Myung-Soon Yang

Astrocytes Induce Hemeoxygenase-1 Expression in Microglia: A Feasible Mechanism for Preventing Excessive Brain Inflammation

Microglia are the major inflammatory cells in the brain, in which microglial inflammatory responses are modulated by interactions with other brain cells. Here, we show that astrocytes, the most abundant cells in the brain, can secrete one or more factors capable of modulating microglial activation by regulating the microglial levels of reactive oxygen species (ROS). Treatment of microglia with astrocyte culture-conditioned media (ACM) increased the expression level and activity of hemeoxygenase-1 (HO-1). ACM also induced nuclear translocation of the nuclear factor E2-related factor 2 transcription factor, increased the binding activity of the antioxidant response element (ARE), and enhanced HO-1 promoter activity in an ARE-dependent manner. Furthermore, treatment with ACM suppressed interferon-γ (IFN-γ)-induced ROS production, leading to reduced inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release. In agreement with these results, mimickers of HO-1 products, such as bilirubin, ferrous iron, and a carbon monoxide-releasing molecule, reduced IFN-γ-induced iNOS expression and/or NO release. Finally, we found that the active component(s) in ACM was heat labile and smaller than 3 kDa. Together, these results suggest that astrocytes could cooperate with microglia to prevent excessive inflammatory responses in the brain by regulating microglial expression of HO-1 and production of ROS.

Impaired inflammatory responses in murine Lrrk2-knockdown brain microglia.

LRRK2, a Parkinsons disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.

Gangliosides activate microglia via protein kinase C and NADPH oxidase

Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)‐1β, tumor necrosis factor‐α (TNF‐α), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL‐1β, TNF‐α, and iNOS in ganglioside‐treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Gö6976, Ro31‐8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC‐α, βII, and δ and NADPH oxidase p67phox translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside‐induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside‐induced activation of NF‐κB, an essential transcription factor that mediates the expression of IL‐1β, TNF‐α, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF‐κB.

Microglia Expressing Interleukin-13 Undergo Cell Death and Contribute to Neuronal Survival In Vivo

How to minimize brain inflammation is pathophysiologically important, since inflammation induced by microglial activation can exacerbate brain damage. In the present report, we show that injection of lipopolysaccharide (LPS) into the rat cortex led to increased levels of interleukin‐13 (IL‐13) and to IL‐13 immunoreactivity, followed by the substantial loss of microglia at 3 days post‐LPS. IL‐13 levels in LPS‐injected cortex reached a peak at 12 h post‐injection, remained elevated at 24 h, and returned to basal levels at day 4. In parallel, IL‐13 immunoreactivity was detected as early as 12 h post‐LPS and maintained up to 24 h; it disappeared at 4 days. Surprisingly, IL‐13 immunoreactivity was detected exclusively in microglia, but not in neurons or astrocytes. Following treatment with LPS in vitro, IL‐13 expression was also induced in microglia in the presence of neurons, but not in the presence of astrocytes or in cultured pure microglia alone. In experiments designed to determine the involvement of IL‐13 in microglia cell death, IL‐13‐neutralizing antibodies significantly increased survival of activated microglia at 3 days post‐LPS. Consistent with these results, the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor‐α (TNF‐α) was sustained in activated microglia and neuronal cell death was consequently increased. Taken together, the present study is the first to demonstrate the endogenous expression of IL‐13 in LPS‐activated microglia in vivo, and to demonstrate that neurons may be required for IL‐13 expression in microglia. Our data strongly suggest that IL‐13 may control brain inflammation by inducing the death of activated microglia in vivo, resulting in an enhancement of neuronal survival.

Interleukin-13 and -4 induce death of activated microglia

When the brain suffers injury, microglia migrate to the damaged sites and become activated. These activated microglia are not detected several days later and the mechanisms underlying their disappearance are not well characterized. In this study, we demonstrate that interleukin (IL)‐13, an anti‐inflammatory cytokine, selectively induces cell death of activated microglia in vitro. Cell death was detected 4 days after the coaddition of IL‐13 with any one of the microglial activators, lipopolysaccharide (LPS), ganglioside, or thrombin. This cell death occurred in a time‐dependent manner. LPS, ganglioside, thrombin, or IL‐13 alone did not induce cell death. Among anti‐inflammatory cytokines, IL‐4 mimicked the effect of IL‐13, while TGF‐β did not. Cells treated with IL‐13 plus LPS, or IL‐13 plus ganglioside, showed the characteristics of apoptosis when analyzed by electron microscopy and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling staining. Electron micrographs also showed microglia engulfing neighboring dead cells. We propose that IL‐13 and IL‐4 induce death of activated microglia, and that this process is important for prevention of chronic inflammation that can cause tissue damage. GLIA 38:273–280, 2002.

Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain

Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)‐injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule‐1, Iba‐1), as well as a general marker for these inflammatory cells (CD11b). CD11b‐immunopositive (CD11b+) cells and Iba‐1+ cells displayed similar behavior in intact and LPS‐injected brain at 6 h after the injection. Interestingly, however, CD11b+ cells and Iba‐1+ cells displayed significantly different behavior at 12 h: Iba‐1+ cells disappeared while CD11b+ cells became round in shape. We found that CD11b/Iba‐1‐double positive (CD11b+/Iba‐1+) ramified microglia died within 6 h after LPS injection. The round CD11b+ cells detected at 12 h were MPO+. These CD11b+/MPO+ cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO+ neutrophils expressed inducible nitric oxide synthase, interleukin‐1β, cyclooxygenase‐2, and monocyte chemoattractant protein‐1, but died within 18 h. CD11b+ cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba‐1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS‐injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain.

Interleukin-13 Enhances Cyclooxygenase-2 Expression in Activated Rat Brain Microglia: Implications for Death of Activated Microglia

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE2 and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-β, neither induced microglial death nor enhanced COX-2 expression or PGE2 or 15d-PGJ2 production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE2 and 15d-PGJ2 induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor γ (ciglitazone) mimicked the effect of PGE2 and 15d-PGJ2, and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor γ antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE2 and 15d-PGJ2, caused microglial death, which terminates brain inflammation.

Wortmannin enhances lipopolysaccharide-induced inducible nitric oxide synthase expression in microglia in the presence of astrocytes in rats.

Microglia, the primary inflammatory cells in the brain, are activated upon brain injury. Activated microglia produce nitric oxide (NO), a major toxin to neuronal cells. It has been reported that astrocytes inhibit microglial activation. In this study, we found that wortmannin, a natural inhibitor of phosphatidylinositol 3-kinase, significantly increased lipopolysaccharide (LPS)-induced NO release and inducible nitric oxide synthase (iNOS) expression in microglia in the presence but not in the absence of astrocytes. In response to LPS even in the presence of wortmannin, iNOS immunoreactivity was detected in microglia but not in astrocytes. These results suggest that astrocytes could regulate microglia-mediated brain inflammation by inhibiting microglial NO release/iNOS expression via a wortmannin-sensitive mechanism.

Multiple mechanisms that prevent excessive brain inflammation

Inflammation of the injured brain has a double‐edged effect. Inflammation protects the brain from infection, but it aggravates injury. Furthermore, brain inflammation is considered a risk factor for neurodegenerative disorders, such as Alzheimers and Parkinsons diseases. Emerging evidence supports the activation of negative regulatory mechanisms during this process to prevent prolonged and extensive inflammation. The inflammatory stimulators themselves or products of inflammatory cells may induce the expression of negative feedback regulators, such as suppressor of cytokine signaling (SOCS)‐family proteins, antioxidant enzymes, and antiinflammatory cytokines. Furthermore, death of activated microglia (major inflammatory cells in the brain) may regulate brain inflammation. Astrocytes, the most abundant cells in the brain, may also act in preventing microglial overactivation. Therefore, we propose that the extent and duration of brain inflammation is tightly regulated through the cooperation of multiple mechanisms to maximize antipathogenic effects and minimize tissue damage.

Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A2, cyclooxygenase, and lipoxygenase

Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000 . J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokine‐induced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI‐P97 and WHI‐P154) had little effect on thrombin‐induced CIS expression. In contrast, cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA2, cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N‐acetyl‐cysteine [NAC] and trolox) reduced thrombin‐induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), products of COX and LO, respectively, potentiated thrombin‐induced CIS expression, indicating that ROS, and PGE2 and LTB4 generated by COX and LO, mediate CIS expression. Since interferon‐γ (IFN‐γ)‐induced GAS‐luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS‐transfected cells compared to control vector‐transfected cells, CIS could have anti‐inflammatory activity. These data suggest that thrombin‐stimulation of ROS and prostaglandin and leukotriene production via the cPLA2, COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.