Experimental Autoimmune Myocarditis in A/J mice Is an Interleukin-4-Dependent Disease with a Th2 Phenotype
2001; Elsevier BV; Volume: 159; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)61685-9
ISSN1525-2191
AutoresMarina Afanasyeva, Yan Wang, Ziya Kaya, Sung‐Min Park, Michael J. Zilliox, Brian Schofield, Susan L. Hill, Noel R. Rose,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoMyocarditis in humans is often associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen. Experimental autoimmune myocarditis (EAM) is induced in mice by immunization with CM. We found that EAM in A/J mice exhibits a Th2-like phenotype demonstrated by the histological picture of the heart lesions (eosinophils and giant cells) and by the humoral response (association of IgG1 response with disease and up-regulation of total IgE). Blocking interleukin (IL)-4 with anti-IL-4 monoclonal antibody (mAb) reduced the severity of EAM. This reduction in severity was associated with a shift from a Th2-like to a Th1-like phenotype represented by a reduction in CM-specific IgG1; an increase in CM-specific IgG2a; an abrogation of total IgE response; a decrease in IL-4, IL-5, and IL-13; as well as a dramatic increase in interferon (IFN)-γ production in vitro. Based on the latter finding, we hypothesized that IFN-γ limits disease. Indeed, IFN-γ blockade with a mAb exacerbated disease. The ameliorating effect of IL-4 blockade was abrogated by co-administration of anti-IFN-γ mAb. Thus, EAM represents a model of an organ-specific autoimmune disease associated with a Th2 phenotype, in which IL-4 promotes the disease and IFN-γ limits it. Suppression of IFN-γ represents at least one of the mechanisms by which IL-4 promotes EAM. Myocarditis in humans is often associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen. Experimental autoimmune myocarditis (EAM) is induced in mice by immunization with CM. We found that EAM in A/J mice exhibits a Th2-like phenotype demonstrated by the histological picture of the heart lesions (eosinophils and giant cells) and by the humoral response (association of IgG1 response with disease and up-regulation of total IgE). Blocking interleukin (IL)-4 with anti-IL-4 monoclonal antibody (mAb) reduced the severity of EAM. This reduction in severity was associated with a shift from a Th2-like to a Th1-like phenotype represented by a reduction in CM-specific IgG1; an increase in CM-specific IgG2a; an abrogation of total IgE response; a decrease in IL-4, IL-5, and IL-13; as well as a dramatic increase in interferon (IFN)-γ production in vitro. Based on the latter finding, we hypothesized that IFN-γ limits disease. Indeed, IFN-γ blockade with a mAb exacerbated disease. The ameliorating effect of IL-4 blockade was abrogated by co-administration of anti-IFN-γ mAb. Thus, EAM represents a model of an organ-specific autoimmune disease associated with a Th2 phenotype, in which IL-4 promotes the disease and IFN-γ limits it. Suppression of IFN-γ represents at least one of the mechanisms by which IL-4 promotes EAM. Myocarditis is a major cause of heart failure and sudden death among adolescents and young adults.1Drory Y Turetz Y Hiss Y Lev B Fisman EZ Pines A Kramer MR Sudden unexpected death in persons less than 40 years of age.Am J Cardiol. 1991; 68: 1388-1392Abstract Full Text PDF PubMed Scopus (365) Google Scholar Although some myocarditis patients recover, many progress to dilated cardiomyopathy, an often fatal condition and a frequent reason for cardiac transplantation.2Brown CA O'Connell JB Myocarditis and idiopathic dilated cardiomyopathy.Am J Med. 1995; 99: 309-314Abstract Full Text PDF PubMed Scopus (84) Google Scholar Many cases of myocarditis in humans are associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen.3Caforio AL Goldman JH Haven AJ Baig KM McKenna WJ Evidence for autoimmunity to myosin and other heart-specific autoantigens in patients with dilated cardiomyopathy and their relatives.Int J Cardiol. 1996; 54: 157-163Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 4Lauer B Schannwell M Kuhl U Strauer BE Schultheiss HP Antimyosin autoantibodies are associated with deterioration of systolic and diastolic left ventricular function in patients with chronic myocarditis.J Am Coll Cardiol. 