Interleukin-10, interleukin-4, and transforming growth factor-beta differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells

The pro-inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co-cultures of rat microglial and astroglial cells, we investigated the effects of the anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1)/beta2, IL-4, and IL-10 on the production of (pro-) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 micrograms/ml, 24 h). IL-10 (10 and 100 ng/ml) and IL-4 (5 and 50 U/ml) suppressed the LPS-induced production of NO, IL-6, and TNF-alpha in a dose-dependent manner, whereas TGF-beta1/beta2 (2 and 20 ng/ml) only suppressed NO production. LPS-induced levels of IL-1beta were suppressed by IL-10, but not by IL-4 and TGF-beta1/beta2. Conversely, co-incubation of the glial cells with LPS and antibodies to TGF-beta1/beta2 selectively enhanced LPS-induced NO production, whereas co-incubation with antibody to IL-10 enhanced LPS-induced production of all pro-inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF-beta1/beta2 and IL-10 are produced by LPS-stimulated glial cell co-cultures. Production of IL-10 in these co-cultures was confirmed by measurement of rat IL-10 by radioimmunoassay. We conclude that anti-inflammatory cytokines affect the production of inflammatory mediators in LPS-activated co-cultures of microglial and astroglial cells differentially.     

Local expression of cytokines in idiopathic inflammatory myopathies

H. Lepidi, V. Frances, D. Figarella-Branger, C. Bartoli, A. Machado-Baeta & J-F. Pellissier (1998) Neuropathology and Applied Biology , 24, 73–79
Local expression of cytokines in idiopathic inflammatory myopathies
The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), are regarded as autoimmune diseases. They are characterized by chronic lymphocytic and macrophagic infiltration in muscle tissue. Of particular importance in understanding the immune response to IIM is the specific pattern of locally produced cytokines. Frozen muscle tissues from IIM (5 DM, 3 PM, and 1 IBM) were used to investigate the cytokine responses. The RT-PCR technique was instrumental to determine the pattern of expression of pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IFN-γ IL-2), and Th2 (IL-4) cytokines. Immunohistochemistry was also used to localize morphologically IFN-γ and IL-4. Our results show that pro-inflammatory cytokines and Th1 cytokines are mainly expressed in IIM. The accumulation of mononuclear inflammatory cells and the inflammatory syndrome in IIM are probably related in part to the production of pro-inflammatory cytokines. Moreover, the pattern of local cytokine expression is consistent with a Th1 immune response related to autoimmune diseases.     

Cytokine regulation of astrocyte function: in-vitro studies using cells from the human brain

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon-γ mainly resulted in increased expression of major histocompatibility complex antigens and co-stimulatory molecules (intercellular adhesion molecule-1, LFA-1α) which mediate astrocyte-T-cell interactions. Another co-stimulatory molecule, B7, was neither expressed nor inducible by IFN-γ and other cytokines. TNF-α and IL-1β were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL-6, IL-8 and colony-stimulating factors), cytokine antagonists (TNF-α soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co-stimulatory molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Transforming growth factor-β₁ was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co-stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal-glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.     

NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines

Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.     

Pro- and anti-inflammatory cytokines regulate the ERK pathway: Implication of the timing for the activation of microglial cells

