Cytokine regulation of CD40 expression in fetal human astrocyte cultures

CD40 can participate in inflammatory processes after binding its cognate ligand (CD40L). We found that fetal human astrocytes constitutively express CD40 mRNA and protein. Upon incubating cultures with proinflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) or with lipopolysaccharide (LPS), CD40 expression was increased. No change in CD40 expression was noted in astrocyte cultures incubated with IL-6, HIV or gp41. Astrocytes also showed increased release of proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 after incubation with CD40L peptide. These observations suggest a role for CD40 in central nervous system (CNS) inflammation and that CD40/CD40L autocrine or paracrine pathways may mediate this role.     

CD14 expression by activated parenchymal microglia/macrophages and infiltrating monocytes following human traumatic brain injury

The immune response in the central nervous system (CNS) is under tight control of regulatory mechanisms, resulting in the establishment of immune privilege. CNS injury induces an acute inflammatory reaction, composed mainly of invading leukocytes and activated microglial cells/macrophages. The generation of this robust immune response requires binding of receptors such as CD14, a pattern recognition receptor of the immune system. CD14, a surface molecule of monocytic cells, is up-regulated after monocyte stimulation and is involved in cellular activation. To examine CD14 expression in human brain lesions we investigated sections of brains obtained at autopsy from 25 cases following closed traumatic brain injury (TBI) and 5 control brains by immunohistochemistry. Detection of CD14 in controls demonstrated constitutive expression by perivascular cells, but not in parenchymal microglial cells, equivalent to known expression pattern of ED2 in rats. Following TBI, numbers of CD14(+) cells in perivascular spaces and in the brain parenchyma increased in parallel within 1-2 days, both at the lesion and in adjacent perilesional areas. The number of CD14(+) cells in perivascular spaces and in the brain parenchyma reached maximum levels within 4-8 days and remained elevated until weeks after trauma. In contrast to activated parenchymal microglia/macrophages, resting parenchymal microglial cells lacked CD14. Thus, early CD14 expression constitutes an essential part of the acute inflammatory CNS response following trauma.     

HIV-1 expression protects macrophages and microglia from apoptotic death

Macrophages and microglia are the predominant cells infected with HIV-1 in the brain, yet the effects of productive HIV infection on the fate of these cells are poorly understood. In this study, we tested the hypothesis that HIV-1 expression influences cell death in infected macrophages and microglial cells. We detected apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) in the cerebral white matter of control and HIV encephalitis (HIVE) brains, and quantitatively analysed apoptotic cells with respect to their location (vessel-associated vs. parenchymal), CD68 expression, and HIV-1 p24 expression. There were more vessel-associated, but not more parenchymal, TUNEL+ cells in HIVE cases as compared to controls. Vessel-associated TUNEL+ cells were primarily endothelial cells (von Willebrand factor+) or macrophages (CD68+). TUNEL+/CD68+ cells were present in both control and HIVE cases in similar frequencies (2.1 +/- 0.7% vs. 1.9 +/- 0.7% of total CD68+ populations, respectively). In HIVE, TUNEL+/p24+ cells were 0.4 +/- 0.2% of the total p24+ cell population, which was lower than the frequency of TUNEL+/CD68+ cells (1.9 +/- 0.7%) in the total CD68+ macrophage population. These results suggest that HIV-1-infected macrophages and microglia are resistant to apoptosis, and may contribute to the formation of a central nervous system viral reservoir.     

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT - PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P<0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder, an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.     

Infiltrating CD14+ monocytes and expression of CD14 by activated parenchymal microglia/macrophages contribute to the pool of CD14+ cells in ischemic brain lesions

CD14, a key pattern recognition receptor of the innate immune system, is a surface molecule on monocytic cells involved in cellular activation. We investigated 18 autopsy cases of focal cerebral infarctions (FCI) by immunohistochemistry to examine CD14 expression following ischemia. Controls confirmed constitutive CD14 expression by few perivascular cells. In contrast to quiescent CD14- parenchymal microglial cells, following ischemia activated microglia/macrophages expressed abundant CD14. In FCI, CD14+ cells increased both in perivascular spaces and in brain parenchyma within 1-2.5 days and remained elevated until late stages. Early CD14 expression suggests an essential part of CD14 in the acute inflammatory response following stroke.     

CD1 expression is differentially regulated by microglia, macrophages and T cells in the central nervous system upon inflammation and demyelination

Expression of CD1 by microglia, macrophages and T cells was investigated ex vivo. In the healthy central nervous system (CNS), resident microglia, macrophages and T cells express levels of CD1 significantly lower than that expressed by splenic macrophages and T cells. During experimental autoimmune encephalomyelitis (EAE), CD1 expression by microglia and the number of CD1+ microglia increase. Macrophages and T cells strongly upregulate CD1 expression in the CNS, but not in the spleen. Whereas the function of CD1 expressed by T cells remains unclear, the expression by microglia and macrophages provides the CNS with a (glyco)lipidic-presenting molecule in an inflammatory and demyelinating environment.     