2000; 35: 11-18Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Treatment of autoimmune myocarditis is challenging, with immunosuppressive therapies giving mixed results.5Mason JW O'Connell JB Herskowitz A Rose NR McManus BM Billingham ME Moon TE A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators.N Engl J Med. 1995; 333: 269-275Crossref PubMed Scopus (1011) Google Scholar The design of better treatment regimens depends on understanding the mechanisms leading to the immune-mediated damage to the heart. To study these mechanisms, we have previously established a murine model of experimental autoimmune myocarditis (EAM) induced by immunization with CM.6Neu N Rose NR Beisel KW Herskowitz A Gurri-Glass G Craig SW Cardiac myosin induces myocarditis in genetically predisposed mice.J Immunol. 1987; 139: 3630-3636PubMed Google Scholar In susceptible mouse strains, such as A/J and BALB/c, EAM represents a model of acute myocarditis with the peak of inflammation in the heart corresponding to day 21 after immunization. Cytokines, such as tumor necrosis factors and interleukin (IL)-1, have been shown to play a key role in the development of EAM.7Lane JR Neumann DA Lafond-Walker A Herskowitz A Rose NR Interleukin 1 and tumor necrosis factor can promote coxsackie B3-induced myocarditis in resistant B10.A mice.J Exp Med. 1992; 175: 1123-1129Crossref PubMed Scopus (177) Google Scholar, 8Smith SC Allen PM Neutralization of endogenous tumor necrosis factor ameliorates the severity of myosin-induced myocarditis.Circ Res. 1992; 70: 856-863Crossref PubMed Scopus (126) Google Scholar, 9Bachmaier K Pummerer C Kozieradzki I Pfeffer K Mak TW Neu N Penninger JM Low-molecular-weight tumor necrosis factor receptor p55 controls induction of autoimmune heart disease.Circulation. 1997; 95: 655-661Crossref PubMed Scopus (68) Google Scholar The role of other cytokines, including those characteristic of Th1 and Th2 immune responses, is unclear. Depending on the cytokine milieu, T-helper lymphocytes can differentiate into two subsets, Th1 and Th2, which represent functionally polarized and mutually antagonistic immune responses.10Liblau RS Singer SM McDevitt HO Th1 and Th2 CD4+ cells in the pathogenesis of organ-specific autoimmune diseases.Immunol Today. 1995; 16: 34-38Abstract Full Text PDF PubMed Scopus (1115) Google Scholar, 11Mosmann TR Sad S The expanding universe of T-cell subsets: th1, Th2, and more.Immunol Today. 1996; 17: 138-146Abstract Full Text PDF PubMed Scopus (3311) Google Scholar Interferon (IFN)-γ and IL-4 are prototypic Th1- and Th2-type cytokines, respectively. IFN-γ stimulates Th1 T-cell development, activates macrophages, induces MHC class I and II expression, promotes delayed-type hypersensitivity reactions, induces Ig class switching to IgG2a in mice, recruits Th1 T cells to the site of the inflammation, plays an important role in clearing intracellular bacteria and intracellular parasites, and exhibits antiviral activity.12Boehm U Klamp T Groot M Howard JC Cellular responses to interferon-γ.Annu Rev Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2451) Google Scholar, 13Finkelman FD Holmes J Katona IM Urban Jr, JF Beckman MP Park LS Schooley KS Coffman RL Mosmann TR Paul WE Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol. 1990; 8: 303-333Crossref PubMed Google Scholar IL-4, on the other hand, stimulates Th2 T-cell development, activates B cells, induces MHC class II expression on B cells, promotes allergic reactions, induces Ig class switching to IgE and IgG1 in mice, recruits eosinophils and Th2 cells to the site of inflammation, and is important in clearing extracellular parasites.13Finkelman FD Holmes J Katona IM Urban Jr, JF Beckman MP Park LS Schooley KS Coffman RL Mosmann TR Paul WE Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol. 1990; 8: 303-333Crossref PubMed Google Scholar, 14Hickey MJ Granger DN Kubes P Molecular mechanisms underlying IL-4-induced leukocyte recruitment in vivo: a critical role for the α4 integrin.J Immunol. 1999; 163: 3441-3448PubMed Google Scholar, 15Nelms K Keegan AD Zamorano J Ryan JJ Paul WE The IL-4 receptor: signaling mechanisms and biologic functions.Annu Rev Immunol. 