Critical Role for Monocyte Chemoattractant Protein-1 and Macrophage Inflammatory Protein-1α in Induction of Experimental Autoimmune Myocarditis and Effective Anti–Monocyte Chemoattractant Protein-1 Gene Therapy
Background— Autoimmune myocarditis is a principal cause of heart failure among young adults and is often a precursor of dilated cardiomyopathy. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) are potent chemotactic factors for mononuclear cells. The inflammatory infiltrate observed in myocardial lesions of myocarditis consists of >70% mononuclear cells. To determine their critical role in the pathogenesis of myocarditis, we inhibited mononuclear cell activation and migration to see if it would affect disease severity and disease prevalence in experimental autoimmune myocarditis (EAM).
Methods and Results— In this report, we demonstrated that blockade of MCP-1 or MIP-1α with monoclonal antibodies significantly reduced severity of myocarditis in BALB/c mice immunized with cardiac myosin. Similar results were obtained when CCR2−/− and CCR5−/− mice were used. In CCR2−/− mice, not only disease severity but also disease prevalence was reduced. To further inhibit mononuclear cell activation and migration, we transfected the mice before inducing EAM with a dominant-negative inhibitor of MCP-1 gene (7ND). This transfection significantly reduced the disease severity, decreased mRNA expression levels, especially of the chemokines RANTES, MIP-2, IP-10, MCP-1, T-cell activation gene 3, and eotaxin in the myocardium, and resulted in a reduction in cardiac myosin-induced interleukin-1 and interleukin-4 and in an increase in interferon-γ and interleukin-10 cytokine production by splenocytes.
Conclusions— Overall, these findings suggest that the chemokines MCP-1 and MIP-1α, acting through their receptors CCR2 and CCR5, are important in the induction of EAM and that inhibition of MCP-1 with 7ND gene transfection significantly reduced disease severity. This strategy may be a new feasible form of gene therapy against autoimmune myocarditis.
Received June 29, 2005; revision received August 12, 2005; accepted September 2, 2005.
The attraction of leukocytes from blood to tissue is essential for inflammation and the host response to infection. This process is partly controlled by chemokines and their receptors. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) are members of the C-C chemokine family, which has been shown to play a major role in the migration of monocytes to an inflammatory focus.1,2 MCP-1 can also attract memory T cells and activated natural killer cells in vitro3 and is key to the development of Th2 responses.4 CCR2, the receptor for MCP-1, is highly expressed on monocytes and activated T cells.5 Similar to MCP-1, MIP-1α can mediate the recruitment of monocytes in several inflammatory diseases.6 CCR5, the major receptor for MIP-1α, is expressed mostly on activated T cells and monocytes/macrophages.5
Inflammation of the heart muscle (myocarditis) is a frequent cause of cardiac failure in young adults. Autoimmunity plays an important role in human myocarditis and contributes to the progression to cardiomyopathy and heart failure.6 Fuse and colleagues7 recently showed increased mRNA levels of MCP-1 in the hearts of rats immunized with cardiac myosin. Also, serum MCP-1 levels of the rats with experimental autoimmune myocarditis (EAM) were significantly elevated from days 15 until 24. In the clinical study, they further showed that serum levels of MCP-1 in patients with acute myocarditis were significantly elevated compared with those of healthy volunteers. However, the precise role of this chemokine in the pathogenesis of myocarditis remains undetermined. To further clarify the role of chemokines such as MCP-1 and MIP-1α and their receptors CCR2 and CCR5 in the pathogenesis of myocarditis and to establish new therapeutic approaches, we first blocked in BALB/c mice immunized with cardiac myosin MCP-1 or MIP-1α by using monoclonal antibodies. We then used CCR2−/− or CCR5−/− mice to further analyze the role of these chemokines and their receptors. Finally, we studied the use of gene therapy to block MCP-1 activity in vivo by using an N-terminal deletion mutant of MCP-1, called 7ND, which lacks the N-terminal amino acids 2 to 8. This mutant MCP-1 has been shown to bind the receptor for MCP-1, CCR2, and block MCP-1–mediated monocyte chemotaxis.8,9 Because the inflammatory infiltrate observed in myocardial lesions of myocarditis consists of >70% mononuclear cells,10 these approaches may also open novel therapeutic strategies to treat myocarditis in humans.