Nogo receptor expression in microglia/macrophages during experimental autoimmune encephalomyelitis progression

Myelin-associated inhibitory factors within the central nervous system(CNS) are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1(NgR1) has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis(MS). In this study, the animal model of MS, experimental autoimmune encephalomyelitis(EAE) was induced in ngr1~(+/+) and ngr1~(–/–) female mice following injection with the myelin oligodendrocyte glycoprotein(MOG_(35–55)) peptide. A fatemap analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively(increasing locomotor disability) from both genotypes, using the CD11 b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1~(+/+) and ngr1~(–/–) mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1~(+/+) and ngr1~(–/–) mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1~(+/+) and ngr1~(–/–) mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of Ng R on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1~(–/–) mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1~(–/–) mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1~(–/–) mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.     

Polarization of macrophages and microglia in inflammatory demyelination

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, and microglia and macrophages play important roles in its pathogenesis. The activation of microglia and macrophages accompanies disease development, whereas depletion of these cells significantly decreases disease severity. Microglia and macrophages usually have diverse and plastic phenotypes. Both pro-inflammatory and antiinflammatory microglia and macrophages exist in MS and its animal model, experimental autoimmune encephalomyelitis. The polarization of microglia and macrophages may underlie the differing functional properties that have been reported. in this review, we discuss the responses and polarization of microglia and macrophages in MS, and their effects on its pathogenesis and repair. Harnessing their beneficial effects by modulating their polarization states holds great promise for the treatment of inflammatory demyelinating diseases.     

TROY and LINGO-1 expression in astrocytes and macrophages/microglia in multiple sclerosis lesions

Nogo constitutes a family of neurite outgrowth inhibitors contributing to a failure of axonal regeneration in the adult central nervous system (CNS). Nogo-A is expressed exclusively on oligodendrocytes where Nogo-66 segment binds to Nogo receptor (NgR) expressed on neuronal axons. NgR signalling requires a coreceptor p75(NTR) or TROY in combination with an adaptor LINGO-1. To characterize the cell types expressing the NgR complex in the human CNS, we studied demyelinating lesions of multiple sclerosis (MS) brains by immunohistochemistry. TROY and LINGO-1 were identified in subpopulations of reactive astrocytes, macrophages/microglia and neurones but not in oligodendrocytes. TROY was up-regulated, whereas LINGO-1 was reduced in MS brains by Western blot. These results suggest that the ternary complex of NgR/TROY/LINGO-1 expressed on astrocytes, macrophages/microglia and neurones, by interacting with Nogo-A on oligodendrocytes, might modulate glial-neuronal interactions in demyelinating lesions of MS.     

Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats

RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.     

Thrombin regulates CD40 expression in microglial cells

Microglial cells are the innate immune cells of the central nervous system and quickly respond to injury by proliferation, cytokine release, and increased cell surface antigen expression. Thrombin is a multifunctional serine proteinase, which has the capability to activate microglial cells. Here, we report that pharmaceutical-grade thrombin dose-dependently increases the expression of CD40 in N9 microglial cells. This effect is blocked by a thrombin inhibitor, mimicked by thrombin receptor-activating peptide and modified by mitogen-activated protein kinase pathway inhibitors. Thrombin-induced CD40 regulation might play a role in diseases with breakdown of the blood-brain barrier such as multiple sclerosis or stroke.     

Brain macrophages and microglia in human fetal hydrocephalus

Whereas several studies have addressed the activation of microglia (the resident mononuclear phagocytes of the brain) and macrophages within the nervous system in experimental animal models of congenital and induced hydrocephalus, little is known of their state of activation or regional distribution in human fetal hydrocephalus. This investigation aimed to address such questions. Ten human fetal cases [20-36 gestational weeks (GW) at postmortem] previously diagnosed with hydrocephalus on ultrasound examination in utero, and 10 non-hydrocephalic controls (22-38 GW at postmortem) were assessed immufcnohistochemically with antibodies directed against MHC class II and CD68 antigens, and lectin histochemistry with Lycopersicon esculentum (tomato lectin). Adjacent sections were also immunoreacted with an antiserum to laminin to detect cerebral blood vessels. Eight out of the 10 hydrocephalus cases showed numerous CD68 and tomato lectin-positive macrophages located at focal regions along the ependymal lining of the lateral ventricles (particularly within the occipital horn). However, only five of these cases demonstrated MHC class II positive macrophages associated with the ventricular lining. Microglial reactivity within periventricular regions could also be identified using the lectin in four cases, two of which were also immunoreactive with CD68 (but not with MHC class II). By comparison, in control cases five out of 10 fetal brains (aged between 20 and 24 GW) showed few or no ependymal or supraependymal macrophages. One case at 28 GW, and cases at 32 and 38 GW (two of which were diagnosed with intrauterine hypoxic-ischemia) did, however, show some MHC class II (CD68 negative) cells located at the ependymal surface. Nevertheless, these were not as numerous or intensely immunoreactive as in the hydrocephalus cases. Microglia interspersed throughout the intermediate zone and circumscribing the basal ganglia were within normal confines in all cases examined. Hydrocephalic cases additionally showed focal regions of hypovascularization or alterations in the structure and orientation of capillaries within periventricular areas, compared to controls. The macrophage response detected at the ependymal lining of the ventricles and within the periventricular area in hydrocephalus may be related both to the severity of hydrocephalus and the age of the fetus.     

