Elsevier

Neurobiology of Aging

Volume 23, Issue 5, September–October 2002, Pages 787-794
Neurobiology of Aging

Cyclooxygenase and 5-lipoxygenase inhibitors protect against mononuclear phagocyte neurotoxicity

https://doi.org/10.1016/S0197-4580(02)00021-0Get rights and content

Abstract

Neuroinflammation and oxidative stress are believed to be contributing factors to neurodegeneration in normal aging, as well as in age-related neurological disorders. Reactive microglia are found in increased numbers in aging brain and are prominently associated with lesions in such age-related degenerative conditions as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). In vitro, stimulated microglia or microglial-like cells secrete neurotoxic materials and are generators of free radicals through their respiratory burst system. Agents that suppress microglial activation are therefore candidates for neuroprotection. We have developed quantitative in vitro assays for measuring neurotoxicity of microglia or other mononuclear phagocytes. Neuronal like SH-SY5Y cells are cultured in supernatants from activated cells of the human monocytic THP-1 line and their survival is followed. Respiratory burst is directly measured on the activated cells. We tested inhibitors of the cyclooxygenase (COX) or the 5-lipoxygenase (5-LOX) pathways as possible neuroprotective agents. The COX pathway generates inflammatory prostaglandins, while the 5-LOX pathway generates inflammatory leukotrienes. We found that inhibitors of both these pathways suppressed neurotoxicity in a dose-dependent fashion. They included the COX-1 inhibitor indomethacin; the COX-2 inhibitor NS-398; the mixed COX-1/COX-2 inhibitor ibuprofen; the nitric oxide (NO) derivatives of indomethacin, ibuprofen and flurbiprofen; the 5-LOX inhibitor REV 5901; and the 5-LOX activating protein (FLAP) inhibitor MK-886. The FLAP inhibitor also reduced respiratory burst activity in a more potent manner than indomethacin. Combinations of COX and 5-LOX inhibitors were more effective than single inhibitors. The data suggest that both COX inhibitors and 5-LOX inhibitors may be neuroprotective in vivo by suppressing toxic actions of microglia/macrophages, and that combinations of the two might have greater therapeutic potential than single inhibitors of either class.

Introduction

The aging process is accompanied by a spectrum of degenerative changes which affect all organs of the body. The brain is particularly vulnerable since neurons are postmitotic cells, and their degeneration results in irretrievable loss of function. The vast majority of elderly patients requiring institutional care suffer from a major brain disorder. Developing methods of protecting neurons from age-related deterioration is clearly an important goal for neuroscience research.

Reactive microglia are a prime focus for attention. They secrete neurotoxic substances when stimulated in vitro, and their respiratory burst system generates large numbers of toxic oxygen free radicals. They are seen in large numbers in association with the lesions of a number of degenerative neurological diseases including Alzheimer’s disease (AD) [35], Parkinson’s disease (PD) [31], amyotrophic lateral sclerosis (ALS) [20] and multiple sclerosis (MS) [9]. They are also seen in increased numbers in the brains of normal elderly individuals [15] as well as aged monkeys [45], [47].

We have developed specific assays to evaluate microglial toxicity in a quantitative fashion [23], [25]. We have also measured their respiratory burst activity directly by polarographic techniques [21], [22]. We have assayed their toxic secretions by exposing neuronal type cells to their culture supernatants. Neurotoxicity is measured through lactate dehydrogenase (LDH) release, or, conversely, neuronal survival is measured by retention of the ability to reduce formazan dyes. These assays are a convenient way of assessing the potency of agents in suppressing microglial toxicity, thereby reducing their apparent capacity to cause neurodegeneration. We have previously reported that non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), demonstrate potency in these assays in a dose-dependent fashion [25]. Such results are consistent with their perceived neuroprotective role in vivo.

