Sung Joong Lee

Adhesion molecule expression and regulation on cells of the central nervous system

Cellular adhesion molecules were initially defined as cell surface structures mediating cell-cell and cell-extracellular matrix (ECM) interactions. Adhesion molecules involved in immune responses have been classified into three families according to their structure: selectins, immunoglobulin (Ig) superfamily, and integrins. It has been well documented that adhesion molecules of these family members (E-selectin, ICAM-1, and VCAM-1) are expressed on brain microvessel endothelial cells in active lesions of multiple sclerosis (MS) brain. In addition, accumulating data show that glial cells can express some of these adhesion molecules upon activation: astrocytes can express ICAM-1, VCAM-1, and E-selectin, and microglia express ICAM-1 and VCAM-1. In vitro studies show that these adhesion molecules are actively regulated by several cytokines which have relevance to MS or experimental autoimmune encephalomyelitis (EAE). In addition, soluble forms of adhesion molecules have been found in the serum and cerebrospinal fluid (CSF) of MS patients, and may be useful diagnostically. Experimental therapy of EAE using antibodies against several adhesion molecules clearly shows that adhesion molecules are critical for the pathogenesis of EAE. Thus far, the function of adhesion molecule expression on brain endothelial and glial cells has not been clearly elucidated. Studies on the possible role of adhesion molecules on brain endothelial and glial cells will be helpful in understanding their involvement in immune responses in the central nervous system (CNS).

NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.

Differential Regulation and Function of Fas Expression on Glial Cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-α or IFN-γ stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-α and IFN-γ induced Fas mRNA by ∼20-fold. STAT-1α and NF-κB activation are involved in IFN-γ- or TNF-α-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-β inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, ∼20% of cells underwent Fas-mediated cell death, which increased to ∼60% when cells were pretreated with either TNF-α or IFN-γ. TGF-β treatment inhibited Fas-mediated cell death of TNF-α- or IFN-γ-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1β (MIP-1β), MIP-1α, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.

ICAM-1-Induced Expression of Proinflammatory Cytokines in Astrocytes: Involvement of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways

ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer’s disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1β and IL-1α protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1α and IL-1β expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

Role of microglial IKKβ in kainic acid-induced hippocampal neuronal cell death

Microglial cells are activated during excitotoxin-induced neurodegeneration. However, the in vivo role of microglia activation in neurodegeneration has not yet been fully elucidated. To this end, we used Ikkbeta conditional knockout mice (LysM-Cre/Ikkbeta(F/F)) in which the Ikkbeta gene is specifically deleted in cells of myeloid lineage, including microglia, in the CNS. This deletion reduced IkappaB kinase (IKK) activity in cultured primary microglia by up to 40% compared with wild-type (Ikkbeta(F/F)), and lipopolysaccharide-induced proinflammatory gene expression was also compromised. Kainic acid (KA)-induced hippocampal neuronal cell death was reduced by 30% in LysM-Cre/Ikkbeta(F/F) mice compared with wild-type mice. Reduced neuronal cell death was accompanied by decreased KA-induced glial cell activation and subsequent expression of proinflammatory genes such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Similarly, neurons in organotypic hippocampal slice cultures (OHSCs) from LysM-Cre/Ikkbeta(F/F) mouse brain were less susceptible to KA-induced excitotoxicity compared with wild-type OHSCs, due in part to decreased TNF-alpha and IL-1beta expression. Based on these data, we concluded that IKK/nuclear factor-kappaB dependent microglia activation contributes to KA-induced hippocampal neuronal cell death in vivo through induction of inflammatory mediators.

Analysis of cellular and behavioral responses to imiquimod reveals a unique itch pathway in transient receptor potential vanilloid 1 (TRPV1)-expressing neurons

Despite its clinical importance, the mechanisms that mediate or generate itch are poorly defined. The identification of pruritic compounds offers insight into understanding the molecular and cellular basis of itch. Imiquimod (IQ) is an agonist of Toll-like receptor 7 (TLR7) used to treat various infectious skin diseases such as genital warts, keratosis, and basal cell carcinoma. Itch is reportedly one of the major side effects developed during IQ treatments. We found that IQ acts as a potent itch-evoking compound (pruritogen) in mice via direct excitation of sensory neurons. Combined studies of scratching behavior, patch-clamp recording, and Ca2+ response revealed the existence of a unique intracellular mechanism, which is independent of TLR7 as well as different from the mechanisms exploited by other well-characterized pruritogens. Nevertheless, as for other pruritogens, IQ requires the presence of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons for itch-associated responses. Our data provide evidence supporting the hypothesis that there is a specific subset of TRPV1-expressing neurons that is equipped with diverse intracellular mechanisms that respond to histamine, chloroquine, and IQ.