1999; 17: 701-738Crossref PubMed Scopus (1269) Google Scholar Extensive studies using other models of autoimmune disease have led to the conclusion that Th1 responses promote autoimmune processes whereas Th2 responses may suppress them.10Liblau RS Singer SM McDevitt HO Th1 and Th2 CD4+ cells in the pathogenesis of organ-specific autoimmune diseases.Immunol Today. 1995; 16: 34-38Abstract Full Text PDF PubMed Scopus (1115) Google Scholar, 11Mosmann TR Sad S The expanding universe of T-cell subsets: th1, Th2, and more.Immunol Today. 1996; 17: 138-146Abstract Full Text PDF PubMed Scopus (3311) Google Scholar The goal of this study was to investigate the balance of Th1 and Th2 responses in the development of EAM. We found that EAM exhibits a Th2-like phenotype both in terms of the histological composition of the heart infiltrate and of the humoral response. Based on these findings, we hypothesized that IL-4 plays a critical role in promoting EAM. Indeed, neutralization of IL-4 with anti-IL-4 monoclonal antibody (mAb) during the course of EAM induction significantly reduced the severity of disease as well as the markers of a Th2 response, and enhanced the production of IFN-γ by splenocytes in vitro. Blocking IFN-γ with a mAb markedly exacerbated disease providing evidence for a limiting effect of IFN-γ on the disease process. We conclude that the suppression of a disease-limiting factor, IFN-γ, represents a plausible pathogenetic mechanism of IL-4 action. Myocarditis was induced in 5- to 7-week-old female A/J mice obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in The Johns Hopkins University School of Medicine conventional animal facility. CM was purified from murine hearts according to the procedure by Shiverick and colleagues.16Shiverick KL Thomas LL Alpert NR Purification of cardiac myosin. Application to hypertrophied myocardium.Biochim Biophys Acta. 1975; 393: 124-133Crossref PubMed Scopus (108) Google Scholar On days 0 and 7, mice received subcutaneous injections of either 250 μg of CM or 100 nmol of a peptide from cardiac α-myosin heavy chain [myhcα(334-352), synthesized by Macromolecular Resources, Colorado State University, Department of Biochemistry, Fort Collins, CO] emulsified in Complete Freund's Adjuvant (CFA) (Sigma Chemical Co., Saint Louis, MO) supplemented with 5 mg/ml of Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI). On day 0, mice received an intraperitoneal injection of 500 ng of pertussis toxin (List Biological Laboratories, Campbell, CA). Mice were sacrificed on day 21. All animal work was approved by the Animal Care and Use Committee of The Johns Hopkins University. Donor A/J mice were immunized with either CM or myhcα(334-352) as described above. On day 21, spleens were aseptically removed and splenocytes were collected. Red blood cells were lysed by incubation with a lysing buffer (Quality Biological Inc., Gaithersburg, MD) for 5 minutes. Splenocytes were cultured for 3 days in RPMI 1640 (Life Technologies, Rockville, MD) with additional supplementation, as we have previously described,17Wang Y Afanasyeva M Hill SL Rose NR Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.Autoimmunity. 1999; 31: 151-162Crossref PubMed Scopus (34) Google Scholar in the presence of 10 μg/ml of CM. After stimulation, splenocytes were washed in phosphate-buffered saline (PBS) and counted by trypan blue exclusion. Recipient female 5- to 7-week-old A/J mice were irradiated (500 rads) 1 day before the transfer. Splenocytes were injected into a tail vein in a dose of 5 × 107 cells/mouse. Control mice received similarly stimulated splenocytes from donors that received adjuvants without CM immunization. Mice were sacrificed on day 14 after transfer. Immediately after euthanasia, mouse hearts were excised, fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Five-μm-thick sections were cut from base to apex and stained with hematoxylin and eosin (H&E). Every fifth section (a total of five sections from each heart) was examined by two independent investigators in a blinded manner. Severity of myocarditis was assessed on a scale from 0 to 5 based on the percentage of the heart section involved: grade 0, no