Induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) are produced by leukocytes and play a role in immune responses. They also function in normal brain physiology as well as in pathological conditions within the central nervous system, where they are produced by brain macrophages (microglia) and brain astrocytes. In this study, we document the ability of human immunodeficiency virus type 1 (HIV-1) to induce TNF alpha and IL-1 in primary rat brain cultures. While productive infection did not occur in these cells, it was not required for cytokine induction. Using monocyte/macrophage-tropic (JRFL) and T-cell-tropic (IIIB) strains of HIV-1, we were able to induce cytokines in both microglia and astrocytes. In addition to whole virus, recombinant envelope proteins also induced these cytokines. The induction of IL-1 and TNF alpha could be blocked by a panel of antibodies recognizing epitopes in the gp120 and gp41 areas of the envelope. Soluble recombinant CD4 did not block TNF alpha and IL-1 production. If TNF alpha and IL-1 can be induced in brain tissue by HIV-1, they may contribute to some of the neurologic disorders associated with AIDS.     

HIV-1, macrophages, glial cells, and cytokines in AIDS nervous system disease

Hallmarks of central nervous system (CNS) disease in AIDS patients are headaches, fever, subtle cognitive changes, abnormal reflexes, and ataxia. Dementia and severe sensory and motor dysfunction characterize more severe disease. Autoimmune-like peripheral neuropathies, cerebrovascular disease, and brain tumors are also observed. Histological changes include inflammation, astrocytosis, microglial nodule formation, and diffuse de- or dysmyelination. Focal demyelination can also be seen. It is clear that AIDS-associated neurological diseases are correlated with greater levels of HIV-1 antigen or genome in tissues. In AIDS dementia, macrophages and microglial cells of the CNS are the predominant cell types infected and producing HIV-1. However, manifestations of the disease make it unlikely that direct infection by HIV-1 is responsible. It seems more likely that the effects are mediated through secretion of viral proteins or viral induction of cytokines that bind to glial cells and neurons. HIV-1 induction of such cytokines as interleukin 1 (IL 1) and tumor necrosis factor-alpha (TNF alpha) may lead to an autocrine feedback loop involving further productive virus replication and induction of other cytokines such as interleukin 6 (IL 6) and granulocyte-macrophage colony-stimulating factor (GMCSF). Interleukin 1 and TNF alpha in combination with IL 6 and GMCSF could account for many clinical and histopathological findings in AIDS nervous system diseases. As HIV-1 infected patients produce elevated levels of IL 1, TNF alpha, and IL 6, it will be important to make a formal connection between the presence of these factors in the CNS, which are all products of activated macrophages, astroglia, and microglia, their in vivo induction directly by virus or indirectly by virus-induced intermediates, and the clinical and pathological conditions seen in the nervous system in this disease.     

Protein kinase C alpha requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor alpha in lipopolysaccharide-stimulated microglia

Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNF alpha, we investigated the regulatory mechanism by which TNFalpha is induced or suppressed in microglia. We found that LPS-induced TNF alpha is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, G?6976, myristoylated pseudosubstrate (20-28), and bisindolylmaleimide. These results suggest that PKC alpha activity as well as p38MAPK activity is associated with TNF alpha induction in LPS-stimulated microglia. The requirement of PKC alpha in LPS-dependent TNFalpha induction was verified in PKC alpha-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKC alpha was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKC alpha levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKC alpha levels are regulated by the p38MAPK activity. We therefore concluded that PKC alpha and p38MAPK are interactively linked to the signaling cascade inducing TNFalpha in LPS-stimulated microglia, and that in this cascade, PKC alpha is requisite for the activation of p38MAPK, leading to the induction of TNF alpha.     