Pro-inflammatory molecules induce glial activation and the release of potentially detrimental factors capable of generating oxidative damage, such as nitric oxide (NO) and superoxide anion (O2.-). Activated glial cells (astrocytes and microglia) are associated to the inflammatory process in neurodegenerative diseases. A strong inflammatory response could escape endogenous control becoming toxic to neurons and contributing to the course of the disease. We evaluated in a hippocampal cells-microglia co-culture model, if the pro-inflammatory condition induced by lipopolysaccharide + interferon-gamma (LPS+IFN-gamma) promoted damage directly or if damage was secondary to glial activation. In addition, we explored the effect of the anti-inflammatory cytokine transforming growth factor-beta1 (TGF-beta1), and pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the regulation of the inflammatory response of microglia. We found that LPS+IFN-gamma-induced damage on hippocampal cultures was dependent on the presence of microglial cells. In hippocampal cultures exposed to LPS+IFN-gamma, TGF-beta1 was induced whereas in microglial cell cultures LPS+IFN-gamma induced the secretion of IL-1beta. TGF-beta1 and IL-1beta but not TNF-alpha decreased the NO production by 70-90%. PD98059, an inhibitor of MAP kinase (MEK), reduced the IFN-gamma-induced NO production by 40%. TGF-beta and IL-1beta reduced the IFN-gamma induced phosphorylation of ERK1,2 by 60% and 40%, respectively. However, the effect of IL-1beta was observed at 30 min and that of TGF-beta1 only after 24 h of exposure. We propose that acting with different timing, TGF-beta1 and IL-1beta can modulate the extracellular signal-regulated kinase ERK1,2, as a common element for different transduction pathways, regulating the amplitude and duration of glial activation in response to LPS+IFN-gamma. Cross-talk among brain cells may be key for the understanding of inflammatory mechanisms involved in pathogenesis of neurodegenerative diseases.     

Oestrogen and progesterone reduce lipopolysaccharide-induced expression of tumour necrosis factor-alpha and interleukin-18 in midbrain astrocytes

Besides microglia, astrocytes exert an important regulatory function in the initiation and control of neuro-inflammatory processes in the central nervous system. Clinical and experimental data suggest that sex steroids are neuroprotective and that neurological/neurodegenerative disorders display sex-specific characteristics. Astroglia is known to respond to toxic stimuli by secretion of distinct pro-inflammatory/apoptotic cytokines. In the present study, we investigated the influence of oestrogen and progesterone on the expression of the cytokines tumour necrosis factor (TNF)-alpha and interleukin (IL)-18 in primary astrocytes obtained from neonatal mouse midbrain and cerebral cortex after the stimulation with lipopolysaccharides (LPS). LPS strongly induced the expression of TNF-alpha in astrocytes from both brain regions and IL-18 in those from midbrain. Oestrogen significantly attenuated LPS-induced TNF-alpha expression in the midbrain glia but not in the cortex glia. Combined treatment with oestrogen and progesterone together diminished LPS-induced IL-18 expression in the midbrain completely. Both steroid effects could be specifically antagonised by the steroid hormone receptor antagonists ICI 182 780 and mifepristone. We conclude that neuroprotective oestrogen and progesterone effects in the midbrain might be in part the consequence of a reduced pro-inflammatory response of astroglia.     

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

9-Cis-retinoic acid suppresses inflammatory responses of microglia and astrocytes

Retinoic acid (RA) regulates a wide range of biologic process, including inflammation. Previously, RA was shown to inhibit the clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The current study investigated the effects of 9-cis-RA on primary mouse microglia and astrocytes, two cell types implicated in the pathology of MS and EAE. The studies demonstrated that 9-cis-RA inhibited the production of nitric oxide (NO) as well as the pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-12 p40 by LPS-stimulated microglia. However, this retinoid had no effect on IL-6 secretion and increased MCP-1 production by LPS-stimulated microglia. In LPS-stimulated astrocytes, 9-cis-RA inhibited NO and TNF-alpha production but had not effect on IL-1beta, IL-6 and MCP-1 secretion. These results suggest that RA modulates EAE, at least in part, by suppressing the production of NO and specific inflammatory cytokines from activated glia and suggests that RA might be effective in the treatment of MS.     

Peroxisome proliferator-activated receptor-alpha and retinoid X receptor agonists inhibit inflammatory responses of astrocytes

