Mice Mercury-Induced Autoimmune Response in Lack of Plasma

Lack of Plasma Protein Hemopexin Dampens Mercury-Induced Autoimmune Response in Mice

This information is current as Sharmila Fagoonee, Cristiana Caorsi, Mirella Giovarelli, of September 30, 2021. Meredin Stoltenberg, Lorenzo Silengo, Fiorella Altruda, Giovanni Camussi, Emanuela Tolosano and Benedetta Bussolati J Immunol 2008; 181:1937-1947; ;

doi: 10.4049/jimmunol.181.3.1937 Downloaded from http://www.jimmunol.org/content/181/3/1937

References This article cites 29 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/181/3/1937.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lack of Plasma Protein Hemopexin Dampens Mercury-Induced Autoimmune Response in Mice1

Sharmila Fagoonee,2* Cristiana Caorsi,†‡ Mirella Giovarelli,†‡ Meredin Stoltenberg,¶ Lorenzo Silengo,* Fiorella Altruda,* Giovanni Camussi,‡§ Emanuela Tolosano,3* and Benedetta Bussolati2,3‡§

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding afﬁnity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear Downloaded from autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4؉T cells isolated from mercury-treated hemopexin-null mice http://www.jimmunol.org/ show reduced IFN-␥-dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN-␥ and, hence, autoimmune responses. The Journal of Immunology, 2008, 181: 1937–1947.

utoimmune diseases are characterized by unknown eti- ratios (4). Finally, on investigating the effect of iron status on ology and may involve ϳ20% of the human population murine systemic lupus erythematosus, Leiter et al. have found that A (1). Representative diseases are systemic lupus erythem- iron supplementation leads to more severe renal disease and in- atosus, rheumatoid arthritis, scleroderma, and type I diabetes mel- creases mortality (5).

litus, and the hallmark of the autoimmune disease is the production Several genes involved in iron handling have been associated by guest on September 30, 2021 of highly specific autoantibodies that recognize molecules con- with T cell abnormalities, including the hemochromatosis gene served throughout evolution (2). Several factors affect the autoim- HFE and the iron transporter NRAMP1 (6). In particular, the latter mune response, including iron-dependent modulation of T cells. has been linked to rheumatoid arthritis, juvenile rheumatoid arthri- Studies in rodents have shown that iron deficiency can lead to tis, and multiple sclerosis. More recently, the haptoglobin ␣2 thymocyte depletion in cortical and medullary regions of the thy- polypeptide has been found to be over-expressed in the serum of mus and a reduced number of spleen T lymphocytes (3). In con- systemic lupus erythematosus patients (7). Haptoglobin is an acute trast, rats loaded with iron have been reported to have an increase phase plasma protein with high binding affinity for hemoglobin in CD8ϩ cells. However, the relationship between iron overload and prevents hemoglobin-derived iron loss. The overexpression in and the immune response is controversial, as other studies have systemic lupus erythematosus patients of the haptoglobin ␣2 shown that transfusional iron overload in thalassemia intermedia polypeptide, which has a higher iron binding capacity than the patients is associated with a blunted response of T cells to mito- other variant, ␣1, suggests that heme-iron availability may mod- gens and with decreases in circulating CD4ϩ cells and CD4/CD8 ulate the autoimmune response. Hemopexin (ϳ10–20 ␮M in plasma) is a 60-kDa, circulating, class I, acute phase reactant with the highest binding affinity to *Department of Genetics, Biology and Biochemistry, and Molecular Biotechnology heme (K Ͻ 1 pM). It binds free heme and mediates its uptake in † d Center; Department of Medicine and Experimental Oncology, University of Turin, liver cells through receptor-mediated endocytosis. Heme is then Turin, Italy; ‡Center for Experimental Research and Medical Studies; §Department of 4 Internal Medicine, San Giovanni Battista Hospital, Turin, Italy; and ¶Department of catabolized by heme oxygenase (HO) into biliverdin, carbon Neurobiology, Institute of Anatomy, University of Aarhus, Århus, Denmark monoxide (CO), and iron. In this way, hemopexin is thought to Received for publication February 22, 2007. Accepted for publication May 29, 2008. prevent heme-iron loss and the proinflammatory effects of free The costs of publication of this article were defrayed in part by the payment of page heme. Studies in vivo on hemopexin-null (Hx-null) mice have con- charges. This article must therefore be hereby marked advertisement in accordance firmed this view. Indeed, Hx-null mice recover less efficiently than with 18 U.S.C. Section 1734 solely to indicate this fact. wild-type controls after an acute hemolytic stress and accumulate 1 This work was supported by the Italian Ministry of University and Research to E.T. and F.A., and by Regione Piemonte to F.A. and B.B. (Grant A141). C.C. is supported iron in proximal tubular cells of the kidney (8). In parallel, the lack by a fellowship from Fondazione Denegri. 2 Address correspondence and reprint requests to Dr. Sharmila Fagoonee, University 4 Abbreviations used in this paper: HO, heme-oxygenase; ANA, antinuclear antibody; of Turin, Via Nizza 52, Turin, Italy. E-mail address: [email protected] or CO, carbon monoxide; HgCl2, mercuric chloride; Hx-null, hemopexin-null; IC, im- Dr. Benedetta Bussolati, Molecular Biotechnology Center, Via Nizza 52, 10126 mune complex; TfR1, transferrin receptor 1; Treg, T regulatory cell. Turin, Italy. E-mail address: [email protected] 3 E.T. and B.B. contributed equally to this work. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 1938 ROLE OF HEMOPEXIN IN AUTOIMMUNITY

