small effect on IgG2a ASCs response to TNP-OVA, and this was only detected at the highest dose examined (Fig. 2H). The IgG2a response to TNP-Ficoll showed a similar induction at this dose but was not significant.
Proportions of PLN Lymphocyte Subpopulations
The proportions of the major lymphocyte subpopulations in PLNs of treated mice were assessed by flow cytometry. Neither RA, when injected alone, altered the size or the relative proportions of any PLN lymphocyte subpopulation (data not shown). HgCl2 had similar effects on lymphocyte subpopulations when injected with or without RAs, although the magnitude of these effects was greater when RA was present (Figs. 3A, 3D, 3G, and 3J). HgCl2-induced increases in PLN cellularity were as a result of significant growth in T- and B-cell subpopulations (Figs. 3A, 3D, 3G, and 3J). The only significant effect on the T:B cell ratio was the decrease seen in mice receiving 5 µg HgCl2 and TNP-OVA. The Th:Tc cell ratio (CD4:CD8) was not significantly altered by exposure to HgCl2 (Fig. 4D).
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Although CdCl2 did not significantly enhance PLN cellularity when injected alone, both T- and B-cell numbers were significantly increased following its coinjection with the RA (Figs. 3B, 3E, 3H, and 3K). These increases were greatest when TNP-OVA was used. When coinjected with TNP-OVA, CdCl2 caused a dose-dependent decrease in the percentages of PLN T cells and an increase in B cells, resulting in a significant reduction in the T:B cell ratio (Fig. 4B). Th cells (CD4) seemed largely responsible for this decrease in the relative proportion of T cells as no equivalent decrease was observed in the Tc (CD8) subpopulation (data not shown). This resulted in a somewhat depressed Th:Tc cell ratio (Fig. 4E). Coinjection of CdCl2 with TNP-Ficoll resulted in a slightly raised T:B cell ratio and a marginally depressed Th:Tc cell ratio (Figs. 4B and 4E).
Of the three metals, PbCl2 had the most marked effects on PLN lymphocyte subpopulations. Qualitatively similar effects were seen with PbCl2 in both the direct assay and the RA-PLNA. PbCl2 vastly increased the numbers of T cells (Th and Tc) and B cells (Figs. 3C, 3F, 3I, and 3L). However, the greatest relative increases were seen in B cell numbers. This caused a highly significant reduction in the T:B cell ratio (Fig. 4C). In addition, Tc cells showed greater population growth when compared to Th cells (Figs. 3I and 3L), resulting in a depressed Th:Tc (CD4:CD8) ratio (Fig. 4F). Interestingly, PbCl2 induced a comparable dose-dependent expansion of T-cell numbers in response to both RAs (Figs. 3C, 3I, and 3L). However, although dose-dependent increases were also seen in the B-cell subpopulation, these increases were much greater in response to TNP-OVA than to TNP-Ficoll (Fig. 3F). The presence of chemical deposits at the injection site, even after 7 days, suggests that macrophages may have had difficulty in clearing the chemical precipitate. Indeed, flow cytometric data confirmed a significant dose-dependent increase in the numbers of PLN cells with the CD3–CD19– phenotype (from 0.9 ± 0.1 x 105 [saline control] to 5.7 ± 0.2 x 105 [PbCl2]). PLN cells of this phenotype are likely to consist of macrophages with some interdigitating dendritic cells and terminally differentiated plasma cells also being present.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aims of this study were to examine the important immune-modulating parameters (i.e., adjuvanticity and immune-sensitizing potential) of PbCl2 and CdCl2 and to analyze the underlying changes in PLN lymphocyte subsets. Effects were compared to those of the well-characterized immunotoxin HgCl2, which has been assessed previously in the RA-PLNA (Albers et al., 1996
, 1998, 1999).
We observed that HgCl2 strongly induced T- and B-cell proliferation in the PLNs of BALB/c mice, 7 days following footpad injection (Fig. 3). ASC responses to coinjected RAs also indicated strong adjuvant (ASC response to TNP-OVA) and immune-sensitizing (IgG responses to TNP-Ficoll) effects of HgCl2 and skewing of immune responses towards Th2 type reactivity (preferential induction of IgG1 ASCs; Fig. 2). These results confirmed the previous findings for HgCl2 in the RA-PLNA and are consistent with this metal's known immune-modulating and immune-sensitizing properties (Albers et al., 1996, 1998, 1999; Ochel et al., 1991; Pelletier et al., 1988).
