fc receptor–mediated inhibitory effect of immunoglobulin therapy

Fc Receptor–Mediated Inhibitory Effect of ImmunoglobulinTherapy on Autoimmune Giant Cell Myocarditis

Concomitant Suppression of the Expression of Dendritic Cells

Keisuke Shioji, Chiharu Kishimoto, Shigetake Sasayama

Abstract—In the present study, the mechanisms and importance of the Fc portion of immunoglobulin in experimental giantcell myocarditis were examined. Giant cell myocarditis was induced in rats by immunization of porcine cardiac myosin.Human intact immunoglobulin (1 g · kg21 · d21) or F(ab9)2 fragments of human immunoglobulin (1 g · kg21 · d21) wereadministered intraperitoneally daily on days 1 to 21. Intact immunoglobulin administration significantly amelioratedmyocarditis, but F(ab9)2 fragments did not. The ribonuclease protection assay revealed that therapy with intactimmunoglobulin, but not F(ab9)2 fragments, suppressed the mRNA expressions of inflammatory and proinflammatorycytokines. Immunohistochemical analysis showed that therapy with intact immunoglobulin, but not F(ab9)2 fragments,suppressed dendritic cell (DC) expression during both the early and the subsequent fulminant phases. Moreover, theearly treatment of intact immunoglobulin until the 11th day or 14th day, when the expression of DCs was completelysuppressed, ameliorated myocarditis. However, the late treatment of intact immunoglobulin beginning on day 15, whenthe expression of DCs had already been completed, failed to ameliorate the condition. An in vitro study showed thatintact immunoglobulin, but not F(ab9)2 fragments, suppressed the lipopolysaccharide-induced interleukin-1b productionassociated with the downregulation of CD32 antigen (Fcg receptor II) expression. Thus, intact immunoglobulin therapymarkedly suppressed myocarditis as a result of Fc receptor–mediated anti-inflammatory action, and the suppression ofthe disease was associated with the suppression of DCs, ie, the suppression of the initial antigen-priming process inexperimental giant cell myocarditis.(Circ Res. 2001;89:540-546.)

Key Words: myocarditisn immunoglobulinn dendritic cellsn cytokinesn Fc receptors

Giant cell myocarditis is frequently fatal. Because thedisease is occasionally associated with various autoim-

mune diseases, autoimmune mechanisms were suggested tobe involved in its pathogenesis.1 The therapeutic efficacy ofhigh-dose immunoglobulin has been reported in inflamma-tory and autoimmune diseases, eg, Kawasaki disease,2 idio-pathic thrombocytopenic purpura,3 and peripartum cardiomy-opathy.4 We have previously reported that immunoglobulintherapy suppresses acute viral myocarditis as a result of ananti-viral effect, an anti-inflammatory effect, and the im-provement of extracellular matrix changes.5,6 Most recently, anovel mechanism of action of immunoglobulin was proposedto be due to anti-inflammatory activities through the inhibi-tory Fc receptors (FcRs).7

It has been suggested that T-cell–mediated autoimmunediseases are the result of inappropriate antigen presentation ofeither a self-antigen or an antigen with the capacity to mimica self-antigen in the peripheral lymphoid tissues.8 In fact,immunosuppressive agents, such as 15-deoxyspergualin, FK-506, and leflunomide, were confirmed to be effective in thesuppression of the initial antigen-priming process in experi-

mental autoimmune myocarditis.9–11 Dendritic cells (DCs)are specialized antigen-presenting cells (APCs) with FcRsthat possess the capacity to activate naive T cells.12 Accord-ingly, DCs appear to play an important role during the initialantigen-priming process of myocarditis.11,13 Therefore, toclarify the FcR-mediated effects, we investigated the effectsof immunoglobulin on autoimmune giant cell myocarditiswith the analyses of immunologic behaviors of DCs andmyocardial cytokines.

