Elsevier

Atherosclerosis

Volume 138, Issue 2, June 1998, Pages 247-253
Atherosclerosis

A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells

https://doi.org/10.1016/S0021-9150(97)00296-7Get rights and content

Abstract

Effect of interleukin 4 (IL-4) on the production of matrix metalloproteinase 1 (MMP-1) by normal and immortalized human intimal smooth muscle cells (SMC) was investigated. The production of the precursors of MMP-1 by intimal SMC was enhanced in a dose-dependent manner by addition of IL-4 to the culture medium, whereas the cytokine also showed an inhibitory effect on DNA synthesis in the cells. In addition, mRNA of IL-4 was found in the atherosclerotic and nonatherosclerotic areas of the intima. Although the production of MMP-1 and the proliferation of SMC are thought to play an important role in reconstruction of the intima during atherogenesis, our results suggest a possible role of IL-4 induced MMP-1 in inhibiting tissue remodeling caused by a variety of arterial disorders including atherosclerosis.

Introduction

In the process of atherosclerosis, remodeling of the arterial intima is a key event in which smooth muscle cells (SMC) and macrophages act through a variety of their phagocytosis and secretory functions [1]. These important cell types, present in a variety of stages of atherosclerosis, release several factors including several kinds of cytokines designed to modulate the function of one another. However, platelet-derived growth factor (PDGF) is thought to be the most essential factor for the atherosclerotic remodeling because it and PDGF-like molecules are secreted by macrophages, SMC, endothelial cells and platelets at the sites of injured endothelial cells [2].

We reported previously that in Kawasaki’s disease, an infantile and acute inflammatory disease of the artery, marked thickening of the intima similar to that of fibrous plaques occurs within several weeks after onset, indicating that immunoregulatory factors are involved in the intimal tissue remodeling of the artery [3]. In almost all tissues, collagens are essential structural proteins; and their degradation, as well as cell proliferation, is precisely controlled by collagenolytic enzymes. Unlike Kawasaki's disease, atherosclerosis is a slowly progressive disease, which generally begins in childhood and does not become clinically manifest until middle-age or later. The essential role of these inflammatory factors, however, is also the case in the remodeling of the arterial wall in atherosclerosis through production and degradation of collagens and cell proliferation. In addition to the stimulatory effect of many factors on SMC proliferation and extracellular matrix production, we described earlier that collagen-degrading enzymes are also responsible for tissue remodeling of the artery 4, 5, 6. As shown in these papers, human aortic SMC synthesis of matrix metalloproteinases (MMPs) occurred in response to PDGF, interleukin 1 (IL-1), epidermal growth factor (EGF) and histamine. Since histamine is produced by mast cells, we added these cells as an important cell type in the group of cell types that participate in the process of atherosclerosis 6, 7.

From the view point of inflammation, T-cells are also often found in the atherosclerotic foci 8, 9. A major cytokine secreted by T-cells and mast cells 10, 11is IL-4, which has been shown to be involved in inflammatory or apoptotic reactions in humans and has profound effects on a variety of cell types that are either stimulatory or inhibitory on the process, including protein expression and cell differentiation [12]. It is also reported that IL-4 strongly inhibits PDGF-induced cell proliferation by blocking the early phase of the cell cycle [13]; whereas, like PDGF, it stimulates cells to produce extracellular matrix protein [14]. Furthermore, it was reported that IL-4 potentially blocks the release of MMP-1 and 92 kDa type IV collagenase (MMP-9/gelatinase B) from alveolar macrophages without altering secretion of tissue inhibitor metalloproteinase [15]. On the other hand, we also reported that retinoic acid, glucocorticoids and prostaglandins are possible anti-atherosclerotic factors because of their inhibitory effects on the production of MMPs and cell proliferation of human SMC, suggesting that a protective system against the remodeling of the arterial wall is engaged in the response of the arterial wall to various kinds of stimuli [16].

Since, with respect to inflammatory responses, the interaction between these migrating T-cells or mast cells and SMC is an important factor for the progression or the inhibition of atherosclerosis, it is necessary to investigate the effect of IL-4 on the production of extracellular matrix-degrading enzymes by human arterial SMC for a better understanding of the process of atherosclerosis. In the present study, we investigated the effect of IL-4 on the production of proMMP-1 and DNA synthesis by human intimal SMC, and the expression of IL-4 mRNA in human intima. Based on the results, we discussed a possible role of IL-4 in the regression of atherosclerosis.

Section snippets

Cell culture of human aortic smooth muscle cells

Human aortic SMC and skin fibroblasts were isolated from human aortas and skin dermal tissue at autopsy [4]. After isolation of the intimal SMC, the cells were incubated in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 2.5 μg/ml of amphotericin B at 37°C in a humidified 5% CO2–95% air mixture. ISS10 cells, which are human aortic immortalized SMC [6], were also used in experiments.

Cloning and human proMMP-1 promoter gene construction

Two oligonucleotide primers designed based

Effect of IL-4 on incorporation of [3H]-thymidine by human aortic SMC and skin fibroblasts

To investigate the proliferative effect of IL-4 on SMC, we examined the incorporation of [3H]-thymidine into IL-4-treated cells in triplicate. After cultivation in serum-free medium for 24 h, 1% serum induced an ≈2-fold up-regulation of DNA synthesis in both SMC and fibroblasts (Fig. 1). The addition of IL-4, however, markedly reduced the incorporation of [3H]-thymidine to a level comparable to that in the medium containing 1% serum.

Immunoblot analysis of proMMP-1 secretion by human aortic SMC and skin fibroblasts in response to IL-4

Three experiments were performed. As shown in Fig. 2, the

Discussion

It has been reported that T-cells 8, 9, 18and mast cells 19, 20, both of which can produce IL-4, also participate in the process of atherosclerosis; although the accumulation of SMC in the intima is the major phenomenon of atherosclerosis, and is accompanied by mononuclear cell infiltration by cells including monocytes/macrophages. T lymphocytes in the lesion site are almost exclusively CD3+, and both helper (CD4+) and suppressor/cytotoxic (CD8+) phenotypes are present at all stages of

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