Female BALB/c mice (6 to 8 weeks of age) were obtained from Charles River Laboratories (Sulzfeld, Germany) and maintained in the conventional animal facility at the University of Heidelberg, and these mice were used in all experiments except the experiments done with CCR2−/− and CCR5−/− mice. CCR2−/− and CCR5−/− mice (both hybrids of C57B1/6 and 129/C57B1/6 respectively, and kindly provided by W.A. Kuziel, University of Texas, Austin) were back-crossed for 6 generations onto the BALB/c background. The CCR2−/+ and CCR5−/+ mice were then interbred to generate CCR2+/+ and CCR5+/+ mice on the same genetic background. The animal work was approved by the Animal Care and Use Committee of the University of Heidelberg.
Antigen Preparation and Induction of Myocarditis
Murine cardiac myosin was purified from pooled mouse hearts through the use of a previously described procedure.11,12 The purified cardiac myosin was emulsified with equal volume of complete Freund’s adjuvant (Sigma) containing 5 mg/mL of Mycobacterium tuberculosis H37Ra (Sigma). Each mouse was injected subcutaneously with 100 μL of emulsion containing 200 μg of cardiac myosin on days 0 and 7.
For the histopathological evaluation, mice were euthanized on day 21, and serial sections were made through the heart. Every fifth section was stained with hematoxylin and eosin. Evidence of myocarditis was evaluated with light microscopy independently and in a blinded manner by 2 pathologists according to a 6-tier scoring system: Grade 0, no inflammation; grade 1, cardiac infiltration in up to 5% of the cardiac sections; grade 2, 6% to 10%; grade 3, 11% to 30%; grade 4, 31% to 50%; and grade 5, >50%. The average score from the pathologist’s readings was taken for statistical analysis with a nonparametric test.
MCP-1 and MIP-1α Blockade
The rat anti-mouse MCP-1 monoclonal antibody (CCL2/JE/MCP-1 mAb, R&D Systems) was used to block MCP-1, and rat anti-mouse MIP-1α mAb (CCL3/MIP-1α mAb, R&D Systems) was used to block MIP-1α. Rat IgG2b mAb from clone A95–1 (PharMingen) was used as an isotype-matched control. BALB/c mice were injected intraperitoneally with MCP-1 mAbs or MIP-1α or isotype-matched control monoclonal antibodies (500 μg of either) in phosphate-buffered saline (PBS; 0.1 mL) on days 12 and 17. These injection times were selected because Fuse and colleagues7 recently showed that serum MCP-1 levels in rats with EAM were significantly elevated from days 15 until 24.
To detect CCR2 knockout alleles, the primer pair of WAK121 (5′-TTCCATTGCTCAGCGGTGCT-3′) and WAK134 (5′-TCAGAGATGGCCAAGTTGAGC-AGA-3′) yield a polymerase chain reaction product of 450 bp.
To detect CCR5 knockout alleles, the primer pair of WAK121 (5′-TTCCATTGCTCAGCGGTGCT-3′) and WAK131 (5′-TGTTTCCTCCTCTAGCCTTCAC-TATG-3′) yield a polymerase chain reaction product of 350 bp.
Plasmid Expression Vectors
Human 7ND complementary deoxyribonucleic acid (cDNA) was constructed by a recombinant chain reaction using wild-type human MCP-1 cDNA (generous gift from T. Yoshimura, National Cancer Institute, Bethesda, Md) as a template and inserted into the BamHI (5′) and NotI (3′) sites of the pcDNA3 expression vector plasmid (Invitrogen) as described previously.13 All sequences were confirmed by sequencing.