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) plays a key role in lipid metabolism and inflammation. Recently, we demonstrated that administration of the PPAR-alpha agonists gemfibrozil and fenofibrate, inhibit the clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we investigated the effects of PPAR-alpha agonists on primary mouse astrocytes, a cell type implicated in the pathology of MS and EAE. Our studies demonstrated that the PPAR-alpha agonists fenofibrate, and WY 14643 inhibited NO production by LPS-stimulated astrocytes in a dose-dependent manner. Additionally, PPAR-alpha agonists inhibited the secretion of the pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 by LPS-stimulated astrocytes. Fenofibrate inhibited NF-kappaB DNA binding activity, suggesting a mechanism by which PPAR-alpha agonists may regulate the expression of genes encoding these pro-inflammatory molecules. Retinoid X receptors (RXRs) physically interact with PPAR-alpha receptors, and the resulting heterodimers regulate the expression of PPAR-responsive genes. Interestingly, a combination of 9-cis RA and the PPAR-alpha agonists fenofibrate or gemfibrozil cooperatively inhibited NO, TNF-alpha, IL-1beta, IL-6, and MCP-1 production by these cells. Collectively, these results raise the possibility that PPAR-alpha and RXR agonists might be effective in the treatment of MS, where activated astrocytes are believed to contribute to disease pathology.     

Modulation of the cytokine network in human adult astrocytes by human herpesvirus-6A

Human herpesvirus-6A (HHV-6A) is a common pathogen whose role in CNS disorders including multiple sclerosis remains controversial. To understand how HHV-6A could influence inflammatory pathways in the CNS, we infected cultured human adult astrocytes and examined the expression of 268 cytokines, chemokines, growth factors and their receptors by gene profiling. HHV-6 infection alone had little effect on the astrocyte gene profile but strongly altered the astrocyte response to proinflammatory cytokines. Under those conditions astrocytes express higher levels of anti-inflammatory mediators including IL-10 and IL-11, chemotactic factors, growth factors and factors controlling type I interferon production. Our data suggest that HHV-6 itself does not evoke a pro-inflammatory response in astrocytes but rather triggers immune modulatory factors in the face of inflammation.     

Role of activated astrocytes in neuronal damage: Potential links to HIV-1-associated dementia

HIV-1-associated dementia (HAD) is an important complication of HIV-1 infection. Reactive astrogliosis is a key pathological feature in HAD brains and in other central nervous system (CNS) diseases. Activated astroglia may play a critical role in CNS inflammatory diseases such as HAD. In order to test the hypothesis that activated astrocytes cause neuronal injury, we stimulated primary human fetal astrocytes with HAD-relevant pro-inflammatory cytokine IL-1beta. IL-1beta-activated astrocytes induced apoptosis and significant changes in metabolic activity in primary human neurons. An FITC-conjugated pan-caspase inhibitor peptide FITC-VAD-FMK was used for confirming caspase activation in neurons. IL-1beta activation enhanced the expression of death protein FasL in astrocytes, suggesting that FasL is one of the potential factors responsible for neurotoxicity observed in HAD and other CNS diseases involving glial inflammation. Our data presented here add to the developing picture of role of activated glia in HAD pathogenesis.     

Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders

Alzheimer and a number of other neurodegenerative diseases are characterized by the presence of reactive microglia and reactive astrocytes in association with the lesions. The classic view that microglia exist primarily in either a resting or activated state needs to be broadened in view of recent results. Resting microglia are in constant activity sampling their surround. Activated microglia may be pro-inflammatory, releasing inflammatory cytokines and other inflammatory mediators, or anti-inflammatory, promoting the healing process. There is evidence that microglial phagocytosis is more powerful in the anti-inflammatory state. Activated astrocytes also have pro-inflammatory and anti-inflammatory properties. In the pro-inflammatory state they release inflammatory cytokines. In the anti-inflammatory state they release various growth factors. In AD and other neurodegenerative diseases, both microglia and astrocytes are in a pro-inflammatory state. From a therapeutic point of view it is desirable to find methods of tipping the balance towards an anti-inflammatory state for both types of glia.     

Functional characterization of substance P receptors on cultured human spinal cord astrocytes: Synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production

Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK-1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin-6 (IL-6), granulocyte-macrophage-CSF, macrophage chemoattractant protein-1 or leukemia inhibitory factor] or prostaglandin E₂ (PGE₂). Interestingly, however, SP selectively potentiated the inducing effect of IL-1β on IL-6 and PGE₂ secretion by spinal cord astrocytes without affecting the IL-1-β-evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor-α (TNF-α) on IL-6 and PGE₂ secretion and that of transforming growth factor-β on PGE₂ secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL-6 and PGE₂ by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases. GLIA 21:183–193, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the immune system in depression and dementia: causal or co-incidental effects?