Materials and Methods

In Vivo Study

ImmunizationAutoimmune myocarditis was induced as previously described.14

Six- to 7-week-old Lewis rats (Shimizu Laboratory Supplies Co,Ltd) were injected subcutaneously in their foot pads with porcinecardiac myosin (1 mg/mL M0531, Sigma Chemical Co) mixed withFreund’s complete adjuvant (FCA) supplemented withMycobacte-rium tuberculosisH37Ra (No. 3113-60, DIFCO) on days 1 and 7.

Original received November 27, 2000; resubmission received April 4, 2001; revised resubmission received July 19, 2001; accepted July 19, 2001.From the Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.Correspondence to Chiharu Kishimoto, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54

Kawaracho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail [email protected]© 2001 American Heart Association, Inc.

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Immunoglobulin Treatment

Experiment IIn the rats immunized with porcine myosin or FCA, intact immuno-globulin (Venoglobulin-IH, a polyethylene glycol–treated humanimmunoglobulin, Welfide Corp) or F(ab9)2 fragments (Gamma-Venin, Aventis Corp) of human immunoglobulin were administeredintraperitoneally daily at a dose of 1 g · kg21 · d21 from day 1 to day15 [intact immunoglobulin and myosin, n56; F(ab9)2 fragments andmyosin, n54] and to day 21 [intact immunoglobulin and myosin,n513; intact immunoglobulin and FCA alone, n54; F(ab9)2 frag-ments and myosin, n510; F(ab9)2 fragments and FCA alone, n54].As determined from previous studies,2,3,5 the dose used was 1 g ·kg21 · d21. Immunoglobulin antigenicity between different speciesdid not appear to be a problem.5,6,15In addition, both agents have thesame chemical structure as the Fab portion of immunoglobulin.Littermate controls were injected with PBS intraperitoneally andkilled on day 15 (myosin, n56; FCA alone, n53) or day 21 (myosin,n516; FCA alone, n54).

Experiment IITo clarify the importance of the suppression of the initial self-antigenprocess by immunoglobulin treatment on the basis of the findings ofexperiment I, in which the expression of DCs, the initiators ofimmune responses, reached maximum at approximately day 15,experiment II was conducted.

Early TreatmentThis protocol aimed to suppress the initial self-antigen–primingprocess during the course of the disease. That is, intact immunoglob-ulin was administered daily to the rats immunized with myosin at thesame dose as given in experiment I from day 1 to day 8 (n55), to day11 (n55), and to day 14 (n54), and the rats were killed on day 21.Control rats were injected with PBS from day 1 to day 8 (n57), today 11 (n56), and to day 14 (n56), and the rats were killed on day21. Two rats in each group were also killed on day 8, day 11, and day14 for the pathological examination.

Late TreatmentThis protocol aimed to investigate the effects of intact immunoglob-ulin on the disease severity after completion of the initial self-antigen–priming process. That is, intact immunoglobulin was admin-istered daily to the rats immunized with myosin at the same dose as

Figure 1. Representative histopathology and immunohistochem-istry for surface markers of myocardial infiltrating cells. A and B,Hematoxylin and eosin (H-E) staining on tissues from rats immu-nized with myosin and injected with PBS. On day 15, severalinfiltrating inflammatory cells (arrow) are observed (A). On day21, extensive injury of myocytes with various kinds of inflamma-tory changes, including multinucleated giant cells (arrow andinset), is observed (B). Original magnification 3100 (inset 3320).C through F, Immunohistochemistry for OX6 (C), ED1 (D), ED2(E), and W3/25 (F) on tissues from rats immunized with myosin,injected with PBS, and killed on day 15. Almost of all the inflam-matory cells in panel A show immunoreactivity for anti-OX6 anti-body (C). ED1-positive cells (D) and ED2-positive cells (E) wereobserved, but only a few cells showed very weak immunoreac-tivity for anti-W3/25 antibody (F). Arrows indicate positive stain-ing cells. Original magnification 3200. All sections are serial.