Gene transfer was done as described before.14 The mice were injected either with 7ND gene or with control plasmid (50 μg in 30 μL PBS) or with 30 μL PBS alone in the femoral muscle using a 27-gauge needle under anesthesia by intraperitoneal injection of pentobarbital. To enhance transgene expression, all mice received electroporation at the injected site.
Detection of Serum Concentrations of Anti–Cardiac Myosin Titers, Cardiac Myosin–Specific Cytokine Production by Splenocytes
The detection of serum concentrations of anti-cardiac myosin titers and cardiac myosin-specific cytokine production by splenocytes was done as previously described.11,12 To detect serum anti–cardiac myosin titers, plates were coated with 100 μL/well of cardiac myosin (5 μg/mL) in bicarbonate buffer (pH 9.6) and left overnight. Mouse secondary antibodies, diluted to 1:1000 for IgG (KPL), IgG1, IgG2a (PharMingen), and IgG2b (Bethyl), were used for detection. Serum samples from test mice were diluted to 1:50, 1:200, 1:800, 1:3200, and 1:12800. Normal mouse serum was used as control. Optical densities were determined at 450 nm. Antibody end point titers for each individual mouse were calculated as the greatest positive dilution of antibody.
For in vitro cytokine production, the splenocytes were cultured at 5×106 per well in Roswell Park Memorial Institute 1640 complete medium in the presence of 30 μg/mL of cardiac myosin or medium alone for 48 hours. Supernatant was collected, aliquoted, and frozen at −20°C. Cytokines were measured by Quantikine cytokine ELISA kits (R&D Systems) according to the manufacturer’s instructions.
RNA Protection Assay
The mCR-5 cytokine receptor multiprobe template set (BD Biosciences Pharmingen) was used to measure mouse mRNAs encoding CCR1, CCR2, CCR1b, CCR3, CCR4, and CCR5. The mCK-5c multiprobe template set (BD Biosciences Pharmingen) was used to measure mouse mRNAs encoding Ltn, RANTES, MIP-1b, MIP-1a, MIP-2, IP-10, MCP-1, T-cell activation gene 3 (TCA-3), and eotaxin. The measurements were performed according to the manufacturer’s guidelines.
Normally distributed data were analyzed by Student’s t test; otherwise, the Mann-Whitney U test was used. Disease prevalence was compared by using a χ2 2-way analysis. Statistical comparison across 3 groups were calculated with the use of ANOVA followed by appropriately conducted multiple comparisons. Probability values of <0.05 were considered significant.
Blocking MCP-1 With Monoclonal Antibodies Reduced the Severity of Disease in EAM
To determine the effect of blocking MCP-1, we induced EAM in BALB/c mice by immunization with cardiac myosin and treated the mice either with MCP-1 mAbs or with the isotype control on days 12 and 17. Compared with the isotype controls, mice treated with MCP-1 mAbs showed a significant decrease in the prevalence and severity of myocarditis (Figure 1). Only 3 of 14 mice treated with MCP-1 mAbs developed myocarditis with lesion grade of 1 and more, whereas no or very little inflammation was detected in the rest of the group. In contrast, all isotype-treated control mice had myocarditis with a lesion grade of 1 and more.
Blocking MIP-1α With Monoclonal Antibodies Reduced the Severity of Disease in EAM
To further study the effect of blocking MIP-1α, EAM was induced in BALB/c mice by immunization with cardiac myosin. They were treated either with MIP-1α mAbs or with the isotype control on days 12 and 17. Compared with the isotype controls, mice treated with MIP-1α mAbs showed a significant decrease in the prevalence and severity of myocarditis (Figure 1). Compared with mice treated with MCP-1 mAbs, the decrease was less but still significant. Four of 8 mice had myocarditis with grade 1 lesions and only 2 of 8 mice treated with MIP-1α mAbs had myocarditis with lesion grade 1.5 and more. In contrast, 9 of 10 mice treated with the isotype-matched control mAb had myocarditis with a lesion grade of 1.5 and more.