of circulating hemopexin may increase the peripheral cell avail- (Sigma-Aldrich). Muscle, skin and heart were treated similarly. Immune ability of heme with the consequent activation of HO-1, which is deposits were analyzed using a Zeiss fluorescence microscope. known to exert an immunomodulatory and cytoprotective effect (9). Serum Ig levels To investigate the role of hemopexin in autoimmune response, Serum IgE levels were determined using an immunoassay for mouse IgE we chose the well-established mouse model of mercury-induced (OPT EIA mouse IgE set; BD Pharmingen). Anti-mouse IgE mAb (cap- autoimmunity characterized by the production of antinuclear Abs ture) was coated on 96-well plates (Falcon) and incubated overnight. Stan- dards and serum (diluted 1:10) were incubated followed by detection with (ANA) and immune complex (IC) deposition in the kidney (10). biotinylated anti-mouse IgE, avidin-HRP, and tetramethylbenzidine sub- We evaluated the susceptibility of Hx-null mice to mercury treat- strate (Sigma-Aldrich). Serum IgG1 and IgG2a levels were determined ment compared with wild-type controls. using an ELISA kit (BD Pharmingen) according to manufacturer’s instruc- We show that the lack of hemopexin dampens mercury-induced tions. Sera were diluted 1/100 in blocking buffer. Absorbance values were autoimmune responses in mice. Hx-null mice produced less ANA measured at 450 nm. and had reduced deposits of IC in the kidney after mercuric chlo- Electron microscopy ride (HgCl2) treatment compared with wild-type mice. Hx-null mice had a reduction in activated T cells and a reduced number of Transmission electron microscopy was performed using Karnowsky’s fix- B lymphocytes in the spleen after treatment with HgCl but not ative with osmium tetroxide-postfixed tissues embedded in epoxy resin 2 according to standard procedures (12). Ultrathin sections were stained with after OVA/CFA immunization, pointing to a specific defect in the uranyl acetate and lead citrate and examined with a Jeol JEM 1010 electron autoimmune response. Interestingly, Hx-null mice had reduced microscope. transferrin receptor 1 (TfR1) expression in T cells under basal Downloaded from conditions possibly dependent on higher heme-derived iron in Intracellular staining of cytokines and flow cytometry these mice. Finally, we demonstrated that IFN-␥-induced activa- ϩ Spleens were dissected and splenocytes were flushed gently out from the tion of STAT1 in CD4 T cells isolated from HgCl2-treated Hx- spleens into DMEM by using a needle. Single cell suspensions were pre- null mice was impaired compared with that of wild-type mice. pared by pipetting several times and subjecting to a Ficoll gradient. The We suggest that hemopexin, by controlling heme-iron availabil- lymphocytes were recuperated, resuspended, counted, and adjusted to a density of 1 ϫ 106 cells/ml. ity to lymphocytes, may participate in the control of autoimmunity http://www.jimmunol.org/ ␥ The mAbs used for the detection of cell surface markers and intracel- by modulating IFN- responsiveness. lular cytokines are documented in Table I. Briefly, the isolated spleen and blood cells were washed with PBS-BSA (0.25%), stained with the appropriate cell surface Ag or the relevant control for 30 min at room temper- Materials and Methods ature, and then washed again and resuspended in PBS-BSA. For intracel- Mice and treatment with HgCl2 lular staining, cells were fixed in 4% paraformaldehyde solution for 10 min at 4°C, permeabilized with TBS plus 1% Triton X-100, and stained with Hx-null mice were generated as previously described (8) and were in a anti-IL-4, anti-IFN-␥, or anti-IL-17 conjugated with PE for 30 min at room genetic 129/Sv background (H-2b). All mice (age matched and 6–8 wk old temperature. Allophycocyanin-conjugated Foxp3 staining was performed at the beginning of each experiment) used were housed in our animal by using a Foxp3 staining set (eBioscience; Insight Biotechnology) ac- facilities, with a 12-h dark/light cycle and access to standard laboratory cording to the manufacturer’s instructions. To measure the levels of phos- by guest on September 30, 2021 chow and tap water ad libitum. At least five animals were used for each phorylated STAT1, cells were fixed immediately after stimulation with 4% experimental point. Hx-null and wild-type mice were injected with a dose paraformaldehyde, permeabilized with ice-cold methanol followed by sa- of 1.6 mg of HgCl (Sigma-Aldrich) per kilogram of body weight s.c. every 2 ponin, and then simultaneously incubated with the anti-phospho(Tyr701)- third day for 4 wk. Additional groups of mice were treated for 6 wk (pro- STAT1-PE or anti-IgG-PE and CD4-allophycocyanin Abs (BD Bio- longed treatment), 4 days, or 1 day. Nontreated mice were used as controls sciences). Cells were analyzed on a FACS (BD Biosciences). Seventy in all experiments. thousand cells were analyzed at each experimental point for intracellular staining vs 30,000 cells for surface staining. PE-, FITC-, and Collection of blood, hearts, skin, spleens, and kidneys allophycocyanin-conjugated anti-IgGs were used as isotype controls where appropriate. After the 4-wk treatment, mice were bled retro-orbitally under anesthesia and sacrificed. Their hearts, skin, spleens, and kidneys were removed asep- ␥ tically and embedded in optimal cryo-preserving tissue compound (OCT) Treatment of splenocytes with IFN- ex vivo (Miles), snap frozen, and stored at Ϫ80°C until sectioning or inclusion in ϫ 6 Mice were treated with HgCl2 for 4 days and 1 10 splenocytes were paraffin after formalin fixation. Blood samples were allowed to clot at 37°C treated with IFN-␥ (100U/ml) for 15 min at 37°C. The reaction was for 30 min and then centrifuged at 3,000 rpm (Microfuge 18 centrifuge; blocked by adding cold PBS and the cells were fixed and processed for Beckman Coulter) for 10 min at room temperature. The serum obtained 701 Ϫ anti-phospho(Tyr )-STAT1 and CD4 staining and flow cytometry anal- was then kept at 20°C until further use. ysis. IFN-␣ (100U/ml) was used as control.

Detection of ANA by immunofluorescence Activation of T cells ex vivo The serum levels of IgG ANA were assayed by using indirect immuno- To analyze cytokine production by T cells ex vivo, 2 ϫ 106 splenocytes ␮ fluorescence. Briefly, 4- m-thick cryostat sections of liver were fixed in were plated in a 24-well, flat-bottom plates containing 1 ml of RPMI 1640 ice-cold acetone, allowed to dry, and incubated with serum from HgCl2- supplemented with 10% FCS and penicillin/streptomycin and either PMA treated mice of both genotypes at serial dilutions followed by FITC-con- (10 ng/ml, final concentration) or ionomycin (500 ng/ml) and monensin (2 jugated goat anti-mouse IgG (Sigma-Aldrich) for 45 min at 37°C. The ␮M) or with monensin (Sigma-Aldrich) alone as control. titers were expressed as the reciprocal value of the highest serum dilution Following incubation for4hat37°C, the cells were harvested, washed, that gave an unequivocal positive reaction. and prepared for flow cytometry. The percentages of IL-4- and IFN-␥- expressing CD4ϩ T cells were determined in the spleens of mice under

Detection of deposits of IgG and C3 in kidneys, muscles, hearts, basal conditions and after treatment with HgCl2. and skin Autometallographic mercury tracing The presence of glomerular deposits of IgG and C3 Abs was detected by direct immunofluorescence performed on mouse tissues rapidly frozen in To trace mercury deposits (i.e. mercury-sulfur nanocrystals) in tissues, au- liquid nitrogen, cut in 3-␮m sections, and incubated in cold acetone fol- tometallographic silver enhancement was performed (13, 14). Briefly, gel- lowed by fixing in 3.5% paraformaldehyde containing 2% sucrose as pre- atin-coated, paraffin-included sections of tissues were placed in an auto- viously described (11). After saturation with PBS-BSA, the sections were metallographic developer containing a mixture of gum arabic, citrate buffer incubated with FITC-conjugated goat anti-mouse IgG or anti-mouse C3 (pH 3.7), hydroquinone, and silver lactate and incubated at 26°C for 60 The Journal of Immunology 1939

Table I. Monoclonal antibodies employed to detect cell surface markers and intracellular cytokines during ﬂow cytometry