Transcriptional regulation of intercellular adhesion molecule-1 in astrocytes involves NF–κB and C/EBP isoforms

ICAM-1 is an inducible cell surface protein that is involved in cell extravasation into inflamed tissues as well as immune responses. ICAM-1 expression is upregulated by proinflammatory cytokines such as TNF-alpha and IL-1beta in numerous cell types including the astrocyte, which functions as an immune effector cell in the central nervous system (CNS). We investigated the mechanism by which the ICAM-1 gene is transcriptionally regulated in astrocytes in response to TNF-alpha and IL-1beta. Human ICAM-1 promoter constructs linked to the reporter gene luciferase were transiently transfected into astrocytes, stimulated with TNF-alpha and IL-1beta, and ICAM-1 promoter activity examined. We determined that binding sites for both NF-kappaB (-186 bp region) and C/EBP (-198 bp region) are involved in TNF-alpha and IL-1beta-mediated ICAM-1 upregulation. Electrophoretic mobility shift assays using antibodies against NF-kappaB and C/EBP isoforms showed that p65 homodimers and p65/p50 heterodimers bind to the NF-kappaB site, and C/EBPdelta homodimers and C/EBPbeta/delta heterodimers bind to the C/EBP site. Transient transfection assays demonstrated that overexpression of p65 could transactivate the promoter activity of ICAM-1 reporter constructs. p50 overexpression had no effect on the basal levels of ICAM-1 transcription, but inhibited, in a dose dependent manner, p65 mediated transcription. Overexpression of C/EBPbeta slightly inhibited basal levels of ICAM-1 promoter activity, however, when C/EBPbeta and p65 were cotransfected, C/EBPbeta completely abolished the transactivating effects of p65. These results demonstrate that cytokine-induced ICAM-1 expression in astrocytes is regulated by interactions between NF-kappaB and C/EBP transcription factors.

Toll-Like Receptors: Sensor Molecules for Detecting Damage to the Nervous System

Toll-like receptors (TLRs) are type I transmembrane signaling molecules that are expressed in cells of the innate immune system. In these cells, TLRs function as pattern recognition receptors (PRR) that recognize specific molecular patterns derived from microorganisms. Upon activation, TLRs trigger a cascade of intracellular signaling pathways in innate immune cells, leading to the induction of inflammatory and innate immune responses, which in turn regulate adaptive immune responses. In the nervous system, different members of the TLR family are expressed on glial cells (astrocytes, microglia, oligodendrocytes, and Schwann cells) and neurons. Recently, increasing evidence has supported the idea that TLRs also recognize endogenous molecules that are released from damaged tissue, thereby regulating inflammatory responses and subsequent tissue repair. These findings imply that TLRs on glial cells may also be involved in the inflammatory response to tissue damage in the nervous system. In this review, we discuss recent studies on TLR expression in the cells of the nervous system and their roles in acute neurological disorders involving tissue damage such as strokes, traumatic spinal cord and brain injuries, and peripheral nerve injuries.

Microglial toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.

Transcriptional regulation of the intercellular adhesion molecule-1 gene by proinflammatory cytokines in human astrocytes

Intercellular adhesion molecule‐1 (ICAM‐1) expression is upregulated by cytokines such as tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and interferon‐γ (IFN‐γ) in numerous cell types including the astrocyte, which functions as an immunoregulatory cell within the central nervous system. We investigated the mechanism by which ICAM‐1 is transcriptionally regulated by proinflammatory cytokines in human fetal astrocytes. TNF‐α and IL‐1β enhanced ICAM‐1 expression at both the mRNA and protein levels, while IFN‐γ had a modest enhancing effect. However, a synergistic response was noted when IFN‐γ was added with either TNF‐α or IL‐1β. Using human ICAM‐1 deletion constructs and linker scanning mutants, we determined that the NF‐κB element (‐186 bp region) is critical for both TNF‐α‐ and IL‐1β‐mediated ICAM‐1 expression, while the IFN‐γ activation sequence (GAS) element at ‐75 bp region is important for IFN‐γ stimulation. The synergistic effect between TNF‐α and IFN‐γ is dependent on both NF‐κB and GAS elements. Upon TNF‐α and IL‐1β stimulation, p65 homodimers and p65/p50 heterodimers bind to the NF‐κB site, and STAT‐1α homodimers bind to the GAS element upon IFN‐γ stimulation. Transient transfection assays demonstrated that overexpression of the p65 protein transactivated the promoter activity of an ICAM‐1 reporter construct, while p50 overexpression inhibited, in a dose‐dependent manner, p65‐mediated ICAM‐1 expression. These data collectively suggest that in human astrocytes, the p65 homodimer is responsible for ICAM‐1 upregulation upon TNF‐α or IL‐1β stimulation, and that IFN‐γ enhancement of ICAM‐1 involves activation of STAT‐1α homodimers. GLIA 25:21–32, 1999.