CCR2−/− and CCR5−/− Mice Show a Reduced Severity of EAM
To examine the role of CCR2, the major receptor for MCP-1, and the role of CCR5, the main receptor for MIP-1α, in the pathogenesis of EAM, we immunized CCR2−/−, CCR2+/+, CCR5−/−, and CCR5+/+ mice with cardiac myosin. Compared with the CCR2+/+ mice, the CCR2−/− mice showed a significant reduction in severity of myocarditis (Figure 2). Only 1 of 14 CCR2−/− mice had myocarditis with a grade 1 and more, whereas 10 of 15 CCR2+/+ mice had myocarditis with grade 1 or greater severity (Figure 3, a through f). The prevalence of EAM was also significantly lower in CCR2−/− mice (33% versus 86%) (Figure 2). Furthermore, interleukin (IL)-1 and IL-4 production by splenocytes was significantly reduced, whereas interferon-γ (IFN-γ) and IL-10 production was increased in CCR2−/− mice (Figure 4).
Similar results were obtained when CCR5−/− mice were used. CCR5−/− mice showed a significant decrease in disease severity. Two of 12 CCR5−/− mice had myocarditis with grade 1 or greater, whereas 8 of 11 CCR5+/+ mice had myocarditis with grade 1 or greater (Figure 2). TNF-α production by splenocytes was significantly reduced in CCR5−/− mice, whereas IL-10 production was increased (Figure 4).
There were no significant differences in autoantibody production (tested for following subclasses of IgG: IgG1, IgG2a, and IgG2b) against cardiac myosin in both groups of mice.
mRNA Expression of Chemokines and Chemokine Receptors in the Myocardium
To look at the expression of different chemokines and chemokine receptors, we measured the mRNA expression levels of the chemokines Ltn, RANTES, MIP-1b, MIP-1a, MIP-2, interferon-inducible protein (IP)-10, MCP-1, TCA-3, and eotaxin, and of the chemokine receptors CCR1, CCR2, CCR1b, CCR3, CCR4, and CCR5 in the myocardium of these mice. We were able to detect mRNA levels for all chemokines tested (Figure I), but the expression of mRNA levels was markedly higher for the chemokines RANTES, MIP-2, IP-10, and MCP-1 in all mice. The mRNA expression levels of RANTES, MIP-2, IP-10, and MCP-1 correlated with the severity of myocarditis. Thus, all mice with lower myocarditis scores showed lower expression of these chemokines compared with mice with higher myocarditis independent of being CCR2- or CCR5-deficient or wild-type mice. Further, we found that primarily the mRNA for the CCR receptors CCR1, CCR2, and CCR5 was expressed in the myocardium of all tested mice (Figure I). There was no mRNA or very low levels for the chemokine receptors CCR1b, CCR3, and CCR4. CCR2−/− and CCR5−/− mice showed lower mRNA expression of the chemokine receptors CCR1 and CCR5 (for CCR2−/− mice) and CCR1 and CCR2 (for CCR5−/− mice) compared with the CCR2+/+ and CCR5+/+ mice. There was no compensatory upregulation of other chemokine receptors when CCR2 receptor or CCR5 receptor knockout mice were used. Similar to the chemokines, the upregulation of mRNA levels of these chemokine receptors were correlated with the severity of inflammation. Mice with lower myocarditis scores showed greater reduction of mRNA of these chemokine receptors compared with mice with higher myocarditis score (data not shown).