Raised in Speciﬁcity Conjugated with Isotype Form Purchased from

Rat Mouse CD4 FITC IgG2b Cedarlane Rat Mouse CD8 PE IgG2b Cedarlane Rat Mouse CD25 PE IgG2b BD Pharmingen Hamster Mouse CD69 PE IgG Abcam Rat Mouse CD44 FITC IgG1 Abcam Rat Mouse CD19 PE IgG2a Serotec Rat Mouse CD4 PE IgG2b Cedarlane Rat Mouse CD71 FITC IgG2a Abcam Rat Mouse IL-4 PE IgG2b BD Pharmingen Rat Mouse IFN-␥ PE IgG1 BD Pharmingen Rat Mouse FoxP3 Allophycocyanin IgG2a eBioscience Rat Mouse CD11b FITC IgG2b Serotec Hamster Mouse CD11c PE IgG1 BD Pharmingen Rat Mouse F4/80 PE IgG2a Serotec Rat Mouse IL-17 PE IgG1 BD Pharmingen Rat Mouse CD4 Allophycocyanin IgG2a BD Pharmingen Mouse Mouse STAT1 (Y701) PE IgG2a BD Pharmingen Rata Unknown Allophycocyanin IgG2a BD Pharmingen Downloaded from Mousea Unknown PE IgG2a BD Pharmingen Rata Unknown FITC IgG2a Dako Rata Unknown PE IgG2a Dako Rata Unknown Allophycocyanin IgG2a eBioscience

a Negative isotope control. http://www.jimmunol.org/ min. After several washings, the sections were counterstained with cresyl Analysis of kidney functionality violet and examined under a light microscope. Creatinine was determined on a Beckman creatinine analyzer (Beckman

Instruments). After treatment for 4 wk with HgCl2, mice were individually Passive Arthus reaction housed for 24 h in metabolic cages and urine was collected for proteinuria For cutaneous Arthus reactions, mice were injected i.v. with 200 ␮lof determination with the Biuret assay. a mixture of 20 mg/ml OVA containing 2.5% Evans blue dye (Sigma- Aldrich), followed by intradermal injection of 60 ␮g/30 ␮l rabbit IgG Statistical analysis

anti-OVA Abs (Cappel) 30 min later. Skin was harvested 4 h later. by guest on September 30, 2021 To assess for edema, the diameter of extravascular Evans blue dye on Results are expressed as mean Ϯ SE. Statistical analyses were performed the reverse side of the skin at site of injection was measured directly. using an unpaired Student’s t test. Skin was fixed in 10% formalin and paraffin-included sections were stained with H&E and the numbers of infiltrating neutrophils and mast cells were determined. Results

HgCl2-induced ANA production and IC deposition was reduced Immunization with OVA/CFA and measurement of serum IgG in Hx-null mice by ELISA Control 129/Sv mice and Hx-null 129/Sv mice were treated

Mice were immunized with a single i.p. injection of 0.2 mg of OVA in with HgCl2 every three days for 4 wk. After that time, the CFA. Ten days later, blood was taken and serum was prepared to measure appearance of an autoimmune disease was evaluated by pro- IgG Abs to OVA by ELISA. Briefly, 96-well plates were coated with a 2% solution of OVA in 0.1 M Tris-HCl buffer. Serum samples were serially duction of ANA and glomerular deposition of IC. diluted in Tris buffer containing 2% bovine ␥-globulin (Sigma-Aldrich) to ANA were barely detectable in both Hx-null and wild-type block nonspecific binding of mouse globulins to the microtitration trays. mice under basal conditions. After HgCl2 treatment, the titers of The samples were incubated at 75 ␮l/well for 1.5 h at room temperature. ANA were significantly reduced in the Hx-null mice compared The wells were then washed five times and incubated with Fc-specific, with controls (Fig. 1A). Generalized and diffuse granular de- peroxidase-conjugated goat Abs to mouse IgG (Cappel). After incubation at room temperature for 1 h, the wells were washed five times and treated posits of IgG were observed by immunofluorescence in the glo- with o-phenylenediamine (100 ␮l/well) for 10 min in darkness. The enzy- merular capillary walls and mesangium and in the basal parts of matic reaction was stopped with 2N H2S04, and absorbance was measured the proximal, distal, and collecting tubules of the wild-type at 490 nm. mice (Fig. 1, B and C). C3 deposition was also present in the glomerular capillary walls, mesangium, Bowman’s capsule, and Western blots and Abs tubular basal membrane (Fig. 1, E and F). A similar deposition T cells were purified from the spleens of mice under basal conditions with of IgG was detected in the cardiac, skin (not shown), and mus- ϩ CD90 microbeads (Miltenyi Biotec) and using MidiMACS magnetic col- cle capillaries (Fig. 1, K and L). By electron microscopy, dense umns (Miltenyi Biotec) as per the manufacturer’s instructions. The column ϩ deposits were seen in the glomerular capillary wall and mesan- was removed from the magnetic field and the CD90 fraction was collected. Cell numbers and viability were determined using trypan blue gial matrix of the renal glomeruli of wild-type mice (Fig. 1, N exclusion. and P). In contrast, the Hx-null mice showed little or no dep- Briefly, total protein extracted from purified T cells was separated by osition of immune complexes or complement in the kidney (Fig. electrophoresis on 10% SDS-polyacrylamide gels, blotted onto nitrocellu- 1, D and G), heart, skin (not shown), and muscle (Fig. 1M) lose membrane (Whatman), and incubated overnight with 1/1000 anti- TfR1 polyclonal Ab (Zymed Laboratories). Membranes were then incu- compared with the wild-type mice after treatment with HgCl2 bated with 1/5000 HRP-conjugated goat anti-rabbit IgG and revealed by for 4 wk. The absence of IC deposits in the Hx-null mice was ECL on a ChemiDoc system (Bio-Rad). also confirmed by electron microscopy (Fig. 1, O and Q). After 1940 ROLE OF HEMOPEXIN IN AUTOIMMUNITY

FIGURE 1. Mercuric chloride (HgCl2)-induced antinuclear Ab production and immune complex deposition was reduced in Hx-null mice after 4 wk of treatment. A,

Quantification of serum ANA. ANA titers were reduced Downloaded from in HgCl2-treated Hx-null mice compared with wild-type p Ͻ 0.01). No differences were noted in basal ,ءء) mice ANA levels. Levels of circulating ANA were measured by indirect immunofluorescence using cryostatic liver sections. Data are expressed as the mean serum titer Ϯ SE. B–M, Direct immunofluorescence of IC deposits. Representative photomicrographs of renal tissue of the http://www.jimmunol.org/ wild-type mice showed generalized and diffuse granular deposits of IgG (B and C) and C3 (E and F)inthe glomerular capillary walls and mesangium. Similar deposition of IgG (K) and C3 (L) was detected in the muscle capillaries of wild-type mice. In Hx-null mice, no deposits of IgG (D) and C3 (G) were observed in the kidney. No IgG deposits were also present in the cardiac muscle (M). Representative photomicrographs of renal

tissue under basal conditions are shown for wild-type by guest on September 30, 2021 (H and I) and Hx-null (J) mice. N–Q: Electron microscopy analysis of kidney. Representative photomicrographs showing presence of dense deposits (arrows) in the mesangial matrix (M) and in the glomerular capillary wall of wild-type mice (N and P). No deposits were observed in the mesangium or in the glomerular capillary walls of Hx-null mice (O and Q). The data are representative of three separate experiments (n ϭ 4/genotype/experiment). Original magniﬁcation: B, E, H, and K, ϫ150; C, D, F, G, I, J, L, and M, ϫ400; N and O, ϫ6000; P and Q, ϫ12,000.