Phagocytic microglia and macrophages in brain injury and repair

AimsPhagocytosis is the cellular digestion of extracellular particles, such as pathogens and dying cells, and is a key element in the evolution of central nervous system (CNS) disorders. Microglia and macrophages are the professional phagocytes of the CNS. By clearing toxic cellular debris and reshaping the extracellular matrix, microglia/macrophages help pilot the brain repair and functional recovery process. However, CNS resident and invading immune cells can also magnify tissue damage by igniting runaway inflammation and phagocytosing stressed—but viable—neurons.DiscussionMicroglia/macrophages help mediate intercellular communication and react quickly to the “find‐me” signals expressed by dead/dying neurons. The activated microglia/macrophages then migrate to the injury site to initiate the phagocytic process upon encountering “eat‐me” signals on the surfaces of endangered cells. Thus, healthy cells attempt to avoid inappropriate engulfment by expressing “do not‐eat‐me” signals. Microglia/macrophages also have the capacity to phagocytose immune cells that invade the injured brain (e.g., neutrophils) and to regulate their pro‐inflammatory properties. During brain recovery, microglia/macrophages engulf myelin debris, initiate synaptogenesis and neurogenesis, and sculpt a favorable extracellular matrix to support network rewiring, among other favorable roles. Here, we review the multilayered nature of phagocytotic microglia/macrophages, including the molecular and cellular mechanisms that govern microglia/macrophage‐induced phagocytosis in acute brain injury, and discuss strategies that tap into the therapeutic potential of this engulfment process.ConclusionIdentification of biological targets that can temper neuroinflammation after brain injury without hindering the essential phagocytic functions of microglia/macrophages will expedite better medical management of the stroke recovery stage.     

Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis

The potential interplay of glial cells with T cells during viral induced inflammation was assessed by comparing major histocompatibility complex molecule upregulation and retention on astrocytes and microglia. Transgenic mice expressing green fluorescent protein under control of the astrocyte-specific glial fibrillary acidic protein promoter were infected with a neurotropic coronavirus to facilitate phenotypic characterization of astrocytes and microglia using flow cytometry. Astrocytes in the adult central nervous system up-regulated class I surface expression, albeit delayed compared with microglia. Class II was barely detectable on astrocytes, in contrast to potent up-regulation on microglia. Maximal MHC expression in both glial cell types correlated with IFN-gamma levels and lymphocyte accumulation. Despite a decline of IFN-gamma concomitant to virus clearance, MHC molecule expression on glia was sustained. These data demonstrate distinct regulation of both class I and class II expression by microglia and astrocytes in vivo following viral induced inflammation. Furthermore, prolonged MHC expression subsequent to viral clearance implies a potential for ongoing presentation.     

中国东北地区CD40及CD40L基因多态性与散发帕金森病的相关性分析

目的 探究CD40基因rs1883832位点及CD40L基因rs1126535位点单核苷酸多态性与帕金森病(Parkinson disease,PD)的相关性。方法 本研究纳入285名中国东北地区汉族健康人和396例PD患者。根据其发病年龄将PD组患者再分为早发PD组(发病年龄≤50岁)和晚发PD组(发病年龄 50岁),收集一般临床资料,提取外周血基因组DNA,利用MALDI-TOF-PEX技术检测rs1883832位点及rs1126535位点多态性分布情况,分析其与帕金森病的相关性。结果 EOPD组与对照组rs1883832位点基因型分布有统计学差异(P 0. 05),等位基因频率在两组间没有统计学差异。PD组和LOPD组与对照组在rs1883832位点及rs1126535位点上,基因型及等位基因型频率无统计学差异。结论 CD40基因rs1883832位点单核苷酸多态性与中国东北地区汉族早发帕金森病相关,T等位基因可能是EOPD的危险因素。