7ND Gene Transfection Decreased the Inflammation in EAM
We determined the effect of 7ND gene transfection and examined whether anti–MCP-1 gene therapy using the 7ND gene construct is effective. Compared with the control plasmid treatment group and PBS group, mice treated with the 7ND gene showed a marked decrease in the prevalence and severity of myocarditis, changes in cytokine profiles, and mRNA expression levels for chemokines (Figure 5, Figure 6⇓, and Figure II). Only 1 of 11 mice treated with the 7ND gene had mild myocarditis with a lesion grade of 1 or more, whereas no or little inflammation was detected in the rest of the group. In contrast, 8 of 12 PBS-treated mice and 8 of 12 control plasmid-treated mice had myocarditis with lesion grades of 1 or more (Figure 5 and Figure II). Cardiac myosin–specific production of IL-1 and IL-4 (compared with control plasmid-treated and PBS-treated mice) by splenocytes were significantly reduced in mice treated with 7ND, whereas IFN-γ and IL-10 (compared with control plasmid-treated and PBS-treated mice) was significantly increased (Figure 6a). Inhibition of MCP-1 with 7ND gene transfection also reduces mRNA expression levels of the chemokines RANTES, MIP-2, IP-10, MCP-1, TCA-3, and eotaxin in the myocardium (Figure 6b).
We have shown an important role for MCP-1 and MIP1-α and their major receptors, CCR2 and CCR5, in the initiation of the autoimmune myocarditis. Not only blocking MCP-1 and MIP1-α by using monoclonal antibodies but also by immunizing CCR2−/− and CCR5−/− mice reduced the severity of myocarditis. Furthermore, we demonstrated successful gene therapy by blocking MCP-1 activity in vivo by using an N-terminal deletion mutant of MCP-1 called 7ND. Because it lacks the N-terminal amino acid 2 to 8 and blocks MCP-1–mediated monocyte chemotaxis,8,9 it is effective in the treatment of EAM in mice. In contrast, the control gene had no significant effect on disease severity. Immunizing CCR2−/− mice and treating BALB/c mice with 7ND resulted in significantly decreased IL-1 and IL-4 and in significantly increased IL-10 and IFN-γ production by splenocytes. In addition, mRNA expression levels of the chemokines RANTES, MIP-2, IP-10, MCP-1, TCA-3, and eotaxin in the myocardium were especially reduced in 7ND-treated mice. Overall, these findings indicated a critical role in the inflammatory response of myocarditis and further suggest that a mutant MCP-1 can be used as a novel therapeutic approach for the treatment of human autoimmune myocarditis.
MCP-1, MIP1-α, and their major receptors, CCR2 and CCR5, play important roles in the pathogenesis of many inflammatory diseases.15–17 MCP-1 mRNA expression has been shown in endomyocardial biopsy specimens from patients with dilated cardiomyopathy, suggesting an important role of this chemokine in the regulation of inflammatory cell infiltration into the myocardium.18
Blocking both MCP-1 or MIP-1α with mAbs before the onset of inflammation, which occurs after day 14 in our model of EAM, led to significant reduction of the severity of myocarditis in BALB/c mice immunized with cardiac myosin. Similar results were obtained when CCR2- and CCR5-deficient mice were immunized. Additionally, in CCR5-deficient mice, the cardiac myosin–specific production of IL-10 by splenocytes was increased and the production of TNF-α was reduced significantly. The production of IL-2 and IFN-γ were also slightly reduced in CCR5-deficient mice, but the differences were not significant. Our results conform with the reported studies that CCR5 is expressed mostly on Th1 lymphoctes.19 In our studies with CCR5-deficient mice, the Th2 cytokine IL-10 was significantly increased and the Th1-associated cytokines tumor necrosis factor-α (TNF-α) significantly and IL-2 and IFN-γ (not significantly) were reduced. Because it is known that the EAM in BALB/c mice is a Th1-induced disease,20 this along with the inhibition of cell migration might be an additional explanation for the reduction in the myocarditis score in CCR5-deficient mice. In CCR2-deficient mice, the cardiac myosin–specific production of IL-1 and IL-4 by splenocytes was reduced significantly, whereas the production of IFN-γ was increased. CCR2 is known to be expressed on both Th1 and Th2 lymphoctes.19 In our previous work, we have shown that IFN-γ and IL-10 are protective cytokines in myocarditis, whereas IL-1 and TNF-α promote inflammation and disease severity in EAM.12,21,22 We further studied the expression of different CCRs during inflammation in the myocardium of mice and questioned whether there is a upregulation of other chemokine receptors in CCR2- or CCR5-deficient mice, and we found that mainly CCR1, CCR2, and CCR5 are expressed in the myocardium, with no or very low expression of mRNA for the receptors CCR3 and CCR4. Further, the CCR2- and CCR5-deficient mice did not show marked upregulation of the other CCRs tested compared with the wild-type mice. Finally, we studied the mRNA expression levels of other chemokines in the myocardium of these mice on day 21 and found that the cytokines RANTES, MIP-2, IP-10, and MCP-1 were especially expressed in the myocardium in all tested mice. The mRNA expression levels of these chemokines correlated with the severity of myocarditis and were independent of CCR2 or CCR5 expression. Thus, the expression of these chemokines was continuously lower in CCR2- and CCR5-deficient mice (with overall mild to no inflammation), but with differences in the wild-type mice by means of higher mRNA expression levels in mice with higher myocarditis score, indicating that these chemokines were mostly released by the myocardium migrating inflammatory cells during the inflammation.
