Original Article
Blockade of macrophage migration inhibitory factor ameliorates experimental autoimmune myocarditis

https://doi.org/10.1016/j.yjmcc.2004.05.016Get rights and content

Abstract

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a critical role in the regulation of macrophage effector functions and T-cell activation. However, its role in the pathogenesis of experimental autoimmune myocarditis (EAM) has remained unresolved. In this study, we studied the role of the MIF in EAM. We investigated the expression of MIF in EAM using enzyme-linked immunosorbent assay, Northern blotting, and immunohistochemistry. Moreover, a neutralizing antibody (Ab) to MIF was injected intraperitoneally from day 0 to 20 (experiment 1), or from day 13 to 19 (experiment 2), after the immunization. Disease severity was estimated by the macroscopic and microscopic findings for the heart, heart weight to body weight ratio, and cellular and humoral immune responses on day 21. Enhanced MIF protein and mRNA expression in the heart tissue and an elevated serum MIF concentration were confirmed in EAM. In experiment 1, the anti-MIF Ab treatment markedly inhibited the onset of EAM. Moreover, a significant reduction in disease severity was also achieved even after the delayed anti-MIF Ab treatment in experiment 2. Furthermore, we demonstrated that MIF blockade decreased the expression of VCAM-1, TNF-α, and IL-1β and the migration of T-cells and macrophages in the EAM heart. These results demonstrate an important role of MIF in the pathogenesis of EAM and suggest that MIF blockade may be a promising new strategy for the treatment of myocarditis.

Introduction

Acute myocarditis is a major cause of sudden death in persons younger than 40 years old [1] and often follows infection by coxsackievirus B3 [2]. The pathogenesis of myocarditis is not fully understood, but there is substantial evidence suggesting that autoimmune responses to heart antigens, particularly cardiac myosin, following viral infection may contribute to the disease process [3].

Experimental autoimmune myocarditis (EAM) in rats is an autoimmune inflammatory cardiac disorder that is an animal model characterized by severe myocardial lesions and multinucleated giant cells. It has been reported that the pathogenesis of the tissue damage in human giant cell myocarditis (GCM) and viral myocarditis resembles that in EAM [4]. Therefore, to understand the mechanisms of myocardial injury and to develop an effective therapeutic means for myocarditis in relation to the autoimmune mechanism, we used this animal experimental model of autoimmune myocarditis.

EAM is a T-cell-mediated autoimmune disease since it can be transferred into naive rats by injection of cultured spleen T-cells from previously immunized syngeneic rats [5]. To induce the disease, primed, autoreactive T-cells must migrate to the heart and recognize the antigen presented by local antigen-presenting cells (APCs). Adhesion and costimulatory molecules expressed on vascular endothelial cells and APCs, respectively, as well as chemokines and cytokines released in response to inflammatory stimuli are intimately involved in both migration and activation of T-cells in the heart [6]. Macrophage migration inhibitory factor (MIF) was originally discovered as a lymphokine derived from activated T-cells that inhibited the random migration of macrophages in vitro and was involved in the mechanism of delayed-type hypersensitivity [7]. Furthermore, it regulates T-cell activation and proliferation [8]. More recently, MIF has been implicated in the pathogenesis of autoimmune disorders such as arthritis [9], glomerulonephritis [10], and even ongoing experimental autoimmune encephalomyelitis (EAE) [11]. Notably, an elevated level of MIF was reported in the heart in the EAM rat [12]. However, very little is known about the role of MIF in the regulation of T-cell responses in the heart and no direct evidence has been provided for its involvement in the pathogenesis of EAM.

In this study, we show that treatment with an anti-MIF antibody (Ab) from the early phase as well as the late phase can significantly inhibit EAM. Furthermore, we demonstrate that MIF blockade decreases the expression of VCAM-1, TNF-α, and IL-1β in the EAM heart and the migration of T-cells and macrophages to this site.

Section snippets

Preparation of rabbit polyclonal Ab against rat MIF

Polyclonal anti-rat MIF serum was generated as described in our previous publications [13]. In brief, the rabbits were inoculated intradermally with 100 μg of MIF emulsified in complete Freund’s adjuvant (CFA) (Difco, Detroit, MI, USA) at weeks 1 and 2, and with 50 μg of MIF diluted in incomplete Freund’s adjuvant (IFA) (Difco) at week 4. The immunoglobulin G (IgG) fraction was prepared using protein A-Sepharose according to the manufacturer’s protocol.

Induction of autoimmune myocarditis

Cardiac myosin was prepared from the

Elevated MIF level in the heart of EAM rat

We examined the MIF protein content by ELISA in the heart tissue (Fig. 1A). ELISA study showed that the MIF protein content slightly but significantly increased in the control IgG-treated EAM heart compared with the normal heart.

Serum concentration of MIF after immunization in EAM

The serum concentration of MIF was analyzed at different times after immunization, using ELISA (Fig. 1B). In the normal rats (day 0), the serum MIF concentration was already detected at around 100 ng/ml. The maximum concentration was obtained 7 d after immunization in

Discussion

Previous studies have shown that EAM in rats can be divided into two distinct stages and these stages are strongly related to the cytokine milieu [18]. First, focal inflammation, mainly consisting of macrophages, and IL-12 expression appear around day 13–14 after immunization. Subsequently, strong inflammation consisting of macrophages and CD4+ T-cells lasts up to day 19 after immunization and leads to myocardial cell destruction. The activation and expansion of antigen-specific T-cells occur

Conclusions

In conclusion, we found that: (1) MIF is expressed by inflammatory cells and cardiac myocytes in pathologic foci of EAM. (2) Anti-MIF Ab treatment from the early phase as well as the late phase could significantly inhibit EAM in a VCAM-1-dependent fashion with suppression of the expression of TNF-α and IL-1β in the heart. (3) Taken together, these results demonstrate an important role for MIF in the pathogenesis of EAM and suggest that MIF blockade may be a promising new strategy for the

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