Molecular consequences of activated microglia in the brain: overactivation induces apoptosis

Microglia, the resident immune cells in the brain, play a pivotal role in immune surveillance, host defense, and tissue repair in the CNS. In response to immunological challenges, microglia readily become activated as characterized by morphological changes, expression of surface antigens, and production of immune modulators that impact on neurons to induce neurodegeneration. However, little is known concerning the fate of activated microglia. In the present study, stimulation of cultured rat primary microglia with 1 ng/mL of the inflammagen lipopolysaccharide (LPS) resulted in a maximal activation as measured by the release of tumor necrosis factor alpha (TNF alpha). However, treatment with higher concentrations of LPS resulted in significantly lower quantities of detectable TNF alpha. Further analysis revealed that overactivation of microglia with higher concentrations of LPS (> 1 ng/mL) resulted in a time- and dose-dependent apoptotic death of microglia as defined by DNA strand breaks, surface expression of apoptosis-specific markers (phosphatidylserine), and activation of caspase-3. In contrast, astrocytes were insensitive to LPS-induced cytotoxicity. In light of the importance of microglia and the limited replenishment mechanism, depletion of microglia from the brain may severely hamper its capacity for combating inflammatory challenges and tissue repair. Furthermore, overactivation-induced apoptosis of microglia may be a fundamental self-regulatory mechanism devised to limit bystander killing of vulnerable neurons.     

Tumor necrosis factor alpha and interleukin-1 alpha inhibit through different pathways interferon-gamma-induced antigen presentation, processing and MHC class II surface expression on astrocytes, but not on microglia

Astrocytes and microglia, two glial cell populations of the CNS, have been described to be involved in many immune processes. We used defined combinations of cytokines, interferon gamma (IFN-gamma)/interleukin-1 alpha (IL-1 alpha) and IFN-gamma/tumor necrosis factor alpha (TNF alpha), to simulate different in vitro immune environments observed in disease or inflammation. In these conditions, we analyzed and compared the regulating effects of these cytokines on cell surface and total expression of MHC II and on the capacity of murine astrocytes and microglia to present peptide and native antigens to specific primed T cells. Neither IL-1 alpha nor TNF alpha affected the IFN-gamma-induced antigen presentation capacity of microglia. Astrocytes, however, were severely impaired in their capacity to present native antigens and, to a minor extent, a peptide antigen. Total expression of MHC II was not affected by these cytokines in microglia, whereas in astrocytes it was reduced by IL-1 alpha and increased by TNF alpha. Both cytokines downregulated MHC II expression at the surface of astrocytes, but not of microglia. This shows that TNF alpha affects the of IFN-gamma-immunocompetent astrocytes to process and present antigen, probably either by altering membrane traffic of MHC II and of antigen and/or enzymatic activities associated with these mechanisms, while IL-1 alpha does so by downregulating MHC II expression. Altogether, our results illustrate how differently astrocytes and microglia react toward a defined, similar immune environment. One type of cell, the astrocytes, downregulate their T-cell stimulation and MHC II trafficking, and probably also their antigen processing, functions while the other, the microglia, maintain their antigen presentation potential.     

N-Acetyl-cysteine inhibition of encephalomyelitis Theiler's virus-induced nitric oxide and tumour necrosis factor-alpha production by murine astrocyte cultures

The pathological mechanisms that cause central nervous system (CNS) dysfunction in most neurological diseases are not well established. Theiler's murine encephalomyelitis virus (TMEV) is known to interact with cells of the CNS and its intracerebral inoculation to susceptible mice strains causes neurological disorders resembling multiple sclerosis (MS). In this study, we reported that primary astrocyte cultures from SJL/J susceptible mice when infected with TMEV released important amounts of nitrites (NO2-) to the culture medium, as measured in the supernatants 24 hours after infection. In addition, we observed an increment in the production of tumour necrosis factor alpha (TNF-alpha) by susceptible SJL/J strain derived astrocytes infected with TMEV. The treatment with the thiolic antioxidant N-acetyl-cysteine partially suppressed the virus-stimulated production of nitric oxide and TNF-alpha, in a dose response fashion. These results indicate that during viral infection astrocytes are an important cellular source of nitric oxide and TNF-alpha, substances which play important roles during CNS inflammatory events. The effects of the antioxidant N-acetyl-cysteine, modulating the production of the above compounds by TMEV-infected astrocytes may be a significant factor in preventing CNS demyelination.     