4 wk of treatment with HgCl2, no increase in serum creatinine (not shown). Prolonged treatment (6 wk) with the heavy metal levels were observed in Hx-null and wild-type mice compared salt did not further alter the autoimmune response of the two with basal levels, nor was there any difference in proteinuria genotypes (not shown). The Journal of Immunology 1941

FIGURE 3. Assessment of inflammatory responses, vascular permeabil- Downloaded from ity, and immune reactions to exogenous Ag. A and B, Passive Arthus reaction-induced recruitment of inflammatory cells in the skin. The reaction was performed as reported in Materials and Methods. Histologic tissue sections stained with H&E showing neutrophil infiltration in the skin of wild-type (A) and Hx-null (B) mice at 4 h after IC challenge are shown. C, Serum OVA-specific IgG responses after 10 days of immunization with

OVA/CFA were determined by ELISA. Bars represent the mean absor- http://www.jimmunol.org/ bance Ϯ SE at different serum dilutions in independently tested mice (n ϭ 5/genotype).

Similar inflammatory responses, vascular permeability, and immune reactions to foreign Ag in wild-type and Hx-null mice To investigate whether Hx-null mice had defects in inflammatory processes and/or vascular permeability, passive Arthus reaction FIGURE 2. Autometallographic silver-enhanced mercury-exposed tis- was performed. In this reaction, OVA plus Evans blue was injected by guest on September 30, 2021 sue. Mice were injected s.c. with HgCl2 for 4 wk and processed for auto- in the tail vein of mice of both genotypes and anti-OVA Abs were metallography as described in Materials and Methods section. Represen- injected s.c. in two sites. There was no difference in inter-genotype tative photomicrographs are shown. Note the equally intense vascular permeability as evaluated by Evans blue dye diffusion autometallographic staining (arrows) in the renal tubules of both wild-type (wild-type mice: 11 mm Ϯ 0.1; Hx-null mice: 10 mm Ϯ 0.2). In (B) and Hx-null (C) compared with the unstained sham control (A). addition, a dense leukocyte infiltration was observed at the site of anti-OVA injection in both wild-type (Fig. 3A) and Hx-null mice (Fig. 3B). These data indicate that the inflammatory response due These data indicate that Hx-null mice were protected from to leukocyte activation by immune deposit formation in the skin

HgCl2-induced autoimmune disease. Thus, experiments investigat- was maintained in the Hx-null mice. ing defects in general immune responses, mercury delivery, or au- Moreover, Hx-null mice immunized with OVA were able to toimmune pathways in Hx-null mice compared with wild-type produce anti-OVA Abs (Fig. 3C) at titers similar to those of wild mice were conducted. type mice. Thus, Hx-null mice were able to respond to a foreign Ag administration by producing Abs, suggesting a specific defect Similar tissue distribution of mercury as revealed by in autoantibody production after mercury exposure. autometallography Because hemopexin is capable of binding divalent metal cations, Characterization of spleen cell populations following HgCl2 e.g., Fe2ϩ,Zn2ϩ, and Co2ϩ, the possibility of differential distri- injections bution of mercury to various tissues between wild-type and Hx- We subsequently analyzed, by flow cytometry, absolute spleen cell null mice could not be ruled out (15). populations under basal conditions and after HgCl2 treatment. Un- Autometallography was applied. The autometallographic tech- der basal conditions, we did not find any statistically significant nique is based on silver enhancement of quantum dots resulting differences either in the total number of splenocytes or in specific from the following: 1) the metabolism of toxic metals in animals cell populations. After treatment with HgCl2 for 4 wk there was an and humans: 2) in vivo, in vitro, and immersion-created zinc-sul- increase in the absolute cell number of the spleen cells of both fur/zinc-selenium nanocrystals; and 3) metal ions liberated from genotypes, but this was only statistically significant in wild-type metal implants and particles (14). Wild-type (Fig. 2B) and Hx-null mice (Fig. 4A). Flow cytometry analysis of the spleen cell popu- (Fig. 2C) mice showed similar intense autometallographic mercury lations revealed a significant increase in CD4ϩ T cells in both staining in the renal tubules of mercury-exposed kidneys after 4 Hx-null and wild-type mice (Fig. 4B). No significant alterations wk of treatment vs the unstained sham control (Fig. 2A). Thus, were noted in CD8ϩ T cell population (Fig. 4C). plasma hemopexin did not control the distribution of mercury to Other spleen populations, i.e., B-cells, macrophages, and den- the tissues after HgCl2 injection in mice. dritic cells, were measured by flow cytometry using, respectively, 1942 ROLE OF HEMOPEXIN IN AUTOIMMUNITY

FIGURE 4. Flow cytometric evaluation of splenic cell populations after 4 wk of treatment with

HgCl2. Splenocytes were isolated and treated as described in Materials and Methods. A, There was an increase in absolute spleen cell numbers of both genotypes, but the difference was statistically signifi- ,p Ͻ 0.01). B and C ,ءء) cant in wild-type mice only Downloaded from Representative graphs show significant increase in splenic CD4ϩ T cells (B) and no significant changes ϩ in CD8 T cells (C) in HgCl2-treated mice of both p Ͻ 0.01). D–G,A ,ءء ;p Ͻ 0.05 ,ء) genotypes significantly larger increase in the number of APCs occurred in wild-type mice after treatment vs Hx- ϩ null ones. The increase in CD19 B cells (D) was http://www.jimmunol.org/ statistically significant in wild-type controls only. Macrophages (E and F) did not change while the increase in CD11cϩ dendritic cells (G) was statisti- .(p Ͻ 0.001 ,ءءء) cally significant in both genotypes Data represent mean percentage Ϯ SE of five separate experiments (n ϭ 4/genotype/experiment). by guest on September 30, 2021

anti-CD19, anti-CD11b, or anti-F4/80 and anti-CD11c Abs (Fig. 4, D Analysis of Th1 and Th2 cytokine production in splenic and G). There was an increase in APC number in both wild-type and CD4ϩ T lymphocytes Hx-null mice after HgCl2 treatment. Interestingly, the increase in Because the Th1/Th2 dichotomy might account for the different CD19ϩ B cells was statistically significant in wild-type mice but not consequences after HgCl2 injection, the role of Th1/Th2 cytokines in the Hx-null ones (Fig. 4D). Macrophages did not change signifi- in this model was investigated on isolated splenocytes. There was cantly after HgCl2 injections (Fig. 4, E and F), whereas absolute den- no significant increase in production of IFN-␥ or IL-4 (represen- dritic cell number increased significantly in both genotypes (Fig. 4G). tative of Th1- and Th2-responses, respectively) by CD4ϩ T cells in Splenic regulatory T cells (Treg) expressing CD4ϩCD25ϩFoxP3ϩ markers, which participate in the regulation of autoreactive T cells, were also analyzed. There was no significant increase in the percent- Table II. Flow cytometric evaluation of Treg populationa age of splenic Treg in wild-type and Hx-null mice after HgCl2 treatment for 4 wk vs the respective basal levels. Results in Table II show Wild-Type Mice Hx-Null Mice the percentage of CD4ϩ T cells expressing CD25 and the percentage ϩ ϩ of CD4 CD25 cells expressing FoxP3. HgCl2 HgCl2 Markers Untreated Treated Untreated Treated To investigate the activation status of CD4ϩ T cells, the expression of CD69 (early activation marker) and CD44 (memory T cell marker) CD4/CD25 8.56 Ϯ 0.58 9.227 Ϯ 0.25 9.36 Ϯ 0.53 7.93 Ϯ 0.75 CD4/CD25/FoxP3 5.68 Ϯ 0.49 4.66 Ϯ 0.11 3.27 Ϯ 0.65 2.62 Ϯ 0.53 were analyzed 1 day and 4 wk, respectively, after treatment with a The percentage of CD4ϩT cells expressing CD25 as well as the percentage of HgCl2. There was a statistically significant increase in the number of ϩ ϩ ϩ ϩ ϩ high CD4 CD25 cells expressing FoxP3 are shown under basal conditions and after 4 wk CD4 CD69 (Fig. 5A) and CD4 CD44 (Fig. 5B) T cells in wild- Ϯ of treatment with HgCl2. Values represent mean percentage SE of five separate type mice compared with basal levels, but not in Hx-null mice. experiments (n ϭ 4/genotype/experiment). The Journal of Immunology 1943 Downloaded from