In the dose range examined, CdCl2 showed little if any immune-stimulating potential when injected alone (Fig. 1). However, a similar dose of CdCl2 stimulated PLN responses to coinjected TNP-OVA (Figs. 1 and 2). These costimulatory effects contrast with the largely immunosuppressive properties reported to date for this metal (Dan et al., 2000; Descotes, 1992; Fujimaki, 1985; Fujimaki et al., 1983). According to the two-signal model of lymphocyte activation, antigen recognition (signal 1) and costimulation (signal 2) are both required for the development of effective adaptive immune responses (Bretscher, 1992). Clearly, the response stimulated by CdCl2 to TNP-OVA suggests an ability to provide costimulation. Therefore, the lack of a PLN response when CdCl2 was injected alone indicates that CdCl2 may not effectively provide signal 1 or antigen recognition. This is supported by its relatively weak effect on ASC responses to TNP-Ficoll (Fig. 2). T-cell cytokines appear essential for isotype switch in responses to such T-cell independent ([TI]; type 2) antigens (Letvin et al., 1981; Mongini et al., 1981). Therefore, IgG production to TNP-Ficoll in this study indicates a metal-induced activation of neoantigen-specific T cells, which then provide soluble help to TNP-Ficoll specific B cells. Thus, in contrast to HgCl2 and PbCl2, our data suggest that CdCl2 is only a weak sensitizer of the immune system, perhaps due to limited generation of neoantigens. Mercury-induced immune sensitization and autoimmunity have been shown to be dependent on the major histocompatability complex haplotype. In the case of Ni2+, its recognition and activation of T cells is reported to be highly dependent on specific residues by the MHC and T-cell receptor (Gamerdinger et al., 2003; Lu et al., 2003). Although crossreactivity with other metals should be possible, this was not detected with Cu, Pd, Co, or Cr (Gamerdinger et al., 2003). Therefore, although Hg, Cd, and Pb share similar physical and chemical properties, subtle differences in coordination chemistries and differing affinities for the same biological ligands could significantly affect their interactions with residues important for immune recognition. However, immune sensitization to CdCl2 needs to be assessed directly in future studies, e.g., by employing a secondary PLNA where an initial footpad exposure is followed several weeks later by a challenge with CdCl2 (Pieters, 2001).
Compared to HgCl2 and CdCl2, PbCl2 had the strongest effects on footpad swelling and PLN cellularity (Fig. 1). Footpad swelling was probably greatly exacerbated by the presence of persistent chemical deposits visible at the highest two doses of PbCl2 (i.e., 40 or 50 µg) that were likely formed due to the precipitation of relatively insoluble Pb salts. These deposits may have led to the constant activation of macrophages with subsequent production of proinflammatory mediators such as cytokines and reactive species. There is some support for this interpretation in that significantly increased numbers of cells with a macrophage-like phenotype (CD3–CD19–) were detected in the PLNs of mice with visible chemical deposits. A similar effect has been reported before for silica (Albers et al., 1997; Bloksma et al., 1995), which is a known activator of macrophages in the PLN. However, silica-induced activation of macrophages appeared inadequate for the development of effective responses to RAs, in particular against TNP-Ficoll (Albers et al., 1997). Therefore, although PbCl2 may activate macrophages, this alone may not be sufficient to effectively switch antibody isotype to IgG production particularly in response to TNP-Ficoll. Furthermore, the intermediate dose of PbCl2 (25 µg) stimulated significant PLN and RA responses in the absence of any visible deposits. PbCl2 caused very strong dose-dependent IgM and IgG responses to both RAs (Fig. 2). This shows that PbCl2 is capable of providing costimulatory signals to antigens and suggests that it can also activate T cells, which could lead to immune sensitization. Indeed, recent studies in our laboratory involving BALB/c mice receiving combined oral and footpad exposures of PbCl2 suggest that immune sensitization to PbCl2 does occur in these mice (Carey, Allshire, and van Pelt, manuscript in preparation). Interestingly, although broadly similar numbers of T cells were induced by PbCl2 to both the RAs, the numbers of B cells induced to TNP-Ficoll were noticeably lower than to TNP-OVA (Fig. 3). This contrasts with HgCl2, which increased B cell numbers similarly for both RAs, and implies that PbCl2 may enhance cognate T/B cell interactions more effectively than noncognate interactions. Consistent with this conclusion, an in vitro study in murine cells showed greater enhancement of antibody responses by lead when physical contact was permitted between B and T cells than when these cells were separated by a porous membrane (McCabe and Lawrence, 1991). Preferential enhancement of cognate interactions by lead could be attributable to its reported effects on the expression of cell surface adhesion molecules (Heidmets et al., 2006; Prozialeck et al., 2002). Enhanced adhesion between cells would likely increase and prolong the interactions between important cell-surface costimulatory molecules such as CD40-CD154 and B7-CD28 interactions. These molecular interactions are important in the generation of autoantibodies in mercury-treated mice and drug-induced type 2 reactions (Bagenstose et al., 2002; Nierkens et al., 2002, 2005). Alternatively, signaling pathways activated directly by cell adhesion molecules could perhaps be interpreted by cells as costimulation.
Like HgCl2, both CdCl2 and PbCl2 tended to skew responses towards IgG1 ASC production, a Th2 isotype (Fig. 2). It is important here to note that the BALB/c strain is not inherently skewed towards Th2 responses. Previous investigators have shown strong Th1 responses in the BALB/c RA-PLNA when examining immune modulation by the diabetogen streptozotocin (Albers et al., 1998). This was characterized by the production of predominantly IgG2a and IgG2b ASCs against TNP-OVA and by the increased occurrence of interferon gamma (IFN 
)–producing T cells. Recent studies suggest that differential effects on signaling by costimulatory molecules (i.e., CD40-CD154 or CD28/CTLA-4 and CD80/CD86) are important in such skewing of responses by LMWCs towards Th1 or Th2 profiles (Nierkens, 2004). Therefore, it will be interesting in future studies to examine the involvement of costimulatory molecules in the development of metal-induced responses such as those seen here.
