Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system
Introduction
Epidemiological and pathological evidence indicates that inflammation contributes to the deterioration in several neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and multiple sclerosis [7], [16], [26], [45], [46], [57], [59], [70]. Elevated levels of inflammatory cytokines in these diseases suggest that they are actively involved in these degenerative processes. Supporting evidence comes from epidemiological studies indicating that gene polymorphisms that enhance expression of certain cytokines increase the risk of AD [38], [47] and PD [48], [69] or modify the age of onset in multiple sclerosis [28], [75].
Microglial cells are significant generators of inflammatory cytokines. They also possess appropriate receptors, which permit them to respond to cytokine stimulation [37]. Microglial activation is beneficial under most circumstances, but overactivation can be damaging to host tissue. Activated microglia may secrete such potentially neurotoxic materials as glutamate, quinolinic acid, proteases, reactive oxygen intermediates and other as yet unidentified toxins [17], [23], [30], [35], [63], [73].
In previous studies we showed that interferon-γ (IFN-γ) stimulates human microglia, or microglia-like human THP-1 cells, to the point where their secretions cause moderate toxicity towards both undifferentiated and retinoic acid-differentiated human neuroblastoma SH-SY5Y cells. This toxicity is enhanced significantly by the addition of bacterial lipopolysaccharide (LPS) [32]. Under such conditions 100% killing of neurons can be achieved.
Since IFN-γ and LPS are not produced by resident brain cells, we undertook this study to determine whether maximum microglial toxicity could be achieved by combinations of inflammatory mediators known to be produced within the brain. We further wished to explore whether endogenous anti-inflammatory products could prevent such toxicity. We show that under the conditions we utilized, human THP-1 monocytic cell toxicity towards SH-SY5Y neuroblastoma cells is not significantly induced by mediators acting alone. A cooperative action of at least two different cytokines is required. Such combinations include IFN-γ with tumor necrosis factor-α (TNF-α); IFN-γ with interleukin (IL) 1α or IL-1β in the presence of TNF-α; and IL-6 with TNF-α. Human microglial cells obtained from surgical tissues were more sensitive than THP-1 cells to inflammatory cytokine stimulation, with a maximal effect occurring after shorter incubation times and with lower cell numbers.
Of the potential inhibitors tested, only IL-4 was able to inhibit THP-1 cell toxicity, while IL-10 was ineffective. Transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF) enhanced toxicity under some experimental conditions.
Section snippets
Reagents
The following human recombinant cytokines were purchased from PeproTech Canada (Ottawa, Ont., Canada): IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-10, TGF-β1 and TNF-α. Human recombinant bFGF (FGF-2) was from Invitrogen (Burlington, Ont., Canada). Bacterial lipopolysaccharide (LPS, from Escherichia coli 055:B5) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were obtained from Sigma (St. Louis, MO, USA). All reagents were of the highest purity available.
Cell culture
The human monocytic THP-1
Results
Previously, we and others have shown that human microglial cells, or human microglial-like THP-1 cells secrete neurotoxic substances when highly activated [10], [17], [32]. In order to determine the potential of various activating agents to induce toxic secretions, we tested combinations of known inflammatory mediators as described under Section 2 as stimulants of THP-1 cells and human microglia.
Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 demonstrate the results obtained by combining various
Discussion
We used THP-1 cells as models of human macrophages and microglia for most of the experiments in this study. THP-1 cells, being derived from human monocytes, have properties comparable to other mononuclear phagocytes [10], [17], [24], [65], [74], [79]. These cells are able to secrete and respond to such cytokines as IL-1α, IL-1β, IL-6, IL-8, IL-12 and TNF-α [54], [72], [79] implying the presence of appropriate cytokine receptors on this cell line. Human adrenergic neuroblastoma SH-SY5Y cells
Acknowledgments
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/CIHR/Astra Zeneca, Canada.
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