Fuse and colleagues7 showed increased mRNA levels of MCP-1 in the hearts of rats immunized with cardiac myosin and significantly elevated serum MCP-1 levels of the rats with EAM from days 15 until 24. Furthermore, Kolattukudy and colleagues23 were able to induce myocarditis in mice by targeted expression of the MCP-1 gene in murine cardiac muscle. Damas and colleagues24 demonstrated that MCP-1 can directly affect cardiomyocytes and showed that MCP-1 can modulate cytokine production within the myocardium by significantly increasing both IL-1b and IL-6 production in cardiomyocytes. They have further shown increased gene expression of the MCP-1 receptor (CCR2) in the failing human myocardium.25 Besides chemotaxis and leukocyte activation, MCP-1 and other chemokines may also regulate several other biological processes with importance to the pathogenesis of heart failure, such as fibrosis, angiogenesis, and apoptosis.26
Determining the appropriate treatment of autoimmune myocarditis still remains a major clinical problem. Immunomodulatory therapies have been used in clinical trials for the treatment of myocarditis.27 However, clinical trials and experimental studies of murine models of myocarditis have yet not established a generally accepted immunosuppressive treatment of autoimmune myocarditis. We obtained promising results by blocking MCP-1 or MIP-1α in vivo. Because MCP-1 and MIP-1α act through different pathways, blocking them together may even have an additive or synergetic effect. Gene therapy to block MCP-1 activity in vivo by using an N-terminal deletion mutant of MCP-1 is another possibility. Recently, Egashira and colleagues28 demonstrated that mutant MCP-1 7ND protein secreted from the transfected skeletal muscle cells into the circulation blood blocks the MCP-1/CCR2 signal pathway in remote target organs or tissue and suppresses monocyte recruitment. This 7ND gene construct has been used successfully in treatment of several other inflammatory diseases.13,14 Further 7ND treatment had no negative effect on the clearance of coxsackievirus B3 in our study, which might be important for future clinical trials, as the myocarditis seen in humans is mostly virus-induced and latent virus may be present (Kaya et al, unpublished data, 2005).