disease; grade 1, up to 10% of the heart section; grade 2, 11 to 30%; grade 3, 31 to 50%; grade 4, 51 to 90%; and grade 5, 90 to 100%. A microscope with a grid was used to estimate the percentage of the heart section involved. Five-μm-thick sections of paraffin-embedded hearts were stained with 0.5% Congo Red in 50% glycine buffer (pH 10)/ethanol for 30 minutes and counterstained with hematoxylin. Murine hearts were collected on day 21 after immunization, embedded in Tissue-Tek OCT (Miles Inc., Elkhart, IN), and frozen at −70°C. Five-μm-thick sections were dehydrated in a desiccator at 37°C for 1.5 hours and then fixed in chilled acetone for 10 minutes. Fixed sections were washed for 5 minutes in each of the three solutions in the following order: 1) PBS; 2) PBS containing 1% normal goat serum and 1.5% hydrogen peroxide; and 3) PBS containing 1% bovine serum albumin. Staining was performed as described.17Wang Y Afanasyeva M Hill SL Rose NR Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.Autoimmunity. 1999; 31: 151-162Crossref PubMed Scopus (34) Google Scholar Incubation with each antibody was for 1 hour at room temperature. An anti-mouse IgG1 mAb (a rat IgG2a isotype) (clone G1-6.5; BD PharMingen, San Diego, CA) was used as a detection antibody. As an isotype control, heart sections were incubated with a rat IgG2a mAb of irrelevant specificity (clone R35-95, BD PharMingen). Peroxidase-conjugated streptavidin (P 0397; DAKO, Carpinteria, CA) in 1:500 dilution in PBS was used as a tertiary antibody. The positive stain was visualized by incubation with 3-amino-9-ethyl-carbazole (no. A-5754; Sigma Chemical Co.) substrate solution for 20 minutes. Mice were bled on days 0, 9, 16, and 21 from the retro-orbital venous plexus using heparinized tubes. Serum levels of CM-specific IgG and its subclasses were determined using CM-coated microtiter plates as we previously described.17Wang Y Afanasyeva M Hill SL Rose NR Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.Autoimmunity. 1999; 31: 151-162Crossref PubMed Scopus (34) Google Scholar Adjusted optical density (OD) was calculated as follows: adjusted OD = (mean OD of a sample − mean OD of a negative control)/(mean OD of a positive control − mean OD of a negative control). Total serum IgE was determined by a specific sandwich enzyme-linked immunosorbent assay (ELISA) using microtiter plates precoated with anti-mouse IgE capture mAb (clone R35-72, BD PharMingen). Rat anti-mouse IL-4 mAb 11B.11 (IgG1) was obtained as a gift from the National Cancer Institute, Frederick, MD. A hybridoma cell line producing rat anti-mouse IFN-γ mAb R4-6A2 (IgG1) was purchased from the American Tissue Culture Collection (Manassas, VA). Rat anti-Escherichia coli (E. coli) β-galactosidase mAb GL113 (IgG1) (the hybridoma cell line was kindly provided by Fred Finkelman, University of Cincinnati, Cincinnati, OH) was used as an isotype control. Hybridoma cell lines were grown by the Core Facility of The Johns Hopkins University. Monoclonal Abs were purified from the concentrated hybridoma supernatants using a HiTrap protein G column (Supelco Chromatography, Bellefonte, PA). Mice were immunized with CM and received anti-IL-4 mAb in a dose of 4 mg of mAb on days −1 and 6 (1 day before each CM injection) and in a dose of 2 mg of mAb on days 2, 9, 12, 15, and 18. Anti-IFN-γ mAb was administered in a dose of 1 mg on days −1, 6, and 12. GL113 mAb was used as an isotype control for both anti-IL-4 and anti-IFN-γ mAbs. All Abs were administered intraperitoneally. Monoclonal Abs were dissolved in a vehicle buffer (50 mmol/L sodium phosphate, 300 mmol/L sodium chloride, pH 6.8, for anti-IL-4 mAb and PBS for anti-IFN-γ and isotype control mAbs). On day 21 after immunization, spleens were removed aseptically. Splenocytes were cultured at the initial cell density of 5 × 106 cells/ml for 48 hours in RPMI 1640 medium (Life Technologies, Inc.) with additional supplementation in the presence of media alone, different concentrations of CM, or Con A (Sigma Chemical Co.) as we previously described.17Wang Y Afanasyeva M Hill SL Rose NR Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.Autoimmunity. 