TABLE 1. Histological Analysis for Experiment I

ImmunizationPeritonealInjection n

HW/BW,mg/g

MacroscopicScore

PericardialEffusion Score

Microscopic Score

Necrosis Infiltration

Treatment from day 1 to day 15for rats killed on day 15

Myosin1FCA PBS 6 3.160.1 0.260.4 0 0.260.4 1.060.0

Myosin1FCA Immunoglobulin 6 3.260.3 0 0 0 0

Myosin1FCA F(ab9)2 fragments 4 3.160.1 0 0 0.360.5 0.560.6

FCA alone PBS 3 3.160.6 0 0 0 0

Treatment from day 1 to day 21for rats killed on day 21

Myosin1FCA PBS 16 4.961.2 1.660.6 1.460.9 2.560.7 2.860.4

Myosin1FCA Immunoglobulin 13 2.960.4*† 0.260.6*† 0.260.6*‡ 0.961.0*† 0.961.0*†

Myosin1FCA F(ab9)2 fragments 10 4.661.1 1.460.8 1.060.9 2.461.1 2.461.1

FCA alone PBS 4 3.160.4 0 0 0 0

FCA alone Immunoglobulin 4 2.860.4 0 0 0 0

FCA alone F(ab9)2 fragments 4 3.060.1 0 0 0 0

n indicates number of rats; HW/BW, ratio of heart weight to body weight. Values are mean6SD.*P,0.01 vs rats immunized with myosin and injected with PBS; †P,0.01 and ‡P,0.05 vs rats immunized with myosin and injected with F(ab9)2 fragments (1-way

ANOVA, Fisher protected least significant difference test).

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in experiment I from day 15 to day 21, and the rats were killed onday 21 (n58). Control rats were injected with PBS during theseperiods and killed on day 21 (n57).

HistopathologyAt euthanasia, macroscopic findings and pericardial effusion weregraded on a scale of 0 to 2, as previously described.14 Microscopicfindings for cellular infiltration and myocardial necrosis were gradedon a scale of 0 to 3, as previously described.14

ImmunohistochemistryImmunohistochemistry for surface markers was performed, as pre-viously described.13 The primary antibodies (Serotec) used were asfollows: OX62 antibody to recognize an integrin or integrin-likemolecule present on DCs andgd T cells,16 V65 antibody tospecifically detectgd T cells,17 OX6 antibody to recognize majorhistocompatibility complex (MHC) class II–expressing cells, includ-ing DCs, monocytes, and B lymphocytes,13 ED1 antibody to detect

inflammatory macrophages,13 ED2 antibody to detect tissue macro-phages,13 W3/25 antibody to detect helper T lymphocytes andmacrophages,13 and OX8 antibody to detect cytotoxic/suppressor Tlymphocytes.13 W3/25 antibody is regarded as being directed againstthe rat homologue of CD4.

Ribonuclease Protection AssaymRNA was extracted from the myocardium by using TRIzol(GIBCO-BRL), and cytokine mRNA levels were measured with

Figure 2. Representative immunohistochemistry for OX62. OX62antibody recognizes DCs and gd T cells. Immunohistochemistryfor OX62 was performed by using rats immunized with myosinand injected with PBS (A and D), treated with intact immuno-globulin (B and E), and treated with F(ab9)2 fragments (C and F).Rats were killed on day 15 (A through C) and day 21 (D throughF). On day 15, several OX62-positive cells are observed (A).Treatment with intact immunoglobulin (B), but not with F(ab9)2fragments (C), suppressed the expression of OX62-positivecells. On day 21, OX62-positive cells are scattered in inflamma-tory foci (D). Treatment with intact immunoglobulin (E), but notwith F(ab9)2 fragments (F), suppressed the expression of OX62-positive cells. Arrows indicate OX62-positive cells. Images are atlow magnification, with high-magnification insets. Original mag-nification 3100 (insets 3320).

