Amyloid-β-induced chemokine production in primary human macrophages and astrocytes

Abstract

In Alzheimer's disease (AD), chemotaxis might be responsible for attracting glial cells towards the neuritic plaque. Using primary monocyte-derived macrophages and primary adult astrocytes as a model, amyloid-beta (Aβ) (1–42) was able to stimulate the production, as measured by RT-PCR, of MIP-1α and MIP-1β mRNA in macrophages and MCP-1 in astrocytes. Cocultures showed in unstimulated as well as in Aβ-stimulated cells an increase in MIP-1α, MIP-1β and MCP-1 mRNA. ELISAs of supernatant samples of stimulated macrophages and astrocytes also showed an increase in MIP-1α and MIP-1β in macrophages and MCP-1 in astrocytes. Stimulated cocultures showed an increase in MIP-1α, MIP-1β and MCP-1 protein levels in contrast to unstimulated cocultures.

Introduction

Alzheimer's disease (AD) is the most abundant form of dementia. Brains of people suffering from AD are characterized by depositions composed of aggregated amyloid-beta (Aβ) proteins and several other molecules (reviewed in Cummings et al., 1998, Dickson, 1997). These depositions are reported to be enclosed by activated microglia (McGeer et al., 1987) and astrocytes Mandybur and Chuirazzi, 1990, Pike et al., 1995. The depositions and the glial cells in their turn are surrounded by degenerating neurons. In total this is called the neuritic plaque, in contrast to the diffuse plaque which is found both in normal and AD brains. There is less Aβ present in these diffuse plaques and Aβ is not as aggregated as in neuritic plaques. Moreover, these diffuse plaques are reported not to be surrounded by microglia and astrocytes and neurons surrounding these diffuse plaques are not degenerating.

The cause of neuronal loss has not been fully determined yet. There are, however, strong suggestions that the Aβ protein can be direct neurotoxic and that surrounding microglia and astrocytes may play an important role in the inflammatory response. Studies have shown that Aβ-stimulation of THP-1 monocytes, rat microglia and primary human macrophages results in the production of an array of pro-inflammatory molecules Ariga and Yu, 1999, Baeuerle and Henkel, 1994, Giulian et al., 1995, Heese et al., 1998, Klegeris et al., 1994, Klegeris et al., 1997, Klegeris and McGeer, 1997, Lorton et al., 1996, Meda et al., 1996, Smits et al., 1999, Tjernberg et al., 1999. There are also several studies reporting the ability of Aβ-stimulated monocytes and macrophages to produce anti-inflammatory cytokines (Szczepanik et al., 2001). Taken together, all these studies have shown the complexity of the situation as it exists in the neuritic plaque. There are numerous glial cells present around each plaque, each residing in its own micro-environment and each capable of producing pro- and/or anti-inflammatory products. It is the balance of pro-inflammatory products and anti-inflammatory products that may be essential in the degenerative process. In AD, it is eventually leading to the degeneration of the neurons surrounding the plaque.

Influencing this balance may help in slowing the disease. A possibility of changing the balance between pro- and anti-inflammatory products is questioning the recruitment of the microglia and astrocytes towards the plaque. There are far more microglia and astrocytes concentrated around these plaques than located elsewhere in the brain. Little is known about the mechanism responsible for attracting so much glial cells. The research presented in this paper focuses on the involvement of proteins with chemotactic capabilities in recruiting these cells to the plaque. One group of chemotactic proteins is the chemokines. Chemokines constitute a superfamily of chemotactic cytokines and are produced by a wide variety of cells, including T-cells, monocytes, endothelial cells, microglia and astrocytes Schall and Bacon, 1994, Schluger and Rom, 1997. In addition to their chemotactic effects in the immune system, chemokines modulate a number of biological responses, including enzyme secretion, cellular adhesion, cytotoxicity, tumor cell growth, degranulation and T-cell activation (Luster, 1998). The chemokines mediate their effects via G protein-coupled receptors of the seven transmembrane domain rhodopsin-type superfamily Murphy, 1996, Wells et al., 1996. These receptors are named CXCR, CCR, CR and CX3CR, respectively.

In many central nervous system (CNS) diseases such as multiple sclerosis (MS), brain trauma, infections or focal ischemia, the blood–brain barrier is breached, and leukocyte infiltration is found at the lesion sites (Ghirnikar et al., 1998). In AD however, the blood–brain barrier is intact and no infiltration of inflammatory cells is present. Because of the continual inflammation in the AD brain, resident CNS cells have to be involved, and indeed many CNS cells are reported to be able to produce chemokines and express chemokine receptors Dorf et al., 2000, Xia and Hyman, 1999. Regarding Aβ-induced chemokine production, however, only a small number of papers have been published. The CC-chemokines MIP-1α and MCP-1 have been found to be produced by Aβ-stimulated monocytes after 48 h of cultivation (Fiala et al., 1998). Also Aβ (25–35)-stimulated U373 astrocytoma cells have been reported to have an increased MCP-1 production and rat astrocytes stimulated with 50 μM Aβ (1–42) for 6 h have been found to produce MCP-1 and RANTES mRNA Johnstone et al., 1999, Prat et al., 2000. In rat astrocytes, the CC-chemokines MIP-1α, MIP-1β, MCP-1 and RANTES mRNA expression can also be induced by either IFNγ or TNF-α.

This paper shows the results of the mRNA production of the CC-chemokines MIP-1α, MIP-1β, MCP-1 and RANTES by primary human monocyte-derived macrophages as a model for microglia cells, by primary adult astrocytes and by cocultured MDMs and astrocytes when stimulated by Aβ (1–42) and Aβ (1–40) for 2, 4, 6 and 8 h. Moreover, chemokines supernatant levels in cultured MDMs, astrocytes and cocultures thereof are determined.