1999; 31: 151-162Crossref PubMed Scopus (34) Google Scholar Levels of IL-4, IL-5, IL-10, IL-12, IL-13, and IFN-γ in the splenocyte culture supernatants were measured using Quantikine murine cytokine ELISA kits (R&D Systems, Minneapolis, MN). Antibody and cytokine data were analyzed using the Student's t-test. The Mann-Whitney nonparametric test was used to compare EAM severity scores between different treatment groups. The strength of the association between CM-specific IgG1 levels and EAM severity was assessed by regression analysis (Statview software, Abacus Concepts, Inc., Berkeley, CA). P values of <0.05 were considered statistically significant. A/J mice were immunized with CM on days 0 and 7. At sacrifice on day 21, hearts were removed for histological examination. Depending on the experiment, the prevalence of myocarditis varied from 50 to 100%. Histologically, myocarditis was characterized by the presence of foci of myocardial infiltration and cardiomyocyte death, involving mainly the subepicardial regions of the myocardium in mild to moderate cases and affecting the full thickness of the myocardium in more severe disease. Myocardial infiltrate often involved the apical area and was frequently accompanied by pericardial infiltration. The heart infiltrate in EAM consisted of many macrophages and CD4+ T cells with some CD8+ T cells and B220+ B cells, as we have previously demonstrated by immunohistochemistry.17Wang Y Afanasyeva M Hill SL Rose NR Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.Autoimmunity. 1999; 31: 151-162Crossref PubMed Scopus (34) Google Scholar Additionally, the heart infiltrate contained many eosinophils (Figure 1, B and C) with a typical eosinophilic cytoplasm and a donut-shaped nucleus (characteristic of mouse eosinophils). The abundance of eosinophils was more prominent in severe cases of myocarditis (lesion scores ≥3). We observed a large number of eosinophils in EAM induced either by active immunization (Figure 1, B and C) or by splenocyte transfer (Figure 1, G and H). Furthermore, EAM induced by immunization with a 19 amino acid peptide from cardiac α-myosin heavy chain [myhcα(334-352)]18Donermeyer DL Beisel KW Allen PM Smith SC Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.J Exp Med. 1995; 182: 1291-1300Crossref PubMed Scopus (101) Google Scholar was characterized by the presence of eosinophils in the heart lesions. A similar histological picture characterized by many eosinophils was observed in a different strain of mice, BALB/c, on CM immunization (data not shown). Another striking feature of the myocardial infiltrate in EAM was the presence of multinucleated giant cells (Figure 1D). Giant cells were typically seen in more severe cases of EAM. The presence of eosinophils suggests a Th2-like phenotype of the disease.19Pakala SV Kurrer MO Katz JD T helper 2 (Th2) T cells induce acute pancreatitis and diabetes in immune-compromised nonobese diabetic (NOD) mice.J Exp Med. 1997; 186: 299-306Crossref PubMed Scopus (199) Google Scholar, 20Kopf M Le Gros G Coyle AJ Kosco-Vilbois M Brombacher F Immune responses of IL-4, IL-5, IL-6 deficient mice.Immunol Rev. 1995; 148: 45-69Crossref PubMed Scopus (123) Google Scholar, 21Nishimura T Iwakabe K Sekimoto M Ohmi Y Yahata T Nakui M Sato T Habu S Tashiro H Sato M Ohta A Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo.J Exp Med. 1999; 190: 617-627Crossref PubMed Scopus (366) Google Scholar Furthermore, macrophage fusion leading to the formation of multinucleated giant cells is induced by IL-4 in both humans22McNally AK Anderson JM Interleukin-4 induces foreign body giant cells from human monocyte/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells.Am J Pathol. 