Figure 3. Quantitative analysis of OX62- and OX6-positive infil-trating cells. OX62 antibody recognizes DCs and gd T cells. OX6antibody recognizes DCs, monocytes, and B lymphocytes. Aand B, Quantitative analysis of OX62. Controls indicate ratsimmunized with myosin and injected with PBS; F(ab9)2 fragment,rats immunized with myosin and treated with F(ab9)2 fragments;intact, rats immunized with myosin and treated with intactimmunoglobulin. Panel A shows results for rats killed on day 15.Treatment with intact immunoglobulin suppressed the expres-sion of OX62-positive cells compared with no treatment (con-trols) and treatment with F(ab9)2 fragments. Treatment withF(ab9)2 fragments did not suppress the expression of OX62-positive cells compared with no treatment (controls). *P,0.05.Panel B shows results for rats killed on day 21. Treatment withintact immunoglobulin suppressed the expression of OX62-positive cells compared with no treatment (controls), but treat-ment with F(ab9)2 fragments did not suppress the expression ofOX62-positive cells compared with no treatment (controls).*P,0.05. C, Quantitative analysis of OX6. Day 15 indicates ratsimmunized with myosin, injected with PBS, and killed on day15; day 21, rats immunized with myosin, injected with PBS, andkilled on day 21. The number of OX6-positive cells is higher onday 21 than on day 15. The ratio of OX62-positive cells to OX6-positive cells in the heart immunized with myosin and injectedwith PBS on day 15 and day 21 was 0.4660.23 and0.04260.029, respectively. Accordingly, in the natural course ofthe disease, the proportion of OX62-positive cells in the MHCclass II–expressing cells is decreased during the fulminantphase (day 21) compared with the early phase (day 15). Quanti-tative analyses were performed by counting all positively stainedcells in 0.2530.25-mm fields. Seven distinct fields per rat werecounted. The number of rats was 3 for all groups. The valuesare expressed as mean6SD.

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RiboQuant Multi-Probe template sets, In Vitro transcription kits, andribonuclease protection assay (RPA) kits (PharMingen) according tothe PharMingen/RiboQuant protocol.18 The NIH Image system wasused to quantify the pixel intensity of macrophage inhibitory factor(MIF) cytokine bands, which were divided by the intensities in theirL32 bands in the same lanes for normalization.

In Vitro StudyU937 cells, human monoblast cells bearing FcRs,19 were stimulatedwith 10 ng/mL lipopolysaccharide (LPS) ofEscherichia coli(Sig-ma). Forty-eight hours later, interleukin (IL)-1b in the medium wasassayed by using antibody-sandwich ELISA.

For the analysis of surface markers, the collected cells wereincubated with an appropriate dilution of primary antibodies: CD16(FcgRIII, Ancell), CD32 (FcgRII, Serotec), and CD64 (FcgRI,Ancell). They were incubated with the fluorescein isothiocyanate–conjugated F(ab9)2 secondary antibody (Serotec), and 13104 cellsper trial were analyzed with a FACScan cytometer (Becton Dickin-son) by use of CELLQuest.

Intact human immunoglobulin or the F(ab9)2 fragments wereadded to the medium 30 minutes before LPS stimulation. The dosesof the agents used in the present study were derived from themethods of Andersson and Andersson.20

Statistical AnalysisValues were expressed as the mean6SD. Statistical analyses of thedata were performed by 1-way ANOVA and were reanalyzed withthe Fisher protected least significant difference test to characterizesignificant differences between groups in the in vitro study andexperiment I; the Studentt test was used in experiment II. A value ofP,0.05 was considered statistically significant.

ResultsFcR-Mediated Inhibitory Effect and Suppressionof Expression of DCs by ImmunoglobulinTreatment In Vivo (Experiment I)

Histopathology and Heart Weight/Body Weight RatioNone of the rats died throughout the entire period. On day 15,the hearts showed a normal appearance macroscopicallyexcept for one of six rats. However, several infiltratinginflammatory cells surrounding small vessels among cardio-myocytes were observed microscopically in all the rats(Figure 1A). In rats treated with intact immunoglobulin, noevidence of myocarditis was shown on day 15. In rats treatedwith F(ab9)2 fragments, several infiltrating inflammatory cellswere observed microscopically. Table 1 shows histologicalanalysis for experiment I.