It is now widely accepted that psychological stress and psychiatric illness can compromise immune function. Furthermore the mechanisms whereby such changes occur are probably associated with the activities of the cytokines and other inflammatory mediators of the immune system which are known to initiate changes in behaviour. This review aims to summarise the experimental and clinical evidence that implicates the pro-inflammatory cytokines in the pathological changes seen in major depression and in Alzheimer's disease (AD). In major depression, evidence is provided to show that both activation (e.g., macrophage activity, acute phase proteins) and inhibition (e.g., natural killer cell activity) of the immune system occur. Many of the behavioural changes seen in depression are simulated by three pro-inflammatory cytokines (IL-1, IL-6 and TNF-alpha), which may produce their impact on the brain by activating cyclooxygenase, nitric acid synthase and corticotrophin releasing factor. Effective antidepressant treatments largely attenuate the immune changes thereby raising the possibility that the normalisation of central biogenic amine function that are conventionally implicated in the cause of depression may be secondary to those of the pro-inflammatory cytokines.With respect to AD, while the cause(s) are unknown, there is both experimental and clinical evidence to suggest that inflammatory processes in the brain caused in particular by TNF-alpha together with the subsequent rise in free radicals, are instrumental in causing the pathological changes which underlie the disease. Evidence in favour of the inflammatory hypothesis is supported by the finding that nonsteroidal anti-inflammatory drugs slow down the progression of the disease.Although, more research is needed into the inter-relationships between the various pro-inflammatory cytokines and the behavioural changes invoked in major depression and AD, the immunological hypothesis has been important in stimulating new concepts regarding the causes of the pathological changes in these diseases and how effective drug treatments may attenuate them.     

Antigen presenting cells treated in vitro by macrophage colony-stimulating factor and autoantigen protect mice from autoimmunity

Macrophage colony-stimulating factor (M-CSF) is a critical cytokine in the development of monocytic lineage and may have immunoregulatory properties. Here we show that peritoneal antigen presenting cells (APCs) treated with M-CSF produced decreased levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-12. These APCs treated with M-CSF+autoantigen peptide significantly suppressed antigen-specific T cell proliferation, induced regulatory CD4(+) and CD8(+) T cells in vitro and in vivo, and significantly suppressed experimental autoimmune encephalomyelitis (EAE). Thus, in vitro treatment of APCs with M-CSF+autoantigen can be a novel therapeutic option for autoimmune diseases.     

Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators

Toll-like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro-inflammatory factors and enhances antigen-presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro-inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti-inflammatory cytokines including interleukin-9 (IL-9), IL-10, and IL-11 and downregulation of the p40 subunit of IL-12 and IL-23. The collective TLR3-induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro-inflammatory reaction.     

Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD: implications for potential therapies

This study was designed to understand the role of inflammatory mediators involved in the neurobiology of childhood adrenoleukodystrophy (cALD) by comparing the differential expression of the inflammatory mediators with metabolite very long chain fatty acids that accumulate in this disease. Histopathological examinations indicated extensive demyelination and accumulation of infiltrates in perivascular cuffs in plaque area (PA) and inflammatory area (IA) compared to normal looking area (NLA) of the cALD brain and controls. The PA had excessive accumulation of cholesterol ester (25-30-fold), VLC fatty acids (8-12-fold), and exhaustive depletion of cholesterol (60-70%) and sphingomyelin (50-55%) in comparison to controls. The mRNA expression of cytokines (IL-1alpha, IL-2, IL-3, IL-6, TNF-alpha, and GM-CSF), chemokines (CCL2, -4, -7, -11, -16, -21, -22, CXCL1, CX3CL1, and SDF-2) and iNOS in IA was significantly increased compared to NLA of the cALD and controls determined by gene array, semiquantitative RT-PCR, and immunohistochemistry. These results indicate that accumulation of VLC fatty acid contents in membrane domains associated with signal transduction pathways may trigger the inflammatory process through activation of resident glial cells (microglia and astrocytes) resulting in loss of myelin and oligodendrocytes.