1995; 147: 1487-1499PubMed Google Scholar and mice.23McInnes A Rennick DM Interleukin-4 induces cultured monocytes/macrophages to form giant multinucleated cells.J Exp Med. 1988; 167: 598-611Crossref PubMed Scopus (238) Google Scholar To further examine the immune response to CM, we measured serum levels of CM-specific antibody. We found that CM-specific IgG (all subclasses) response was positively associated with disease (P < 0.0001). Further, by examining the IgG subclasses we found that CM-specific IgG1 (Figure 2, A and C) but not IgG2a (Figure 2, B and D), or any other IgG subclass (data not shown) correlated with the presence of disease on CM immunization. Day 9 is the earliest time point at which we could detect CM-specific antibody responses. On day 9, IgG2a, IgG2b, and IgG3 responses were still absent whereas the IgG1 response was already detectable in those mice that developed disease (Figure 2, A and B). IgG1 levels were also positively associated with the severity of disease (Figure 2E) (P value for the regression coefficient was 0.003). On the other hand, CM-specific IgG2a tended to be lower in mice with more severe disease (data not shown). A positive association between disease and CM-specific IgG1 was present when EAM was induced using a smaller dose of CM, 100 instead of 250 μg (data not shown). It has been reported that IgG1 production in mice is enhanced by IL-4, whereas IgG2a response is associated with IFN-γ production.13Finkelman FD Holmes J Katona IM Urban Jr, JF Beckman MP Park LS Schooley KS Coffman RL Mosmann TR Paul WE Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol. 1990; 8: 303-333Crossref PubMed Google Scholar As a surrogate measure of a Th2 to Th1 ratio, we examined an IgG1 to IgG2a ratio and found that it correlated with both the presence and severity of EAM (P value for the regression coefficient was 0.003). The addition of pertussis toxin to the treatment regimen may skew the immune response toward Th2. To exclude the possibility that the predominance of IgG1 response in our model was simply a pertussis toxin-associated phenomenon, we repeated the experiment omitting pertussis toxin from the treatment regimen. Histologically identical EAM was still induced in the absence of pertussis toxin, although the disease was less severe. Most important, the IgG1 response remained the predominant response and was associated with disease (Figure 2F). By examining Ab responses in mice immunized with myhcα(334-352) peptide, we found that disease is associated with both CM-specific IgG1 (Figure 2G) and myhcα(334-352)-specific IgG1 (Figure 2H). In these mice there was no association of disease with other IgG subclasses. We also found the same association of CM-specific IgG1 with disease in EAM induced by splenocyte transfer, in which recipients received no adjuvants (data not shown). This further supports the conclusion that the predominance of IgG1 response in EAM is not dependent on adjuvants. We found that IgG1 is deposited along the cardiomyocytes in the diseased heart (Figure 1, E and F) but not in the normal heart. In addition, IgG1-positive cells were found in the cardiac lesions. These cells, which are most likely to be IgG1-producing plasma cells, were present in clusters throughout the heart infiltrate. By contrast, there was a very little deposition of IgG2a in the heart in EAM (data not shown). Because IL-4 also drives IgE response,13Finkelman FD Holmes J Katona IM Urban Jr, JF Beckman MP Park LS Schooley KS Coffman RL Mosmann TR Paul WE Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol. 1990; 8: 303-333Crossref PubMed Google Scholar we measured the serum levels of total IgE in EAM and found that they are increased on CM immunization (Figure 4A, day 21 versus day −1 and day 9). Total IgE response started rising after day 9, was present on day 16, and further increased until day 21 when the experiment was terminated. However, total IgE response did not significantly correlate with the presence of disease (data not shown). Measurement of CM-specific IgE production was not possible because of the lack of sensitivity of our ELISA method. Thus, both the histological findings and the humoral immune response in EAM are consistent with a Th2 type of immune response, implicating IL-4 as a pathogenetic factor in this disease. Based on the presence of eosinophils and giant cells in the heart infiltrate, the strong association of CM-specific IgG1 with disease, and the up-regulation of the IgE response, we hypothesized that IL-4 is involved in the development of EAM. Therefore, we examined the role of IL-4 in EAM by treating mice with anti-IL-4 mAb during the course of disease induction. A/J mice were immunized with CM as described previously. In addition, mice received