Research reportIntegrin Mac-1 and β-amyloid in microglial release of nitric oxide
Introduction
While the β-amyloid protein associated with Alzheimer's disease (AD) has been well characterized biochemically, its primary biological function and mode of action in AD has not been determined. β-Amyloid occupies the core of neuritic plaques which are made of degenerative neurites. Microglial cells, in their reactive form, are found closely associated with matured neuritic plaques 5, 23; however, very little information has been available on the effects of the β-amyloid present in the neuritic plaques on microglial cells.
In the rodent, microglial cells release the free radical nitric oxide 7, 8, 25, 43. This may have significance in the pathogenesis of AD given that nitric oxide release from microglia is accompanied by a marked reduction in neuronal cell survival in vitro [13]. The inducible nitric oxide synthase (iNOS) found in microglia is calcium- and calmodulin-independent and must be induced by agents such as bacterial cell wall lipopolysaccharide (LPS) or IFN-γ13, 19, 39, 43. We have shown that cultured microglial cells respond to IFN-γ when co-stimulated with either β1–40 or β25–35 to release nitric oxide, while only β1–40 induces significant nitric oxide release from microglial cells by itself [20]. These findings were also demonstrated, in part, by Meda et al. [27]who reported a synergistic effect between β25–35 and IFN-γ in triggering nitric oxide and tumor necrosis factor-α (TNF-α) release from microglia. Similarly, it was shown that rodent peritoneal macrophages without priming with IFN-γ are activated by β1–40 but not by β25–35 [24]. The results of these studies support the notion that β-amyloid may be responsible for activation of microglia and subsequent nitric oxide release in vivo. At present, the triggering mechanisms involved in microglial stimulation for release of nitric oxide have not been documented.
Our hypothesis is that a microglial receptor through which β-amyloid might trigger nitric oxide release is the leukocyte integrin Mac-1. The Mac-1 receptor is a likely choice given that, when bound by appropriate ligands in the presence of a co-stimulatory factor such as IFN-γ, Mac-1 activates macrophages and neutrophils to produce reactive mediators including reactive oxygen intermediates and nitric oxide 14, 15, 17, 28, 32, 33. Coincidentally, β25–35 in combination with IFN-γ stimulates iNOS activity and the release of nitric oxide from microglia [20]. Mac-1 belongs to a family of adhesion molecules composed of a heterodimer between an α and β subunit found exclusively on leukocytes or cells of this lineage. Each of these adhesion molecules shares an identical β subunit (CD18) and is distinguished by its α subunit designated CD11a, CD11b, and CD11c for LFA-1, Mac-1, and p150,95α, respectively [22]. Microglial cells express Mac-1 and in AD, Mac-1 expression is upregulated in reactive microglial cells associated with the neuritic plaques 1, 31. Recently it was reported that antibody to CD18 was found to stimulate nitric oxide production and inducible nitric oxide synthase mRNA expression in alveolar macrophages [29]. In the present studies, we investigated a potential role for CD11b and CD18 of Mac-1 in β-amyloid-mediated release of nitric oxide from cultured hippocampal microglia.
Section snippets
Synthesis and biotinylation of β-amyloid peptide
β-Amyloid (β25–35) was synthesized and purified by the University of Iowa Protein Structure Facility (Iowa City, IA). β25–35 was dissolved in sterile tissue culture water (Sigma) or 0.1 M PBS (for Mac-1 binding studies) in a stock concentration of 1 mg/ml (pH 7.2) and aliquots were stored at −20°C. β25–35 was biotinylated according to a protocol obtained from Vector Laboratories. Briefly, β25–35 was dissolved in 100 mM HEPES buffer, pH 8.5, at a concentration of 2 mg/ml, then 400 μl
The effect of Wt.3 and OX42 on nitric oxide release from microglia
Antibodies specific for antigen-binding sites on Mac-1 are known to elicit a number of cell responses including respiratory burst activity. Therefore, before observing the effects of these antibodies on β-amyloid-mediated release of nitric oxide from microglia, it was necessary to determine what, if any, effect Wt.3 or OX42 had on nitric oxide release from microglia. In this experiment, nitric oxide levels were measured in the culture media of microglia exposed to Wt.3 or OX42. We first
Discussion
Microglial cell interactions with β-amyloid were studied using a synthetic β25–35 peptide. β25–35 corresponds to amino acids 25–35 of the 42 amino acid β-amyloid protein found in the neuritic plaques and has been shown to be a neurotoxic portion of β-amyloid 26, 41, 42. While β1–40, a larger fragment of the 42 amino acid β-amyloid protein, may be used in future studies, we choose to limit the scope of this study to the effects of the β25–35 peptide only. Our previous study demonstrated that β
Acknowledgements
Special thanks to Cathy Martens (Department of Veterinary Anatomy) and Bruce Pesch (National Animal Disease Center) for their technical support. This study was supported by a professional advancement grant from the Iowa State University Graduate College, Graduate Student Senate, and Department of Veterinary Anatomy.
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