Regulation of microglial activities by glial cell line derived neurotrophic factor

Much attention has been paid to the ability of glial cell line-derived neurotrophic factor (GDNF) to protect neurons from neurotoxic insults in the central nervous system (CNS). However, little is known about GDNF action on CNS glia that also can express GDNF receptor systems. In this study, we examined the effects of GDNF on primary rat microglia that function as resident macrophages in the CNS and as the source of proinflammatory mediators upon activation. We found that treatment of primary rat microglia with GDNF had no effect on the secretion of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), but it increased the nitric oxide (NO) production to some extent. In addition, GDNF increased the enzymatic activity of superoxide dismutase (SOD), the gene expression of surface antigen intercellular adhesion molecule-1 (ICAM-1), the production of the integrin alpha5 subunit, and the phagocytotic capability in primary rat microglia. Furthermore, inhibition of mitogen-activated protein kinase (Erk-MAPK) in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. In vivo evidence also showed that amoeboid cells with integrin alpha5 or with ED1 immunoreactivity appeared in GDNF-treated spinal cord tissues at the lesion site 1 week post spinal cord injury (SCI). Furthermore, inhibition of Erk-MAPK in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. Taken together, our results indicate that GDNF has a positive regulatory effect on microglial activities, such as phagocytosis and the upregulation of adhesion molecules.     

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up‐regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial‐derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation‐mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV‐2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial‐derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up‐regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS‐induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 566–584, 2016     

Peroxisome proliferator-activated receptor alpha agonists as therapy for autoimmune disease

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPAR gamma ligands, which include the naturally occurring PG metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. The PPAR alpha agonists, gemfibrozil, ciprofibrate, and fenofibrate, have an excellent track history as oral agents used to treat hypertriglyceridemia. In the present study, we demonstrate that these PPAR alpha agonists can increase the production of the Th2 cytokine, IL-4, and suppress proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1-11, as well as reduce NO production by microglia. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine secretion of human T cell lines by inhibiting IFN-gamma and promoting IL-4 secretion. These results suggest that PPAR alpha agonists such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis.     

Neuroprotective Effect of 6-Paradol in Focal Cerebral Ischemia Involves the Attenuation of Neuroinflammatory Responses in Activated Microglia

Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia.     

The suppressive effects of recombinant human tumor necrosis factor-alpha on normal and malignant myelopoiesis: synergism with interferon-gamma

The modulation of growth of normal and leukemic myeloid progenitor cells in soft agar cultures by recombinant human tumor necrosis factor-alpha (TNF alpha) and recombinant human interferon-gamma (IFN gamma) was investigated. TNF alpha inhibited colony formation of all colony types representing different maturational stages of normal progenitor cells committed to the myeloid lineage with different orders of sensitivity. Blast-type colonies derived from patients with acute myelogenous leukemia were more sensitive to TNF alpha inhibition than progenitor cells purified from normal bone marrow or bone marrow from patients with stable-phase chronic myelogenous leukemia. The response of most colony types to IFN gamma was poor. However, when IFN gamma was administered together with TNF alpha, synergistically enhanced antiproliferative effects were detected in all colony types tested. The antiproliferative action of IFN gamma on myelopoiesis was enhanced in culture by the presence of autologous monocytes, presumedly by inducing endogenous production of TNF alpha. However, TNF alpha seemed to act directly on the progenitor cells themselves to suppress their clonal growth, rather than involving accessory marrow elements such as monocytes and/or T lymphocytes.     

Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-beta, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.     

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.     