On day 21, 11 of 16 hearts showed severe and diffusediscolored myocarditis with massive pericardial effusion.Extensive injury of the myocytes with inflammatory changesand multinucleated giant cells were observed microscopically(Figure 1B). Treatment with intact immunoglobulin, but notF(ab9)2 fragments, reduced the severity of the disease, asassessed by measuring the heart weight/body weight ratio andhistological scores.

Immunohistochemistry of Surface MarkersOn day 15, in rats immunized with myosin and injected withPBS, almost of all the infiltrating inflammatory cells showedimmunoreactivity for anti-OX6 antibody (Figure 1C). ED1-positive cells (Figure 1D) and ED2-positive cells (Figure 1E)were observed, but only a few cells showed very weak

Figure 4. RPA for mRNAs of Th1, Th2, and proin-flammatory cytokines. A, Rats killed on day 15. Inrats immunized with myosin and injected with PBS,mRNA expression of MIF was enhanced by 3.1-fold relative to intact hearts. However, treatmentwith intact immunoglobulin reduced the mRNAexpression of MIF (1.1-fold relative to intacthearts). B, Rats killed on day 21. In rats immunizedwith myosin and injected with PBS, mRNAs of Th1cytokines (such as IL-18), Th2 cytokines (such asIL-6 and IL-10), and proinflammatory cytokines(such as MIF, IL-1a, IL-1b, and IL-1Ra) were mark-edly upregulated compared with intact heartmRNAs. Treatment with immunoglobulin, but notF(ab9)2 fragments, reduced the expression of cyto-kine mRNAs. IFN-g indicates interferon-g. L32 is ahousekeeping gene. A representative finding of 3distinct experiments is shown.



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immunoreactivity for anti-W3/25 (Figure 1F) or anti-OX8(data not shown) antibody. Several OX62-positive cellsinfiltrated into the perivascular cardiac tissue (Figure 2A); allwere DCs, because no anti–V65-positive cells were detected(data not shown). Accordingly, MHC class II–positive my-eloid cells, including DCs, play a pivotal role during the earlyphase. Treatment with intact immunoglobulin (Figures 2Band 3A), but not F(ab9)2 fragments (Figures 2C and 3A),reduced the number of OX62-positive cells.

On day 21, OX62-positive cells were scattered in inflam-matory foci (Figure 2D), and only a few V65-positive cellswere detected (data not shown). The number of OX6-positivecells was markedly increased (Figure 3C). The ratio ofOX62-positive cells to OX6-positive cells in the heart immu-nized with myosin and injected with PBS on day 15 and day21 was 0.4660.23 and 0.04260.029, respectively. Accord-ingly, in the natural course of thedisease, the proportion ofOX62-positive cells in the MHC class II–expressing cells wasdecreased during the fulminant phase (day 21) compared withthe early phase (day 15). Treatment with intact immunoglobu-lin (Figures 2E and 3B), but not F(ab9)2 fragments (Figures 2Fand 3B), reduced the numbers of OX62-positive cells.

Ribonuclease Protection AssayOn day 15 (Figure 4A), the mRNA expression of cytokines inintact hearts immunized with FCA alone was only for MIF,which is released as a proinflammatory cytokine.21 In ratsimmunized with myosin and injected with PBS, mRNAexpression of MIF was enhanced (by 3.1-fold relative tointact hearts). However, in rats immunized with myosin andtreated with intact immunoglobulin, the mRNA expression ofMIF was not enhanced (1.1-fold relative to intact hearts).