anti-IL-4 mAb (11B.11) intraperitoneally on days −1, 2, 6, 9, 12, 15, and 18. A control group received equivalent amounts of isotype control mAb (GL113). Additionally, we included a control group that received the equivalent volumes of a vehicle buffer and a control group that received CM immunization alone. All mice were sacrificed on day 21. We found that the anti-IL-4 treatment significantly reduced the severity of myocarditis (Figure 3 and also see Figure 7A). The prevalence of severe disease (grade ≥ 3) was 20% in the anti-IL-4-treated group and 80% in the isotype control group. In repeated experiments, the severity grade of 4 or higher was never observed in any of the anti-IL-4-treated mice. All of the control groups (isotype control, vehicle buffer control, and the group that received CM without any other treatment) did not differ in terms of disease prevalence or severity. The reduction in severity observed in the anti-IL-4-treated group was associated with smaller numbers of eosinophils present in the heart infiltrate.Figure 7IL-4 blockade reduces and IFN-γ blockade exacerbates the severity of EAM. A: Normal heart is from an untreated A/J mouse. CM-immunized mice received anti-IL-4 (11B.11 rat IgG1 anti-mouse IL-4 mAb), or anti-IFN-γ (R4-6A2 rat IgG1 anti-mouse IFN-γ mAb), or the isotype control mAb (GL113 rat IgG1 anti-E. coliβ-galactosidase mAb) as described in Figure 3, Figure 6. Mice were sacrificed on day 21. Hearts shown have EAM severity grades that correspond to the median grade from each group (anti-IL-4 heart has grade 1, isotype control heart has grade 3, and anti-IFN-γ heart has grade 4). Original magnification, ×15. H&E stain. B: A representative heart from the anti-IFN-γ-treated group (left) compared to a control mouse heart with no disease (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We examined the humoral immune response for changes associated with the reduction in severity of EAM on anti-IL-4 treatment. First, we measured the levels of total IgE and found that anti-IL-4 treatment abrogated the total IgE response (Figure 4A). This result confirmed the effectiveness of the blockade of IL-4 because there is a very strong association between the IgE levels and IL-4 activity. Next, we examined IgG subclasses and found that anti-IL-4 treatment significantly suppressed CM-specific IgG1 levels early during the course of EAM (day 9), but this suppression became nonsignificant later (day 21) compared to the control groups (Figure 4B). The fact that the anti-IL-4-treated group had a substantial IgG1 response on day 21 was consistent with incomplete abrogation of the disease by anti-IL-4 treatment; there was still a positive correlation between the severity of disease and IgG1 levels (P value for the regression coefficient was 0.007). Anti-IL-4-treated mice had higher levels of CM-specific IgG2a on day 21 compared to the control groups (Figure 4C). Anti-IL-4-treated mice also had higher levels of specific IgG2b and IgG3 but the differences were not statistically significant (data not shown). This increase in the IgG2a response can be explained by the fact that IL-4 and IFN-γ have an antagonistic relationship. Neutralization of IL-4 leads to the up-regulation of IFN-γ and therefore enhances IFN-γ-mediated effects such as the production of IgG2a antibody. To examine the effect of anti-IL-4 treatment on cytokine production, we collected splenocytes from the individual mice on day 21 after immunization and cultured them for 48 hours in the presence of CM. Splenocyte supernatants were examined for the presence of different cytokines. Anti-IL-4 treatment decreased IL-4, IL-5, and IL-13 production by splenocytes in response to CM (Figure 5, A–C) and dramatically enhanced the ability of the splenocytes to produce IFN-γ (Figure 5D) as compared to the control groups. We also looked at levels of IL-10 and IL-12 in the supernatants and found no difference between the anti-IL-4-treated group and the control groups (data not shown). IL-12 levels were below the lower limit of detection in the majority of mice. Taken together, our findings demonstrate that anti-IL-4 treatment resulted
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