Environmental pollutants such as heavy metals are increasingly being implicated in the induction and exacerbation of a range of immune-mediated conditions, including allergy, hypersensitivity, and autoimmunity (Dayan, 1990). Since they stimulate isotype switch to IgG1 production against innocuous levels of the unrelated antigens TNP-OVA and TNP-Ficoll (Fig. 2), lead, cadmium, and mercury may play a role in such conditions. For example, allergy has been defined as "disease following a response by the immune system to an otherwise innocuous antigen" (Janeway Jr. et al., 2004). Allergy is characterized by the creation of IgE to antigens. This requires the production of Th2 cells that produce interleukin 4 (IL-4) and IL-13 (Finkelman et al., 1988, 1990). Similarly, isotype switch to IgG1 production requires Th2 cytokine profiles (Snapper et al., 1988). Therefore the stimulation of IgG1 production to innocuous levels of TNP-OVA is broadly analogous to enhanced IgE production against environmental antigens, the basis for allergy. Indeed, previous RA-PLNA studies have shown that Hg strongly stimulates the production of IgE ASCs (Albers et al., 1999).
The stimulation of IgG1 responses to TNP-Ficoll by all three metals indicates that each is also capable (although relatively weakly for Cd) of activating neoantigen-specific lymphocytes that can cause metal hypersensitivity (Fig. 2). Such cells may initiate allergic or autoimmune responses through linked recognition and epitope spreading or through noncognate (i.e., bystander) stimulation of allergen-specific or autoreactive TI antigen–specific B cells (Vos et al., 2000).
The immune-stimulating properties of Cd and Pb observed in the present study are consistent with some previous reports (Bernard et al., 1987; El-Fawal et al., 1999; Waterman et al., 1994) but are at variance with most of the existing data (Cook et al., 1975; Descotes, 1992; Ewers et al., 1985; Exon et al., 1986; Fischbein et al., 1993; Fujimaki et al., 1983; Ritz et al., 1998; Sarasua et al., 2000; Sata et al., 1997; Yucesoy et al., 1997 ). The PLNA is used for the detection of potential to induce both acute local sensitization and autoimmune reactions following subcutaneous injection of the compound. The results to date with the PLNA show that most chemicals known to induce autoimmune or allergic reactions in man induce positive PLN responses (Pieters, 2001). However, additional validation studies are required to confirm the predictive power of the PLNA and RA-PLNA with regard to systemic immune modulation (Ravel and Descotes, 2005).
Environmental and occupational heavy-metal exposures are characteristically prolonged and at low levels, which can result in (oral) tolerance (Chen et al., 1995) as has been shown for numerous immune sensitizing chemicals (Cavani et al., 2000; Dubois et al., 2003; Galliaerde et al., 1995). The contradictory effects observed between the various studies may therefore be explained in part by variations in study design (dose, frequency, and route of exposure). HgCl2 is a well-established autoimmunogen, whereas gold salts have long been used in the treatment of rheumatoid arthritis but are also associated with adverse immune reactions, such as hypersensitivity and exacerbation of autoimmunity. In a recent study, Layland et al. (2004) demonstrated that both mercury and gold prime two distinct CD4+ T-cell populations; CD4+CD25+ immune stimulatory T cells and CD4+CD25– immune-regulatory/suppressor T cells. It has been postulated that it is the balance between these two populations of CD4+ T cells which largely determines the net immune-modulating effects and that this balance is dependent on the route and frequency of exposure (Carey et al., 2005). Therefore, although this study demonstrates that both Cd and Pb have immune-sensitizing potential, the outcome following exposure to the metals will vary and depend on a number of factors including dose, route, and frequency of exposure.
In summary, this is the first report of immune modulation by Cd and Pb in the RA-PLNA and confirms the effects of HgCl2. That hypersensitivity reactions to Cd have not been reported before may be due to this metal's relatively weak ability to provide signal 1 (i.e., antigen recognition) for adaptive immune responses. This study also supports an immune-sensitizing role for PbCl2 and suggests that it may stimulate cognate T/B cell interactions more effectively than noncognate interactions. We conclude that acute exposure to lead, cadmium and mercury may cause immune-sensitizing effects.
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NOTES |
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1 Present address: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037.
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ACKNOWLEDGMENTS |
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This work was supported by the Higher Education Authority, of Ireland, under the Programme for Research in Third Level Institutions, Cycle 2, as part of the National Development Plan. The authors gratefully acknowledge Dr. Raymond Pieters and colleagues (Institute for Risk Assessment Sciences, Utrecht University, the Netherlands) for their assistance with method establishment. The authors also thank Peter O'Keeffe for his invaluable technical assistance.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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