On day 21 (Figure 4B), in intact hearts immunized withFCA alone and injected with PBS, mRNA expression ofcytokines was detected only for MIF. In rats immunized withmyosin and injected with PBS, mRNAs of Th1 cytokines(such as IL-18), Th2 cytokines (such as IL-6 and IL-10), andproinflammatory cytokines (such as MIF, IL-1a, IL-1b, andIL-1Ra) were markedly upregulated, and mRNA expressionof IL-12p35, IL-12p40, and interferon-g was slightly upregu-lated. Treatment with immunoglobulin, but not F(ab9)2 frag-ments, reduced the expression of cytokine mRNAs.

FcR-Mediated Inhibitory Effect ofImmunoglobulin In VitroIL-1b production was increased by LPS stimulation in U937cells (0.760.8 pg/mL for controls, n54; 9.161.5 pg/mL forLPS, n54 [P,0.01]). Intact immunoglobulin (7.061.4pg/mL for 0.6 mg, n54 [P5NS]; 5.561.7 pg/mL for 6.0 mg,n54 [P,0.05]), but not F(ab9)2 fragments (8.961.5 pg/mLfor 0.4 mg, n54 [P5NS]; 8.865.0 pg/mL for 4.0 mg/mL,n54 [P5NS]), suppressed LPS-induced IL-1b production ina dose-dependent manner (Figure 5A).

In U937 cells, CD32 was expressed and upregulated byLPS stimulation (Figure 5B). The expression of CD16 andCD64 was not varied from the baseline at any time point (datanot shown). Intact immunoglobulin downregulated LPS-induced CD32 expression, and the median fluorescenceintensity (MFI) relative to the control was decreased by the

treatment (143626% for LPS, n54; 101618% for LPS plusintact immunoglobulin, n54 [P,0.05]). However, the LPS-induced CD32 expression was not changed by the treatmentof F(ab9)2 fragments [143626% for LPS, n54; 125611% forLPS plus F(ab9)2 fragments, n54 (P5NS)].

Importance of Suppression of Initial Self-AntigenPriming Process by Intact ImmunoglobulinTreatment (Experiment II)

Early TreatmentThe treatment from day 1 to day 8 failed to decrease theseverity of myocarditis (Table 2). Treatment with intact

Figure 5. Effects of immunoglobulin on LPS-induced IL-1b pro-duction and CD32 (FcgRII) expression. A, Analysis of IL-1b pro-duction. IL-1b production was increased by LPS stimulation inU937 cells. Intact immunoglobulin, but not F(ab9)2 fragments,suppressed LPS-induced IL-1b production in a dose-dependentmanner. The values are expressed as mean6SD of 4 indepen-dent experiments. *P,0.05 vs IL-1b production stimulated withLPS without intact immunoglobulin treatment. B, Representativeflow cytometric analysis of CD32 expression. In graph a, CD32was expressed in nonstimulated U937 cells (dotted line, MFI100%). In graph b, CD32 expression was upregulated by LPSstimulation (solid line, MFI 148%). In graph c, LPS-inducedCD32 expression (solid line) was downregulated by intact immu-noglobulin treatment (heavy solid line, MFI 109%). In graph d,LPS-induced CD32 expression (solid line) was not changed byF(ab9)2 fragments (heavy solid line, MFI 123%). Background flu-orescence intensity (shadow area) was obtained through theprocedure without primary antibodies. The x-axis shows fluo-rescence intensity (arbitrary units). The y-axis shows cellnumber. Experimental values are expressed as the percent-age of MFI relative to the control value. The findings weresimilar and are representative of 4 separate experiments.FL1-H indicates fluorescence intensity.