FIGURE 5. Expression of activation markers on CD4ϩ T cells after Materials and treatment with HgCl2. Mice were treated as described in http://www.jimmunol.org/ Methods. A, Percentages of splenic CD4ϩCD69ϩ T cells evaluated 1 day after treatment with HgCl2 showed a significant increase in wild-type mice -p Ͻ 0.05). B, Percentages of splenic CD4ϩCD44high T cells eval ,ء) only uated 4 wk after treatment with HgCl2 showed a significant increase in wild-type mice only with respect to basal level. The difference in ϩ high CD4 CD44 T cells between HgCl2-treated wild-type and Hx-null mice p Ͻ 0.05). Bars represent mean Ϯ SE of ,ء) was statistically significant three separate experiments (n ϭ 4/genotype/experiment). by guest on September 30, 2021 FIGURE 6. Evaluation of total IgG1, IgG2a, and IgE from serum after either genotype after treatment with HgCl2 for 4 wk (Table III)as treatment with HgCl2. The levels of total IgG1, IgG2a, and IgE from serum shown by flow cytometry experiments. Ex vivo treatment of iso- were measured as described in Materials and Methods section. A and B, lated splenocytes with PMA and ionomycin induced IL-4 and Analysis at different time points after HgCl injections showed that there ϩ 2 IFN-␥ expression in CD4 T cells in both Hx-null and wild-type was no increase in IgG1 (A) and IgG2a (B) production in Hx-null mice mice before and after treatment with HgCl2 (data not shown). To compared with wild-type controls. The increase in serum IgG1 and IgG2a confirm the cytokine profile, quantitative RT PCR was performed in HgCl2-treated wild-type mice was statistically significant after 4 days p Ͻ ,ءء ;p Ͻ 0.05 ,ء) and returned to basal levels after 4 wk of treatment on spleen total RNA of 10-day HgCl2-treated mice from both genotypes using assays-on-demand probes for IL-4 and IFN-␥ (Ap- 0.01). C, Serum IgE significantly increased in both genotypes 10 days after ;p Ͻ 0.05 ,ء) HgCl treatment and returned to basal levels 4 wk later plied Biosystems). Again, no difference in Th1 and Th2 cytokine 2 -p Ͻ 0.01). No significant differences in IgE were noted between wild ,ءء ϩ expression was noted (not shown). Yet another CD4 T cell sub- type and Hx-null mice. Values represent mean Ϯ SE of three separate set, Th17, implicated in immune regulation, was analyzed in the experiments (n ϭ 4/genotype/experiment). spleen, but no increase in IL-17-producing CD4ϩ cells was noted after HgCl2 treatment in both Hx-null and wild-type mice compared with nontreated mice (Table III). Determination of serum levels of IgG1, IgG2a, and IgE after

HgCl2 injections ϩ Table III. Flow cytometric analysis of cytokine expression by CD4 T IgG1, IgG2a, and IgE were measured from the serum of mice cells in wild-type and Hx-null mice under basal conditions and after treated for different time periods with HgCl2 as parameter for B treatment with HgCl2 for 4 wk was performed as described in Materials and Methodsa cell activation. Interestingly, there was a statistically significant increase in IgG1 and IgG2a after 4 days of treatment in wild-type mice but not in Hx-null mice (Fig. 6, A and B). In contrast, a Untreated HgCl2 Treated statistically significant increase in IgE 10 days after HgCl2 injec- Wild-Type Hx-Null Wild-Type Hx-Null tion was seen in both genotypes (Fig. 6C). Four weeks after treat- Markers Mice Mice Mice Mice ment, these Igs returned to baseline levels. CD4/IL-4 1.81 Ϯ 0.61 1.12 Ϯ 0.62 0.73 Ϯ 0.18 0.66 Ϯ 0.18 CD4/IFN-␥ 13.94 Ϯ 3.34 13.58 Ϯ 3.43 11.03 Ϯ 0.51 9.73 Ϯ 3.23 CD4/IL-17 1.00 Ϯ 0.35 1.41 Ϯ 0.68 1.17 Ϯ 0.21 1.27 Ϯ 0.44 Spleen cell characterization following OVA/CFA injection a Th1, Th2, and Th17 cells were analyzed for the production of IFN-␥ , IL-4, and To analyze whether the differences in absolute spleen cell numbers IL-17, respectively. The percentages of CD4ϩ T cells expressing the different cytokines are shown. Values represent mean percentage Ϯ SE of three separate experi- and T cell activation status observed between wild-type and Hx- ϭ ments (n 4/genotype/experiment). null mice were specific to HgCl2 injection, we also characterized 1944 ROLE OF HEMOPEXIN IN AUTOIMMUNITY

C). There were no statistically signiﬁcant changes in IL-4 and in IFN-␥ in either genotype 10 days after OVA/CFA treatment (not shown). Overall, OVA/CFA treatment revealed no differences between wild-type and Hx-null mice.

