Differential neutrophil infiltration contributes to regional differences in brain inflammation in the substantia nigra pars compacta and cortex.

Brain inflammation is a suggested risk factor for neurodegenerative disease. Interestingly, severe inflammation in the substantia nigra pars compacta (SNpc) accelerates the onset and progression of Parkinson's disease. In this study, we examined the underlying mechanisms of severe inflammation in the SNpc by comparing the inflammatory process with that in the cortex. In intact brain, the densities of CD11b(+) microglia were similar in the SNpc and cortex. However, lipopolysaccharide injection enhanced the CD11b(+) cell number in the SNpc, but not in the cortex. Previously, we reported that CD11b and myeloperoxidase (MPO) double-positive neutrophils infiltrate the SNpc following LPS injection (GLIA 55:1577-88). Notably, the MPO(+) neutrophil number increased dramatically in the SNpc, but only slightly in the cortex. The extent of neutrophil infiltration appeared to correlate with neuronal damage. We confirmed that loss of neurons in the SNpc was significantly reduced in neutropenic rats versus normal rats following LPS injection. In addition, the densities of astrocytes were much lower in the intact SNpc, compared with the cortex. Furthermore, after LPS injection, damage of endothelial cells and astrocytes, and blood-brain barrier (BBB) permeability was more pronounced in the SNpc. These results collectively suggest that excessive neutrophil infiltration and environmental factors, such as lower astrocyte density and higher BBB permeability, contribute to severe inflammation and neuronal death in the SNpc.

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-1beta, 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 PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) 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 PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma 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, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.

Ionizing radiation induces astrocyte gliosis through microglia activation.

The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.

Anti-inflammatory roles of retinoic acid in rat brain astrocytes: Suppression of interferon-gamma-induced JAK/STAT phosphorylation.

The anti-inflammatory effect of retinoic acid (RA) has been investigated for several decades. However, the underlying mechanisms responsible for this effect are largely unknown. In this study, we demonstrate that 9-cis-RA (cRA) and all-trans-RA (tRA) inhibit interferon-gamma (IFN-gamma)-induced inflammatory responses in astrocytes. In primary cultured rat brain astrocytes and C6 astroglioma cells, both cRA and tRA decreased IFN-gamma-induced expression of interferon regulatory factor-1. Both RA isoforms also reduced IFN-gamma-induced activation of signal transducers and activators of transcription (STAT)1, STAT3, Janus kinase (JAK)1, and JAK2. This inhibitory effect was significant when cells were pre-treated with RA prior to IFN-gamma. Furthermore, the effect of pre-treated RA was abolished in the presence of cycloheximide, indicating a requirement for de novo protein synthesis. Suppressors of cytokine signaling (SOCS), which are negative regulators of the JAK/STAT pathway, may be candidate mediators of the anti-inflammatory function of RA. Both cRA and tRA induced SOCS3 mRNA expression. These results suggest that RA induces an anti-inflammatory effect by suppressing the activation of the JAK/STAT pathway in IFN-gamma-treated astrocytes. SOCS3 may be at least one of the mechanisms that mediate the anti-inflammatory roles of RA.

Nordihydroguaiaretic acid inhibits IFN-gamma-induced STAT tyrosine phosphorylation in rat brain astrocytes.

The Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal cascades are major pathways that mediate the inflammatory functions of interferon-gamma (IFN-gamma), an important pro-inflammatory cytokine. Therefore, regulation of JAK/STAT signaling should modulate IFN-gamma-mediated inflammation. In this study, we found that nordihydroguaiaretic acid (NDGA), a well-known lipoxygenase (LO) inhibitor, suppressed IFN-gamma-induced inflammatory responses in brain astrocytes. In the presence of NDGA, interferon regulatory factor-1 expression was significantly reduced. Expression of monocyte chemotactic protein-1 and interferon-gamma inducible protein-10 mRNA in response to IFN-gamma was significantly suppressed in the presence of NDGA, as was tyrosine-phosphorylation of JAK and STAT. However, the 5-LO products, leukotriene B(4) (LTB(4)) and leukotriene C(4), were not detected in cells treated with IFN-gamma, indicating that the effect of NDGA seemed to be independent of 5-LO inhibition. In addition, two other 5-LO inhibitors (Rev5901 and AA861) did not mimic the effect of NDGA, and the 5-LO metabolites, 5-hydroxyeicosatetraenoic acid and LTB(4), were unable to reverse NDGA-driven suppression of STAT activation or affect basal STAT phosphorylation. Taken together, these results suggest that NDGA regulates IFN-gamma-mediated inflammation through mechanisms unrelated to the inhibition of 5-LO.

Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes.

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-gamma-induced inflammatory responses in rat astrocytes. We revealed that IFN-gamma rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-gamma-responsive transcriptional responses. Compared with levels in control cells, IFN-gamma-induced IFN-gamma-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-gamma-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-gamma-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-gammaRalpha subunit and IFN-gamma-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-gammaRalpha and augment the interaction of IFN-gammaR with either STAT1 or STAT3 in response to IFN-gamma. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-gamma-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-gamma-responsive inflammatory signaling in rat astrocytes.

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 A(2) (cPLA(2)), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA(2), 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 E(2) (PGE(2)) and leukotriene B(4) (LTB(4)), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE(2) and LTB(4) generated by COX and LO, mediate CIS expression. Since interferon-gamma (IFN-gamma)-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 cPLA(2), COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.

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)-1beta, tumor necrosis factor-alpha (TNF-alpha), 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-1beta, TNF-alpha, and iNOS in ganglioside-treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Go6976, Ro31-8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC-alpha, betaII, and delta and NADPH oxidase p67(phox) 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-kappaB, an essential transcription factor that mediates the expression of IL-1beta, TNF-alpha, 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-kappaB.

Thrombin induces suppressor of cytokine signaling 3 expression in brain microglia via protein kinase Cdelta activation.

Microglia (brain macrophages) are activated upon brain damage. In this study, we demonstrated that thrombin, a pro-inflammatory stimulator of microglia, induced expression of suppressors of cytokine signaling (SOCS) in microglia. RT-PCR analysis and Northern blot analysis showed that thrombin induced SOCS3 mRNA expression. Further experiments indicated SOCS3 expression was not affected by cycloheximide, indicating thrombin directly stimulated SOCS3 transcript expression without de novo protein synthesis. We investigated whether PKCdelta played a role in thrombin-stimulated SOCS3 expression. We found that thrombin activated PKCdelta, and the specific inhibitor of PKCdelta, rottlerin, significantly suppressed thrombin-stimulated SOCS3 expression. In thrombin-pretreated cells, microglial activation-induced by another inflammatory stimulator, lipopolysaccharide, was attenuated compared to that in non-pretreated cells. These results suggest thrombin induce not only proinflammatory mediators but also negative feedback regulators of inflammation, SOCS, which prevent prolonged inflammatory reactions in microglia.