ARTICLE |
Correspondence to: Cheuk-Man Yu, Dept. of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. E-mail: cmyu@cuhk.edu.hk
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Summary |
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Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI. (J Histochem Cytochem 51:625631, 2003)
Key Words: macrophage migration, inhibitory factor, macrophages, myocytes, myocardial infarction, gene expression
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Introduction |
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MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) was originally described as a product of T-cells that inhibits the migration of macrophages in vitro and promotes macrophage accumulation in the skin delayed-type hypersensitivity reaction (
Recently, we have demonstrated that, in patients with acute myocardial infarction (AMI), MIF levels were significantly elevated and were higher than those in patients with reversible myocardial ischemia (
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Materials and Methods |
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Induction of AMI in Rats
Left coronary artery occlusion was performed in 12-week male SpragueDawley rats as previously described (
In Situ Hybridization
A 420-bp Xba I/BamH I fragment of mouse MIF cDNA in the Bluescript SK+ vector (Stratagene; La Jolla, CA), which is highly homologous to the rat sequence (
Immunohistochemistry
Double immunostaining of MIF expression by infiltrating macrophages in infarcted or non-infarcted heart tissues was performed on 4-µm paraffin sections of formalin-fixed heart with a microwave-based, multiple immunoenzymatic staining (
Western Blotting Analysis and EIA Detection of Plasma MIF Level
Total cellular protein was extracted by 50 mM Tris-HCl, pH 8, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM PMSF and a mini-complete proteinase inhibitor cocktail (Roche). Western blotting of MIF has been previously described (
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Frozen heart tissue samples were homogenized and RNA was extracted by the acidphenolguanidiniumthiocyanate method ( mRNA was detected by RT-PCR using TATA-box binding protein (TBP) as an internal control. The primers used were MIF, 5' CCA GGA CCG CAA CTA CAG CAA 3' (forward) and 5' GGG CTC AAG CGA AGG TGG AAC 3' (reverse); IL-1ß, 5' CCT TCT TTT CCT TCA TCT TTG 3' (forward) and 5' ACC GCT TTT CCA TCT TCT TCT 3' (reverse); TNF-
, 5' CGT CGT AGC AAA CCA CCA AGC 3'; and TBP, 5' ACC CTT CAC CAA TGA CTC CTA TG 3' (forward) and 5' ATG ATG ACT GCA GCA AAT CGC 3' (reverse). PCR was performed at 94C for 45 sec, 58C for 40 sec, and 72C for 45 sec for 29 cycles. Validations of the semiquantitative RT-PCR were performed using normal rat left ventricular cDNA to ensure that there was no interaction between two pairs of primers, and the reaction condition was standardized for different amounts of cDNA (
Statistical Analysis
The differences in mean value among the various rat groups were analyzed by one-way analysis of variance (ANOVA) with Scheffe's correction or paired sample t-test where appropriate, using a statistical software program (SPSS for Windows, ver. 10.0; SPSS, Chicago, IL). All data were expressed as mean ± SD. A p value <0.05 was considered statistically significant.
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Results |
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The systolic and diastolic blood pressure remained normal and did not differ between sham and AMI animals throughout the experiments (systolic: sham, 113 ± 12 vs 107 ± 13 mmHg, p=NS; diastolic, sham 93 ± 14 vs 88 ± 13 mmHg, p=NS). There was no difference in the body weight between the groups over the 2-week time course (sham 395 ± 48 vs 411 ± 50 g, p=NS).
Myocardial MIF mRNA and Protein Expression in Normal Hearts and Animals with AMI
Weak MIF mRNA and protein were constitutively expressed by myocardium in both normal and sham-operated rats (Fig 1a, Fig 1b, Fig 2a, Fig 2b, Fig 3, and Fig 4). In contrast, by in situ hybridization and RT-PCR, MIF mRNA was markedly expressed by myocytes in the infarcted region, peaking at 6 hr after AMI (p<0.001) (Fig 1c and Fig 3; Table 1). Upregulation of MIF mRNA by myocytes remained high throughout the study period (Fig 1c1g). In the noninfarcted area, myocardial MIF mRNA was also upregulated (p<0.01), although to a lesser extent compared to the infarcted region (Fig 1h and Fig 3; Table 1). The MIF protein expression in the infarcted region did not show an early peak but increased gradually over the 2-week study period (Fig 2 and Fig 4; Table 1). Meanwhile, ELISA showed that the MIF level in serum was significantly increased at 6 hr (p<0.001) (Table 1). This may reflect a rapid secretion of the stored cytoplasmic MIF from the damaged myocytes after AMI.