Respiratory burst response of microglia/macrophages could be relevant to AD since it has been shown that components of the NADPH oxidase enzyme complex responsible for the respiratory burst are upregulated and activated in AD [46], [50], [51]. Furthermore in vitro experiments show that Alzheimer amyloid β protein can activate NADPH-dependent oxidase, which is the key enzyme responsible for the respiratory burst [8].

The arachidonic acid substrate for COX is also the substrate for lipoxygenase (LOX). In the periphery, the 5-lipoxygenase (5-LOX) pathway generates leukotrienes which are known to be inflammatory mediators [12], [42]. The role of the 5-LOX pathway in brain is almost unexplored. If products of 5-LOX activity are significant activators of microglia, or if they are harmful to neurons, then inhibitors of 5-LOX might have neuroprotective effects. Since 5-LOX and COX are companion pathways using arachidonic acid as their common substrate (see Fig. 1), inhibitors of the two pathways might have additive, or even synergistic neuroprotective effects when used in combination.

Here, we review some of our previous data and present new data on the effects of inhibitors of the COX and 5-LOX pathways on these toxicity assays. The COX inhibitors chosen were the selective COX-1 inhibitor indomethacin, the selective COX-2 inhibitor NS-398, and the mixed COX-1/COX-2 inhibitor ibuprofen. In addition, the nitric oxide (NO) derivatives of the well known NSAIDs (NO-NSAIDs), indomethacin, ibuprofen and flurbiprofen, were tested before and after esterase treatment. These recently developed NO-NSAIDs have reduced gastrointestinal toxicity compared with their non-esterified counterparts [14]. It is therefore important to assess their relative anti-inflammatory activity compared with their parent compounds. The selective 5-LOX inhibitor REV 5901 and MK-886, the selective inhibitor of 5-LOX activating protein (FLAP) were chosen to evaluate the 5-LOX pathway. The data indicate that 5-LOX inhibitors may be neuroprotective agents in vivo, and that combinations of COX and 5-LOX inhibitors might be more effective than either type of agent alone. NO derivatives of NSAIDs acted in a highly comparable fashion to their parent drugs, and their activity in this assay increased only marginally by esterase treatment.

Section snippets

Reagents

The 5-LOX inhibitor REV 5901 was from Cayman Chemical (Ann Arbor, MI), the FLAP inhibitor MK-886 was from Biomol (Plymouth Meeting, PA), the NSAIDs ibuprofen and indomethacin were from Sigma (St. Louis, MO), the specific COX-2 inhibitor N-(2-cyclohexyloxyl-4-nitrophenyl) methanesulfonamide (NS-398) was from Tocris Cookson (St. Louis, MO). The following NO-NSAIDs were obtained from NICOX (Sophia Antipolis, France): NO-indomethacin (NCX 530), NO-ibuprofen (NCX 2103), and NO-flurbiprofen (NCX

Results

Stimulation of THP-1 cells by a combination of LPS and IFN-γ leads to the secretion of neurotoxins by these cells. Preliminary experiments showed that these toxins effectively killed neuronal SH-SY5Y cells that were either undifferentiated, or differentiated with retinoic acid treatment for 2 or 4 days. A standard experimental protocol using undifferentiated cells was chosen for the study of drug effects since under these conditions the spontaneous death rate of neuronal cells was somewhat

Discussion

Our results show that inhibitors of the 5-LOX pathway have similar effects to inhibitors of the COX pathway in suppressing toxic functions of activated human monocytic THP-1 cells, which were used as a model for human microglia/macrophages. The specific 5-LOX inhibitor REV 5901 and the FLAP inhibitor MK-886 both reduced neurotoxic secretions of monocytic THP-1 cells in a dose-dependent fashion, but were less potent than COX inhibitors (Fig. 2, Fig. 3). Interestingly, it appeared that

Acknowledgements

This work was supported by a grant from the Jack Brown and Family Alzheimer’s disease Research Fund, and by a grant from the Alzheimer Society of Canada/Astra Zeneca/CIHR.

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