Effect of IFN-␥ treatment on CD4ϩ T lymphocytes isolated from

HgCl2-treated mice To study whether an excess of unbound heme in Hx-null mice might affect iron homeostasis in T lymphocytes, we analyzed the expression of the heme-degrading enzyme HO-1 and that of TfR1 on T cells isolated from the spleens of Hx-null and wild-type mice. As expected, HO-1 expression was higher in the T cells of Hx-null mice than in those of wild-type animals, even if the difference did not reach statistical significance (not shown). Moreover, Hx-null splenic T cells showed a significantly reduced TfR1 expression compared with wild-type mice (Fig. 8, A and B). In agreement with Western blot data, flow cytometry analysis demonstrated a signif- ϩ icantly lower TfR1 (CD71) expression in Hx-null CD4 T cells Downloaded from compared with that of wild-type mice (Fig. 8C). FIGURE 7. Increase in absolute spleen lymphocyte numbers and T cell Based on previously reported results showing that iron may af- activation following OVA injection. Mice were treated and lymphocytes fect IFN-␥ responsiveness through the TfR1-mediated down-reg- were isolated as described in Materials and Methods. A, Absolute number ulation of IFN-␥R2 expression at the cell membrane (16), we rea- ϩ ϩ of T lymphocytes (CD4 and CD8 ) as well as that of B lymphocytes soned that an excess of heme in Hx-null mice might have the same (CD19ϩ) showed similar increases in both genotypes after 10 days of im- ϩ ϩ effect. To test this hypothesis, we treated human ST4 T cells with http://www.jimmunol.org/ munization with OVA. B, Percentage of CD4 CD69 T cells evaluated 1 increasing amounts of hemin and then verified their responsiveness day after treatment with OVA showed a significant increase in both geno- to IFN-␥ by measuring STAT1 phosphorylation following 15 min p Ͻ 0.05). C, Percentage of ,ء) types compared with basal level ϩ high of incubation with IFN-␥. Interestingly, hemin at 2.5 and 5 ␮M CD4 CD44 T cells evaluated 10 days after treatment showed a signif- ␥ p Ͻ significantly inhibited IFN- -induced STAT1 activation (not ,ءءء) icant increase in both genotypes with respect to basal level 0.001). Values in A represent mean increase with respect to basal level Ϯ shown). Finally, STAT1 phosphorylation was measured in splenic ϩ SE of three separate experiments (n ϭ 4/genotype/experiment). Data in B CD4 T cells. The endogenous levels of phosphorylated STAT1 in ϩ and C represent mean percentage Ϯ SE of three separate experiments (n ϭ CD4 T cells isolated from wild-type and Hx-null mice, measured 4/genotype/experiments). by flow cytometry, were undetectable. We thus treated mice of by guest on September 30, 2021 both genotypes with HgCl2 for 4 days and then determined the responsiveness of splenic CD4ϩ T cells to IFN-␥ by measuring the response to OVA/CFA immunization. Ten days after treatment STAT1 phosphorylation following 15 min of incubation with with OVA/CFA, there was a similar increase in T and B lympho- 100U/ml IFN-␥. Interestingly, Hx-null CD4ϩ T cells showed a cytes in both genotypes, the increase being more pronounced in the statistically significant reduction in the activation of STAT1 after B cell population (Fig. 7A). Moreover, both CD4ϩCD69ϩ and IFN-␥ treatment compared with that of wild-type mice. CD4ϩ T CD4ϩCD44high T cells increased in the same manner following cells from both genotypes displayed the same levels of STAT1 OVA/CFA treatment in Hx-null and wild-type mice (Fig. 7, B and phosphorylation in response to IFN-␣, which was used as a control (Fig. 9).

Discussion In this study, we show that 129/Sv wild-type mice elicited an autoimmune response to s.c. injection of the heavy metal mercury as reﬂected by ANA production and IgG and C3 deposits in several organs. Intriguingly, the absence of hemopexin in mice of the same strain dampened the autoimmune manifestations. Hx-null mice produced signiﬁcantly less autoantibodies as judged by lower ANA titers in serum and had reduced IC deposits in the kidneys and in other organs compared with wild-type mice after chronic

treatment with HgCl2. FIGURE 8. Hx-null mice have lower expression of TfR1 in splenic T The differences in the autoimmune response cannot be ascribed cells. A and B, T lymphocytes were purified and total protein was extracted to differences in mercury distribution, inflammatory response, as described in Materials and Methods. Representative Western blotting or the ability to produce Abs after challenge with an exogenous (A) shows the level of TfR1 expression in purified splenic T cells from four Ag. This led to the conclusion of a specific defect in the autoim- wild-type (first four lanes from the left) and four Hx-null mice (last four mune response to the chronic injection of HgCl in the knockout lanes from the left) under basal conditions. Actin was used as loading 2 mice. Because mercury is a lymphoproliferative agent (17), the control. Hx-null mice had lower TfR1 expression compared with wild-type mice as shown by the densitometric analysis of the TfR1/actin ratio (B). C, absolute cell numbers of the different splenic populations were determined in the two genotypes before and after 4 wk of treat- Lower TfR1 (CD71) expression in Hx-null mice was confirmed by flow ϩ cytometry analysis of CD4ϩCD71ϩ T cells under basal conditions. Mean ment. There was a significant increase in CD4 T cells in both ϩ ϩ percentages of splenic CD4 CD71 T cells are shown. Values represent genotypes after HgCl2 injections. The APCs also increased in mean Ϯ SE of three separate experiments (n ϭ 4/genotype/experiment). number in both genotypes, but the increase was significantly larger The Journal of Immunology 1945

lute T and B cell number was observed in wild-type and Hx-null mice, demonstrating that the difference in B cell proliferation after

HgCl2 treatment was speciﬁc to this autoimmune stimulus. The

reduced increase in the number of B cells in HgCl2-treated Hx-null mice with respect to wild-type mice might explain why Hx-null mice produce significantly fewer autoantibodies. Moreover, Hx-null mice failed to activate CD4ϩ T cells as demonstrated by the lack of induction of CD4ϩCD69ϩ and CD4ϩCD44high T cell populations. Again, this impairment is specific to the autoimmune stimulus, because T cell activation after OVA/CFA treatment is similar in Hx-null and wild-type mice. This notion is supported by previous observations in mice deficient for CD28 and CD40 ligand (18). These mice exhibited reduced ϩ high numbers of CD4 CD44 T cell after HgCl2 injections and had complete lack of all features of mercury-induced autoimmunity. Due to the reduced activation of T cells, Hx-null T and B lymphocytes might have weaker interactions which could justify the lower production of autoantibodies after mercury exposure. The percentage of CD4ϩCD25ϩFoxP3ϩ Treg or CD4ϩCD17ϩ Downloaded from

T cells did not change after chronic HgCl2 injections in the respective genotypes, thus excluding the involvement of Treg or Th17 populations in the pathogenesis of mercury-induced autoimmunity in our models. Cytokines produced by CD4ϩ T cells have been shown to play crucial roles in the development of mercury-induced autoimmune http://www.jimmunol.org/ manifestations (19). This point was addressed by our present studies by analyzing IFN-␥ and IL-4 expression, assessed as representative cytokines of the Th1 and Th2 subsets, respectively. The expression of IFN-␥ and IL-4 in CD4ϩ T cells was similar in Hx-null and wild-type mice under basal conditions and did not

change after HgCl2 treatment. Thus, another recently identiﬁed T cell subset, Th17, was analyzed. Th17 participates in various dis- ϩ

eases like rheumatoid arthritis and multiple sclerosis (20). CD4 T by guest on September 30, 2021 cell- specific production of IL-17 in the spleen was measured by flow cytometry, and no induction in the production of IL-17 was noted in either genotype compared with mice under basal conditions. These results show that the lack of hemopexin does not influence the expression of IL-4, IFN-␥, and IL-17 in the CD4ϩ T cells, at least in the 129/Sv genetic background, after chronic treat-

ment with HgCl2. In contrast, measurements of total IgG isotypes and IgE in se-

rum at different times after HgCl2 treatment showed a transient and signiﬁcant increase in IgG1 and IgG2a after 4 days as well as in IgE after 10 days, indicating both Th1- and Th2-type (Th0) responses in wild-type mice. This is in concordance with data from