Murine microglial cells produce and respond to interleukin-18

Interleukin (IL)-18 (interferon-gamma-inducing factor or IL-1gamma) belongs structurally to the IL-1 cytokine family and shares biological properties with IL-12. Expression, intracellular signaling, and functional relevance of IL-18 within the CNS are mostly unknown. We show that IL-18 protein is synthesized within mouse brain, preferentially during early postnatal stages, and that microglial cells but not astrocytes are a potential source. IL-18 is produced by cultured microglia on exposure to lipopolysaccharide (LPS). Microglia also express major components of the IL-1/IL-18 receptor system. On IL-18 stimulation, microglial IL-1 receptor-associated kinase (IRAK) can be coprecipitated with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) but not with IL-1 receptor type I, indicating that IRAK recruits TRAF6 during IL-18 signaling. IL-18 inhibits the LPS-induced release of IL-12 and attenuates that of TNF-alpha, whereas the production of IL-6 and macrophage inflammatory protein-1alpha is only marginally affected. IL-18 may play a role during CNS development and can be produced by activated microglia, thus probably contributing to immune and inflammatory processes in the brain.     

Vesnarinone Represses the Fibrotic Changes in Murine Lung Injury Induced by Bleomycin

We investigated the potential usefulness of vesnarinone, a novel cytokine inhibitor, for the treatment of lung fibrosis using a murine model of bleomycin (BLM)-induced pulmonary fibrosis. Mice were fed a control diet (n=42), or a diet containing low (n=42) or high (n=42) dose of vesnarinone. Dietary intake of vesnarinone minimized the BLM toxicity as reflected by significant decreases in numbers of inflammatory cells, KC, and soluble TNF receptors in the bronchoalveolar lavage fluid. A quantitative evaluation of histology demonstrated significantly mild lung parenchymal lesions in BLM-treated mice fed with diet containing high dose of vesnarinone than in the control diet group. Consistent with the histopathology, hydroxyproline levels in lung tissue from BLM-treated mice fed with diet containing vesnarinone were significantly lower than that from mice fed with control diet. We concluded that vesnarinone inhibits BLM-induced pulmonary fibrosis, at least in part, by the inhibition of acute lung injuries in the early phase.     

Thalidomide treatment reduces tumor necrosis factor alpha production and enhances weight gain in patients with pulmonary tuberculosis.

BACKGROUND: The monocyte-derived cytokine, tumor necrosis factor alpha (TNF alpha), is essential for host immunity, but overproduction of this cytokine may have serious pathologic consequences. Excess TNF alpha produced in pulmonary tuberculosis may cause fevers, weakness, night sweats, necrosis, and progressive weight loss. Thalidomide (alpha-N-phthalimidoglutarimide) has recently been shown to suppress TNF alpha production by human monocytes in vitro and to reduce serum TNF alpha in leprosy patients. We have therefore conducted a two-part placebo-controlled pilot study of thalidomide in patients with active tuberculosis to determine its effects on clinical response, immune reactivity, TNF alpha levels, and weight. MATERIALS AND METHODS: 30 male patients with active tuberculosis, either human immunodeficiency virus type 1 positive (HIV-1+) or HIV-1-, received thalidomide or placebo for single or multiple 14 day cycles. Toxicity of the study drug, delayed type hypersensitivity (DTH), cytokine production, and weight gain were evaluated. RESULTS: Thalidomide treatment was well tolerated, without serious adverse events. The drug did not adversely affect the DTH response to purified protein derivative (PPD), total leukocyte, or differential cell counts. TNF alpha production was significantly reduced during thalidomide treatment while interferon-gamma (IFN gamma) production was enhanced. Daily administration of thalidomide resulted in a significant enhancement of weight gain. CONCLUSIONS: The results indicate that thalidomide is well tolerated by patients receiving anti-tuberculosis therapy. Thalidomide treatment reduces TNF alpha production both in vivo and in vitro and is associated with an accelerated weight gain during the study period.