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Myocardial MIF Expression and Macrophage Accumulation in the Infarcted Region
As shown in Fig 2, there was no macrophage infiltration in the normal, sham-operated, and noninfarcted heart tissues. In contrast, a significant increase in the number of macrophages was seen at day 1 (p<0.05) and continued to increase throughout the experimental time course (p<0.001) (Fig 2d2g, Table 1). Infiltrating macrophages were localized exclusively within the infarcted region, where strong MIF expression was found.
MIF Expression by Infiltrating Macrophages
Marked upregulation of MIF mRNA and protein was also found in infiltrating macrophages in the infarcted region. As shown in Fig 1, strong MIF mRNA was found in the macrophages in the infarcted region compared to myocytes in the noninfarcted area (Fig 1d1h). This was further confirmed by double immunostaining, showing that the ED1+ macrophages were co-expressing MIF within the infarct region (Fig 2d2g, Table 1), indicating that the activated macrophages are also the major source of MIF during AMI. Correlation analysis showed that upregulation of myocardial MIF correlated closely with the number of infiltrating macrophages in the infarcted lesions (r=0.61; p<0.01).
Expression of Interleukin-1ß and Tumor Necrosis Factor- in Normal Hearts and Animals with AMI
In the infarcted region, IL-1ß level was significantly elevated by more than ninefold in the first day of AMI (p<0.01) (Table 1). The level decreased very rapidly afterwards and was no different from the control level from 3 days to 2 weeks. A similar, although smaller surge of IL-1ß level was observed in the noninfarcted area on day 1 (p<0.05), which decreased afterwards. For TNF- level, it followed a very similar time course of change to that of IL-1ß, becoming elevated by >20-fold on day 1 in the infarcted region (p<0.001) and decreasing rapidly thereafter (Table 1). In the noninfarcted region, similar changes were observed, although to a much lesser extent than that of the infarcted region.
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Discussion |
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This study demonstrated, for the first time, that upregulation of MIF is involved in AMI. In normal heart, the pre-formed MIF was constitutively and weakly expressed by myocytes. After AMI, MIF mRNA was rapidly upregulated in myocytes within hours, preceding the macrophage infiltration in the infarcted region on day 1. The increase in serum MIF level after AMI and loss of cytoplasmic MIF protein in myocytes suggests that MIF may be rapidly secreted from damaged myocytes. Alternatively, ischemic myocytes may not be able to synthesize MIF protein which, in general, is a later process than mRNA synthesis. These may explain a late and smaller peak observed for protein expression in the infarcted region. On the other hand, the elevated serum MIF level may also be a result of systemic response, although it was not observed in patients with unstable angina in a previous study (
Macrophages, which had originally been considered to be the target of MIF action, were identified as one of the major sources of MIF production during AMI, being evident from day 1 onwards. Indeed, almost all infiltrating macrophages expressed MIF in the infarcted region. Macrophage-derived MIF may both initiate and amplify the inflammatory process during AMI by recruiting more macrophages, neutrophils, and T-cells to the site of inflammation by upregulating the adhesion molecules such as ICAM-1 (. Such changes were more dramatic in the infarcted region, where myocyte damage was obvious after cellular hypoxia.
It has been shown that myocyte damage after AMI is mediated by an inflammation-like response. This was supported by the observation in animal studies that infiltration of inflammatory cells, such as macrophages and neutrophils, in the infarct zone was evident early after AMI (, inducible nitric oxide synthase, and cellular activation (
In conclusion, MIF is rapidly and markedly upregulated in myocytes during AMI. Upregulation of MIF by myocytes contributes significantly to macrophage accumulation, and further expression of MIF by infiltrating macrophages may perpetuate the inflammatory response, which may augment the extent of ischemia-induced myocyte damage.
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Acknowledgments |
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Supported by a research grant from CRCG, University of Hong Kong, Hong Kong (HKU7325/99M).
Received for publication May 22, 2002; accepted November 7, 2002.
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