FIGURE 9. STAT1 phosphorylation is reduced in HgCl2-treated Hx- other groups showing that both types of responses are involved in ϩ null mouse CD4 T cells after IFN-␥ (IFN-g) stimulation ex vivo. Mice the pathogenesis of mercury-induced autoimmunity (21). Hx-null were treated with HgCl2 for 4 days. Splenocytes were isolated from these mice had no change in serum IgG1 or IgG2a levels but had a ␥ ␣ mice and treated with IFN- (100 U/ml), or IFN- (IFN-a; 100U/ml) as significant increase in IgE levels, pointing to a Th2-type response control for 15 min at 37°C. Activation of STAT1 was evaluated by flow in these mice after treatment with HgCl . These experiments sug- cytometry analysis using an anti-phospho(Tyr701)-STAT1 Ab as indicated 2 in Materials and Methods. A gate was created around the population that gested that Hx-null mice, after HgCl2 injections, lack a response to stained positive for CD4. Representative dot plots are shown (A–F). A and IFN-␥, which is crucial for class switching to IgG2a (22). B, Basal STAT1 phosphorylation in CD4ϩ T cells obtained from both In accordance with this, Kono and coworkers have shown that genotypes. C and D, IFN-␥-induced STAT1 activation was significantly cell responsiveness to IFN-␥ especially in the early phase of dis- ϩ reduced in CD4 T cells isolated from Hx-null mice compared with those ease induction, rather than IL-4/IFN-␥ imbalance, is crucial in de- p Ͻ 0.05). E and F, No significant difference termining susceptibility to mercury-induced autoimmunity (23). In ,ء) from wild-type ones ␣ between wild-type and Hx-null mice was observed using IFN- (100U/ml) agreement with this view, IFN-␥-null as well as IFN-␥R-null or G, The data and means obtained in three separate experiments are shown IFN regulatory factor-1-null mice showed significant protection (n ϭ 2/genotype/experiment). against mercury-induced autoimmune lesions (17). Recently, it has been shown that iron regulates T lymphocyte in wild-type mice with respect to basal level, especially in CD19ϩ sensitivity to IFN-␥. In particular, iron uptake mediated by a trans- B cells and CD11cϩ dendritic cells compared with Hx-null mice. ferrin receptor delivers a signal that leads to IFN-␥R2 internaliza- In contrast, after OVA/CFA treatment, a similar increase in abso- tion, thus attenuating activation of the IFN-␥/STAT1 pathway 1946 ROLE OF HEMOPEXIN IN AUTOIMMUNITY Downloaded from FIGURE 10. Lack of hemopexin causes refractoriness of CD4ϩ T cells to IFN-␥ (IFNg). Schematic representation of CD4؉ T cells of wild-type and Hx-null mice are shown. IFN-␥ binds to IFN-␥R1 (IFNgR1) (binding chain), and the IFN-␥R2 (transducing chain) is recruited to the complex. JAK1 and JAK2 bound to the receptor complex transactivate each other and phosphorylate IFN-␥ R1, generating a docking site for STAT1. Phosphorylated STAT1 dimerizes and translocates to the nucleus where it activates IFN-␥-regulated genes. IFN-␥ R2 expression at the plasma membrane is regulated by the TfR1 level, the latter being controlled by intracellular iron through the iron regulatory proteins. In wild-type mice, extracellular heme does not contribute to the intracellular iron pool because it is bound by hemopexin and transported to the liver. In Hx-null mice, unbound heme is taken up by the cell and, consequently, is metabolized by enzyme HO-1 into biliverdin, CO, and iron. The increase in the iron pool results in TfR1 mRNA degradation and lower http://www.jimmunol.org/ .␥-protein expression at the plasma membrane. This causes the down-regulation of IFN-␥R2 expression, inducing refractoriness of CD4؉ T cells to IFN

(16). In contrast, the iron chelating agent desferroxamine up-reg- unbound heme is taken up by the cell and, consequently, is me- ulates IFN-␥R2 surface expression and reinstates IFN-␥/STAT1 tabolized by enzyme HO-1 into biliverdin, CO, and iron. The in- activation. We have previously shown the importance of he- crease in the iron pool results in TfR1 mRNA degradation and mopexin in recuperating heme-derived iron from the circulation lower protein expression at the plasma membrane. This causes the -In Hx-null mice, it is expected that heme-iron is not recovered down-regulation of IFN-␥R2 expression in CD4؉ T cells impair .(8) by the liver but taken up by other tissues, including lymphocytes. ing STAT1 phosphorylation, thus inducing refractoriness to by guest on September 30, 2021 In this study we show that Hx-null T cells have significantly IFN-␥. This model is supported by work from other groups show- reduced TfR1 expression compared with wild-type mice. Because ing that Th1-mediated effect or function is particularly sensitive to TfR1 is mainly regulated posttranscriptionally by iron through the changes in iron homeostasis (29). iron regulatory protein system (24), our data indicate that Hx-null In conclusion, our data show a dampened activation of CD4ϩ T T cells are indeed iron overloaded compared with those of wild- cells in Hx-null mice that could be responsible for reduced auto- type mice, likely because these take up the excess heme. In agree- immune response after mercury exposure. This possibly led to re- ment with this, a slight increase in HO-1 in Hx-null T lymphocytes duced B cell proliferation and differentiation and a lower amount was observed. The increase in HO-1 activation in lymphocytes has of autoantibody production and hence reduced the amount of sys- been previously reported to suppress the autoimmune response and temic IC deposits in Hx-null mice. to limit T cell activation (25). The interplay between TfR1 and Thus, hemopexin modulation may be a new potential therapeu- IFN-␥R2 during CD4ϩ T cell activation is not yet clear, but it has tic tool in the treatment of autoimmune disorders. been shown that TfR1 can physically associate with TCR in the immunological synapse (26, 27) and that TCR engagement also Acknowledgments induces a rapid copolarization of IFN-␥R with TCR (28). More- We thank Nanni Passerini for creatinine measurement and proteinuria eval- over, Regis et al. have recently demonstrated that TfR1 expression uation, Laura Morando for performing transmission electron microscopy, ␥ at the plasma membrane modulates the IFN- R level, thus regu- and Veronica Fiorito for help with some flow cytometry experiments. Vale- lating the responsiveness of T cells to IFN-␥ (16). ria Poli, Franco Novelli, and Gabriella Regis are acknowledged for pro- Importantly, ex vivo treatment of splenocytes with IFN-␥ viding ST4 cells and for helpful advice on STAT1/IFN-␥ experiments. showed that there was a statistically significant reduction in ϩ STAT1 phosphorylation in CD4 T lymphocytes isolated from Disclosures HgCl -treated Hx-null mice compared with that of HgCl -treated 2 2 The authors have no financial conflict of interest. wild-type mice. Moreover, in vitro treatment of human ST4 T cells with hemin followed by IFN-␥ stimulation demonstrated that STAT1 phosphorylation is reduced, supporting our hypothesis that References free heme might affect IFN-␥ signaling. 1. Cervera, R. 2001. The epidemiology and significance of autoimmune diseases in health care. Scand. J. Clin. Lab. Invest. Suppl. 235: 27–30. Our data suggest that the blunted T cell activation in Hx-null 2. Utz, P. J., T. J. Gensler, and P. Anderson. 2000. Death, autoantigen modifications, mice is accounted for by their refractoriness to IFN-␥ according to and tolerance. Arthritis Res. 2: 101–114. the model presented in Fig. 10. In wild-type mice, extracellular 3. Kuvibidila, S., M. Dardenne, W. Savino, and F. Lepault. 1990. Influence of iron-deficiency anemia on selected thymus functions in mice: thymulin biological heme does not contribute to the intracellular iron pool because it is activity, T-cell subsets, and thymocyte proliferation. Am. J. Clin. Nutr. 51: bound by hemopexin and transported to the liver. In Hx-null mice, 228–232. The Journal of Immunology 1947