There are a number of mechanisms by which blocking MCP-1 may affect the development of myocarditis. Although the pathogenesis of myocarditis has not yet been fully elucidated, the migration of antigen-specific T cells in the myocardium is considered to be the initial process. Subsequently, large numbers of inflammatory cells infiltrate into the myocardium, including non–antigen-specific T cells, B cells, macrophages, and neutrophils that elicit full-blown myocarditis. In the latter process, chemoattractants play a major role in the recruitment of inflammatory cells. Fuse et al7 demonstrated an abrupt onset and serum levels of MCP-1 elevated simultaneously with the onset of the disease. The expression of MCP-1 mRNA also coincided with the onset of EAM. MCP-1 is a potent chemotactic factor for mononuclear cells. Thus, the inflammatory infiltrate observed in myocardial lesions of myocarditis consists of >70% mononuclear cells.10
Proinflammatory cytokines are important in the development of autoimmune myocarditis. Administration of IL-1 or TNF-α promoted virus- and myosin-induced myocarditis in genetically resistant B10.A mice.22 In addition, when A/J mice are infected with CB3 and treated with an IL-1 receptor antagonist, myocardial injury is diminished.29 Thus, IL-1 and TNF-α are critical in the pathogenesis of autoimmune myocarditis. Both cytokines can upregulate major histocompatibility complex class I and class II expression in the cardiac interstitium and on myocytes, possibly inducing or enhancing inflammation. Cardiomyocytes stimulated in vitro by MCP-1 are able to produce proinflammatory cytokines such IL-1,24 which in turn may reduce cardiomyocyte contractility and increase cardiac hypertrophy.30,31 IL-1 in turn may induce MCP-1 production in several cell types.32 Accordingly, in CCR2−/− mice immunized with cardiac myosin and in mice treated with 7ND, there was significantly less infiltration, including all cell types, in the heart sections, significantly reduced cardiac myosin-specific production of IL-1 and IL-4 and increased production of IL-10 and IFN-γ. Recently, we showed that IL-4 promotes the disease in EAM and IFN-γ limits it. Suppression of IFN-γ represents at least one of the mechanisms by which IL-4 promotes EAM.33 Lack of IFN-γ due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Treatment of mice with recombinant IFN-γ suppressed the development of myocarditis. Thus, IFN-γ has a disease-limiting effect in EAM.21 IL-10 is primarily produced by monocytes and T cells and plays an important role in controlling cell-mediated and inflammatory responses, including EAM.12 IL-10 also suppresses inflammatory responses in cardiomyocytes by reducing TNF-α–stimulated release of MCP-1. Although TNF-α has no effect on IL-10 and only modest effects on IL-1 and IL-6 levels in adult rat cardiomyocytes, this cytokine is a potent inducer of MCP-1 in these cells.24 However, there is strain and species-specific difference in myocarditis with regard to Th1 and Th2 and the promotion of cardiac inflammation.20,33,34
There appears to be no compensatory upregulation of other chemokines when blocking MCP-1 with the 7ND construct. In contrast, mice treated with 7ND showed significant reduction in other chemokines such as RANTES, MIP-2, IP-10, MCP-1, TCA-3, and eotaxin, all chemokines that can be produced by different inflammatory cells locally during an inflammation and thus aggravate inflammation. Therefore, reducing the migration of inflammatory cells by blocking MCP-1 not only leads to reduced inflammatory cells in the myocardium but also reduced production of additional chemokine production in the myocardium.
Overall, these findings suggest that MCP-1 and MIP-1α, acting through their receptors CCR2 and CCR5, are important in the induction of EAM and that inhibition of MCP-1 with 7ND gene transfection significantly reduced the disease severity. This strategy may be a new feasible form of gene therapy against autoimmune myocarditis.
This work was supported by the Deutsche Herzstiftung and in part by the National Institutes of Health grants HL67290 and HL70729. The authors thank Florian Leuschner, Theresa Tretter, and Özay Kaya for critically reading the manuscript.
The online-only Data Supplement, which contains Figure I and Figure II, can be found with this article at http://circ.ahajournals.org/cgi/content/full/112/22/3400/DC1.
Rollins BJ. Chemokines Blood. 1997; 90: 909–928.
Carr MW, Roth SJ, Luther E, Rose SS, Springer TA. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A. 1994; 91: 3652–3656.
Zhang YJ, Rutledge BJ, Rollins BJ. Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis: identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis. J Biol Chem. 1994; 269: 15918–15924.
Zhang Y, Rollins BJ. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer. Mol Cell Biol. 1995; 15: 4851–4855.
Kaya Z, Dohmen KM, Wang Y, Schlichting J, Afanasyeva M, Leuschner F, Rose NR. Cutting edge: a critical role for IL-10 in induction of nasal tolerance in experimental autoimmune myocarditis. J Immunol. 2002; 168: 1552–1556.