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immunoglobulin from day 1 to both day 11 and day 14decreased the severity of myocarditis, as assessed by measur-ing the heart weight/body weight ratio and histologicalscores. On day 8, OX62-positive cells were rarely detected inrats injected with PBS or in rats treated with intact immuno-globulin. On day 11, only a few OX62-positive cells hadinfiltrated the cardiomyocytes in rats injected with PBS butnot the rats treated with intact immunoglobulin. On day 14,several OX62-positive cells were observed among cardio-myocytes in rats injected with PBS but not in rats treated withintact immunoglobulin. Accordingly, the early treatment ofintact immunoglobulin from day 1 to day 11 or day 14, butnot day 8, suppressed the expression of DCs, ie, the initialantigen-priming process, leading to the suppression ofmyocarditis.

Late TreatmentTreatment with intact immunoglobulin did not decrease theseverity of myocarditis (Table 2). Thus, the late treatmentafter the completion of DC expression did not cause areduction in the severity of myocarditis.

DiscussionIn the present study, it was shown that intact immunoglobu-lin, but not F(ab9)2 fragments, markedly suppressed both theseverity of the disease and cytokine mRNA expression inexperimental giant cell myocarditis in vivo. Intact immuno-globulin, but not F(ab9)2 fragments, suppressed LPS-inducedIL-1b production associated with downregulation of CD32expression in U937 cells in vitro. Accordingly, FcR-mediatedinhibitory effects actually play an important role in thereduction of inflammatory cytokine production in vitro and invivo. In addition, intact immunoglobulin, but not F(ab9)2

fragments, suppressed the DC expression during both theearly and fulminant phases in vivo. An additional experiment

confirmed that the suppression of DC expression during theearly phase of the disease, when DCs operate actively, causeda reduction in myocarditis. The effect of immunoglobulinmay be associated with the suppression of the expression ofDCs, ie, the suppression of the initial antigen-priming processin experimental giant cell myocarditis.

FcgRs act as trigger molecules for inflammatory, allergic,endocytic, and inhibitory activities of immune effectorcells.7,22 It has been reported that F(ab9)2 fragments do notameliorate experimental allergic encephalomyelitis in rats15

and that the anti-inflammatory activity of immunoglobulin ismediated through the inhibitory FcR.7 In the present study, anin vivo study confirmed that intact immunoglobulin admin-istration, but not the administration of F(ab9)2 fragments,completely suppressed the severity of the disease and themRNA expression of cytokines (Figure 4). An in vitro studyshowed that intact immunoglobulin, but not F(ab9)2 frag-ments, suppressed LPS-induced the IL-1b production associ-ated with the downregulation of CD32 (FcgRII) expression. Itis suggested that mRNA expression of cytokines may beblocked not only by anti-cytokine antibodies included inimmunoglobulin used in the present study but also by theanti-inflammatory action mediated through the inhibitoryFcR. The former possibility may be slight, because F(ab9)2

fragments did not suppress mRNA expression of cytokines,which theoretically may possess anti-cytokine antibodies. Weconfirmed that the anti-inflammatory action of intact immu-noglobulin in murine myocarditis induced by encephalomyo-carditis virus was due to the reduction of the plasma level ofinterferon-g and soluble intercellular adhesion molecule-1but not to antiviral effects.6 Accordingly, the present studyadded further information that intact immunoglobulin treat-ment suppressed not only viral myocarditis but giant cellmyocarditis by the anti-inflammatory action.

TABLE 2. Histological Analysis for Experiment II

ImmunizationPeritonealInjection n

HW/BW,mg/g

MacroscopicScore

PericardialEffusion Score

Microscopic Score

Necrosis Infiltration

Early treatment (rats killed on day 21)

Treatment from day 1 to day 8

Myosin1FCA PBS 7 4.260.7 1.960.4 1.160.9 2.660.5 2.660.5

Myosin1FCA Immunoglobulin 5 3.760.6 1.460.9 1.060.7 1.661.1 2.260.8


Myosin1FCA PBS 6 5.861.3 2.060.0 1.760.5 2.860.4 2.860.4

Myosin1FCA Immunoglobulin 5 3.661.0* 1.061.0* 0.660.9* 1.461.1* 1.461.1*


Myosin1FCA PBS 6 5.460.6 2.060.0 1.760.5 2.860.4 2.860.4

Myosin1FCA Immunoglobulin 4 3.860.4† 0.861.0* 0.561.0* 1.361.0† 1.560.6†

Late treatment (rats killed on day 21)


