Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system

https://doi.org/10.1016/j.neurobiolaging.2004.06.012Get rights and content

Abstract

Neuroinflammatory processes are thought to be a significant factor in the pathology of a number of degenerative neurological diseases. A variety of cytokines influence inflammatory levels. Here we show that a cooperative action of two or more cytokines is required to induce significantly human microglial and monocytic THP-1 cell toxicity towards SH-SY5Y neuroblastoma cells. Such toxicity was induced by the following combinations: interferon-γ (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. Toxicity induced by the various stimulatory combinations was not accompanied by an increased nitrite production. Of the potential inhibitors tested, IL-4 downregulated the toxic action of microglia when applied to THP-1 cells either before stimulation or 24 h after stimulation. Toxicity was not inhibited by IL-10, and was even enhanced by transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF). These data suggest that antagonists of cytokine receptors, as well as inhibitors of their intracellular pathways may be effective anti-inflammatory agents.

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.

References (80)

  • G.H. Jeohn et al.

    Synergistic neurotoxic effects of combined treatments with cytokines in murine primary mixed neuron/glia cultures

    J Neuroimmunol

    (1998)
  • O.H. Kantarci et al.

    A population-based study of IL-4 polymorphisms in multiple sclerosis

    J Neuroimmunol

    (2003)
  • I.M. Kerr et al.

    Of JAKs, STATs, blind watchmakers, jeeps and trains

    FEBS Lett

    (2003)
  • Y. Kitamura et al.

    Interleukin-4 inhibited mRNA expression in mixed rat glial and in isolated microglial cultures

    J Neuroimmunol

    (2000)
  • A. Klegeris et al.

    Activation of macrophages by Alzheimer β amyloid peptide

    Biochem Biophys Res Commun

    (1994)
  • A. Klegeris et al.

    Regulation of glutamate in cultures of human monocytic THP-1 and astrocytoma U-373 MG cells

    J Neuroimmunol

    (1997)
  • S. Kumar et al.

    Intracellular signaling pathways as a target for the treatment of rheumatoid arthritis

    Curr Opin Pharmacol

    (2001)
  • F. Licastro et al.

    Interleukin-6 gene alleles affect the risk of Alzheimer's disease and levels of the cytokine in blood and brain

    Neurobiol Aging

    (2003)
  • M.U. Martin et al.

    Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family

    Biochim Biophys Acta

    (2002)
  • P.L. McGeer et al.

    Association of interleukin-1β polymorphisms with idiopathic Parkinson's disease

    Neurosci Lett

    (2002)
  • M. McMillian et al.

    Selective killing of cholinergic neurons by microglial activation in basal forebrain mixed neuronal/glial cultures

    Biochem Biophys Res Commun

    (1995)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

    J Immunol Methods

    (1983)
  • R.E. Mrak et al.

    Glial cytokines in Alzheimer's disease: review and pathogenic implications

    Hum Pathol

    (1995)
  • S. Pahlman et al.

    Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation

    Cell Differ

    (1984)
  • L.C. Platanias et al.

    Signaling pathways activated by interferons

    Exp Hematol

    (1999)
  • J. Prieto et al.

    Regulated expression of integrins and other adhesion molecules during differentiation of monocytes into macrophages

    Cell Immunol

    (1994)
  • A.B. Roberts

    TGF-β signaling from receptors to the nucleus

    Microbes Infect

    (1999)
  • J. Sanceau et al.

    Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NFκB, and Sp1 transcription factors

    J Biol Chem

    (1995)
  • T. Schulte et al.

    Polymorphisms in the interleukin-1 alpha and beta genes and the risk for Parkinson's disease

    Neurosci Lett

    (2002)
  • E. Stylianou et al.

    Interleukin-1

    J Biochem Cell Biol

    (1998)
  • A.M. Szczepanik et al.

    IL-4, IL-10 and IL-13 modulate Aβ(1–42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line

    J Neuroimmunol

    (2001)
  • K. Vandenbroeck et al.

    Occurrence and clinical relevance of an interleukin-4 gene polymorphism in patients with multiple sclerosis

    J Neuroimmunol

    (1997)
  • D.G. Walker et al.

    Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia

    Neurobiol Aging

    (2001)
  • F.X. Zhang et al.

    Bacterial lipopolysaccharide activates nuclear factor-κB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

    J Biol Chem

    (1999)
  • L. Attisano et al.

    Signal transduction by the TGF-β superfamily

    Science

    (2002)
  • G. Chen et al.

    TNF-R1 signaling: a beautiful pathway

    Science

    (2002)
  • H. Chen et al.

    Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease

    Arch Neurol

    (2003)
  • C.A. Colton et al.

    Microglial contribution to oxidative stress in Alzheimer's disease

    Ann NY Acad Sci

    (2000)
  • C.K. Combs et al.

    Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of β-amyloid and prion proteins

    J Neurosci

    (1999)
  • D. Groot CJA et al.

    Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization

    J Neurosci Res

    (1997)
  • Cited by (51)

    • Dietary fats modulate neuroinflammation in mucin 2 knock out mice model of spontaneous colitis

      2022, Biochimica et Biophysica Acta - Molecular Basis of Disease
    • Pattern recognition receptors mediate pro-inflammatory effects of extracellular mitochondrial transcription factor A (TFAM)

      2018, Molecular and Cellular Neuroscience
      Citation Excerpt :

      The pro-inflammatory CNS effects of TFAM, shown here, warrant further investigation of this mitochondrial DAMP in neuroinflammatory conditions. To investigate the mechanisms underlying the pro-inflammatory activity of extracellular TFAM, we returned to the previously reported human in vitro cell culture studies using THP-1 cells to model human microglia (Klegeris et al., 2005; Gouveia et al., 2017). TFAM alone induced the release of MCP-1 from THP-1 cells (Fig. 6).

    View all citing articles on Scopus
    View full text