4. Guglielmo, P., F. Cunsolo, T. Lombardo, G. Sortino, R. Giustolisi, E. Cacciola, sensitivity to the IFN-␥/STAT1 signaling pathway in vitro and in vivo. Blood and E. Cacciola. 1984. T-subset abnormalities in thalassaemia intermedia: pos- 105: 3214–3221. sible evidence for a thymus functional deficiency. Acta Haematol. 72: 361–367. 17. Pollard, K. M., P. Hultman, and D. H. Kono. 2005. Immunology and genetics of 5. Leiter, L. M., K. R. Reuhl, S. P. Racis, Jr., and A. R. Sherman. 1995. Iron status induced systemic autoimmunity. Autoimmun. Rev. 4: 282–288. alters murine systemic lupus erythematosus. J. Nutr. 125: 474–484. 18. Pollard, K. M., M. Arnush, P. Hultman, and D. H. Kono. 2004. Costimulation 6. Bowlus, C. L. 2003. The role of iron in T cell development and autoimmunity. requirements of induced murine systemic autoimmune disease. J. Immunol. 173: Autoimmun. Rev. 2: 73–78. 5880–5887. 19. Jiang, Y., and G. Moller. 1995. In vitro effects of HgCl2 on murine lymphocytes: 7. Pavon, E. J., P. Munoz, A. Lario, V. Longobardo, M. Carrascal, J. Abian, ϩ A. B. Martin, S. A. Arias, J. L. Callejas-Rubio, R. Sola, et al. 2006. Proteomic I. Preferable activation of CD4 T cells in a responder strain. J. Immunol. 154: analysis of plasma from patients with systemic lupus erythematosus: Increased 3138–3146. presence of haptoglobin alpha2 polypeptide chains over the alpha1 isoforms. 20. Rohn, T. A., G. T. Jennings, M. Hernandez, P. Grest, M. Beck, Y. Zou, M. Kopf, Proteomics 6 (Suppl. 1): S282–S292. and M. F. Bachmann. 2006. Vaccination against IL-17 suppresses autoimmune 8. Tolosano, E., E. Hirsch, E. Patrucco, C. Camaschella, R. Navone, L. Silengo, and arthritis and encephalomyelitis. Eur. J. Immunol. 36: 2857–2867. F. Altruda. 1999. Defective recovery and severe renal damage after acute hemo- 21. Hu, H., G. Moller, and M. Abedi-Valugerdi. 1999. Mechanism of mercury-in- lysis in hemopexin-deficient mice. Blood 94: 3906–3914. duced autoimmunity: both T helper 1- and T helper 2-type responses are in- 9. Bach, F. H. 2006. Heme oxygenase-1 and transplantation tolerance. Hum. Im- volved. Immunology 96: 348–357. munol. 67: 430–432. 22. Coffman, R. L., H. F. Savelkoul, and D. A. Lebman. 1989. Cytokine regulation of immunoglobulin isotype switching and expression. Semin. Immunol. 1: 55–63. 10. Martinsson, K., and P. Hultman. 2006. The role of Fc-receptors in murine mer- 23. Kono, D. H., D. Balomenos, D. L. Pearson, M. S. Park, B. Hildebrandt, cury-induced systemic autoimmunity. Clin. Exp. Immunol. 144: 309–318. P. Hultman, and K. M. Pollard. 1998. The prototypic Th2 autoimmunity induced 11. Camussi, G., C. Tetta, M. C. Deregibus, F. Bussolino, G. Segoloni, and by mercury is dependent on IFN-␥ and not Th1/Th2 imbalance. J. Immunol. 161: A. Vercellone. 1982. Platelet-activating factor (PAF) in experimentally induced 234–240. rabbit acute serum sickness: role of basophil-derived PAF in immune complex 24. Hentze, M. W., M. U. Muckenthaler, and N. C. Andrews. 2004. Balancing acts: deposition. J. Immunol. 128: 86–94. molecular control of mammalian iron metabolism. Cell 117: 285–297. 12. Herrera, M. B., B. Bussolati, S. Bruno, L. Morando, G. Mauriello-Romanazzi, 25. Chora, A. A., P. Fontoura, A. Cunha, T. F. Pais, S. Cardoso, P. P. Ho, L. Y. Lee, F. Sanavio, I. Stamenkovic, L. Biancone, and G. Camussi. 2007. Exogenous R. A. Sobel, L. Steinman, and M. P. Soares. 2007. Heme oxygenase-1 and carbon Downloaded from mesenchymal stem cells localize to the kidney by means of CD44 following acute monoxide suppress autoimmune neuroinflammation. J. Clin. Invest. 117: tubular injury. Kidney Int. 72: 430–441. 438–447. 13. Danscher, G., and B. Moller-Madsen. 1985. Silver amplification of mercury sul- 26. Davis, R. J., M. Faucher, L. K. Racaniello, A. Carruthers, and M. P. Czech. 1987. fide and selenide: a histochemical method for light and electron microscopic Insulin-like growth factor I and epidermal growth factor regulate the expression localization of mercury in tissue. J. Histochem. Cytochem. 33: 219–228. of transferrin receptors at the cell surface by distinct mechanisms. J. Biol. Chem. 14. Danscher, G., and M. Stoltenberg. 2006. Silver enhancement of quantum dots 262: 13126–13134. resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, 27. Salmeron, A., A. Borroto, M. Fresno, M. J. Crumpton, S. C. Ley, and B. Alarcon. in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) 1995. Transferrin receptor induces tyrosine phosphorylation in T cells and is http://www.jimmunol.org/ metal ions liberated from metal implants and particles. Prog. Histochem. Cyto- physically associated with the TCR ␨-chain. J. Immunol. 154: 1675–1683. chem. 41: 57–139. 28. Batista, A., J. Millan, M. Mittelbrunn, F. Sanchez-Madrid, and M. A. Alonso. 15. Mauk, M. R., F. I. Rosell, B. Lelj-Garolla, G. R. Moore, and A. G. Mauk. 2005. 2004. Recruitment of transferrin receptor to immunological synapse in response Metal ion binding to human hemopexin. Biochemistry 44: 1864–1871. to TCR engagement. J. Immunol. 172: 6709–6714. 16. Regis, G., M. Bosticardo, L. Conti, S. De Angelis, D. Boselli, B. Tomaino, 29. Weiss, G. 2002. Iron and immunity: a double-edged sword. Eur. J. Clin. Invest. P. Bernabei, M. Giovarelli, and F. Novelli. 2005. Iron regulates T-lymphocyte 32 (Suppl. 1): 70–78. by guest on September 30, 2021