Myosin1FCA PBS 7 5.661.0 2.060.0 1.460.8 2.660.5 3.060.0

Myosin1FCA Immunoglobulin 8 5.160.5 2.060.0 1.860.5 2.560.5 2.860.5

n indicates number of rats. Values are mean6SD. Early treatment of intact immunoglobulin from day 1 to both day 11 and day 14 suppressed the initialantigen-priming process, leading to the suppression of myocarditis. However, the late phase (the phase after completion of antigen-priming) treatment of intactimmunoglobulin did not cause the reduction of severity of myocarditis.

*P,0.05 and †P,0.01 vs rats immunized with myosin injected with PBS (Student t test).



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The present study provided evidence that almost of all theinfiltrating inflammatory cells were MHC class II–positivemyeloid cells, including DCs during the early phase. Theproportion of DCs in the MHC class II–expressing cells washigher during the early phase compared with during thefulminant phase (Figure 3). DCs are functionally specializedAPCs and efficient stimulators of B and T cells. Mature DCsexpress high MHC class II molecules that are 10- to 100-foldgreater on DCs than on other APCs, such as B cells andmonocytes.8,12 Intact immunoglobulin administration, but notthe administration of F(ab9)2 fragments, may suppress theactivation of DCs because immature DCs are well equippedto capture antigens and have antigen-capturing Fcg and Fcereceptors12; ie, exogenous native and intact immunoglobulinmay bind to FcRs on DCs and prevent internalization of theantigen, and as a result, intact immunoglobulin with the Fcportion may prevent DCs from processing antigens to formMHC peptide complexes. The results of experiment II con-firmed that the early treatment of immunoglobulin by thetime DCs infiltrated to cardiomyocytes caused a reduction inmyocarditis. Accordingly, the effect of immunoglobulin maybe associated with the suppression of the initial antigen-priming process in experimental giant cell myocarditis.

We have previously reported that immunoglobulin sup-presses coxsackievirus B3 myocarditis by an antiviral anti-body included in the agent.5 However, immunoglobulintreatment failed to ameliorate myocarditis in an Interventionin Myocarditis and Acute Cardiomyopathy With ImmuneGlobulin (IMAC) trial of human myocarditis.23 One reasonmay be that patients with dilated cardiomyopathy withnoninflammatory causes occupied a large part of that trial.From the present findings, it may be that immunoglobulintherapy, if it is initiated early, is effective against human giantcell myocarditis, a disease with no present effective therapyother than transplantation.1 In conclusion, the present studyprovided evidence that intact immunoglobulin therapy mark-edly suppressed myocarditis because of the FcR-mediatedanti-inflammatory action and the concomitant suppression ofthe initial antigen-priming process in experimental autoim-mune myocarditis. The findings of the present study mayyield important insights into both the clinical use of thistherapy for human immune or autoimmune myocarditis andthe future studies of FcR-mediated therapy for immune orautoimmune diseases.

AcknowledgmentsThis study was supported in part by research grants from theConference on Coronary Artery Disease, Japanese Education ofScience and Welfare (Nos. 08877110 and 0947016), the KanaeShinyaku Foundation, and Japan Cardiovascular Research Founda-tion. We thank Drs Hajime Nakamura and Junji Yodoi, Institute forVirus Research, Kyoto University, for helpful discussions andcritical comments on the manuscript.

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Keisuke Shioji, Chiharu Kishimoto and Shigetake SasayamaGiant Cell Myocarditis: Concomitant Suppression of the Expression of Dendritic Cells

Mediated Inhibitory Effect of Immunoglobulin Therapy on Autoimmune−Fc Receptor

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fc receptor–mediated inhibitory effect of immunoglobulin therapy

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