Ni W, Egashira K, Kitamoto S, Kataoka C, Koyanagi M, Inoue S, Imaizumi K, Akiyama C, Nishida KK, Takeshita A. New anti-monocyte chemoattractant protein-1 gene therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2001; 103: 2096–2101.
Shimizu S, Nakashima H, Masutani K, Inoue Y, Miyake K, Akahoshi M, Tanaka Y, Egashira K, Hirakata H, Otsuka T, Harada M Anti-monocyte chemoattractant protein-1 gene therapy attenuates nephritis in MRL/lpr mice. Rheumatology (Oxford). 2004; 43: 1121–1128.
Rao AR, Quinones MP, Garavito E, Kalkonde Y, Jimenez F, Gibbons C, Perez J, Melby P, Kuziel W, Reddick RL, Ahuja SK, Ahuja SS. CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease. J Immunol. 2003; 171: 4875–4885.
Ni W, Kitamoto S, Ishibashi M, Usui M, Inoue S, Hiasa K, Zhao Q, Nishida K, Takeshita A, Egashira K. Monocyte chemoattractant protein-1 is an essential inflammatory mediator in angiotensin II-induced progression of established atherosclerosis in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol. 2004; 24: 534–539.
Bruhl H, Cihak J, Schneider MA, Plachy J, Rupp T, Wenzel I, Shakarami M, Milz S, Ellwart JW, Stangassinger M, Schlondorff D, Mack M. Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells. J Immunol. 2004; 172: 890–898.
Bonecchi R, Bianchi G, Bordignon PP, D’Ambrosio D, Lang R, Borsatti A, Sozzani S, Allavena P, Gray PA, Mantovani A, Sinigaglia F. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med. 1998; 187: 129–134.
Smith SC, Allen PM. Myosin-induced acute myocarditis is a T cell-mediated disease. J Immunol. 1991; 147: 2141–2147.
Afanasyeva M, Wang Y, Kaya Z, Stafford EA, Dohmen KM, Sadighi Akha AA, Rose NR. Interleukin-12 receptor/STAT4 signaling is required for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway. Circulation. 2001; 104: 3145–3151.
Lane JR, Neumann DA, Lafond-Walker A, Herskowitz A, Rose NR. Interleukin 1 or tumor necrosis factor can promote Coxsackie B3-induced myocarditis in resistant B10.A mice. J Exp Med. 1992; 175: 1123–1129.
Damas JK, Eiken HG, Oie E, Bjerkeli V, Yndestad A, Ueland T, Tonnessen T, Geiran OR, Aass H, Simonsen S, Christensen G, Froland SS, Attramadal H, Gullestad L, Aukrust P. Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure. Cardiovasc Res. 2000; 47: 778–787.
Weber KS, Nelson PJ, Grone HJ, Weber C. Expression of CCR2 by endothelial cells: implications for MCP-1 mediated wound injury repair and in vivo inflammatory activation of endothelium. Arterioscler Thromb Vasc Biol. 1999; 19: 2085–2093.
Egashira K, Koyanagi M, Kitamoto S, Ni W, Kataoka C, Morishita R, Kaneda Y, Akiyama C, Nishida KI, Sueishi K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy inhibits vascular remodeling in rats: blockade of MCP-1 activity after intramuscular transfer of a mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis. FASEB J. 2000; 14: 1974–1978.
Lane JR, Neumann DA, Lafond-Walker A, Herskowitz A, Rose NR. Role of IL-1 and tumor necrosis factor in coxsackie virus-induced autoimmune myocarditis. J Immunol. 1993; 151: 1682–1690.
Gulick T, Chung MK, Pieper SJ, Lange LG, Schreiner GF. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness. Proc Natl Acad Sci U S A. 1989; 86: 6753–6757.
Yamamoto T, Eckes B, Mauch C, Hartmann K, Krieg T. Monocyte chemoattractant protein-1 enhances gene expression and synthesis of matrix metalloproteinase-1 in human fibroblasts by an autocrine IL-1 alpha loop. J Immunol. 2000; 164: 6174–6179.