Serum amyloid A primes microglia for ATP-dependent interleukin-1\u3b2 release

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1\u3b2 (IL-1\u3b2), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1\u3b2 release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

The role of P2X7 in pain and inflammation

The P2X7 purinoceptor is unique amongst the P2X receptor family in that its activation is able to stimulate the release of mature, biologically active interleukin-1β (IL-1β), as well as a variety of other proinflammatory cytokines. Coupled with the predominate localisation of this receptor to immunocytes of haemopoetic origin, this receptor is an obvious candidate to play a major and pivotal role in processes of pain and inflammation. Using genetically modified animals that lack the P2X7 receptor, several investigators have shown that these mice do indeed demonstrate a blunted inflammatory response, and fail to develop pain following both inflammatory and neuropathic insult. These animals also show altered cytokine production in response to inflammatory stimulus, which is far broader than merely modulation of IL-1β release. In this short article, we review the role of the P2X7 receptor in modulating the release of cytokines and other mediators, and discuss the findings made from P2X7 receptor-deficient animals. As well as highlighting outstanding questions regarding this intriguing receptor, we also speculate as to the potential therapeutic benefit of P2X7 receptor modulation

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X7 knockout mice or in cells pretreated with a P2X7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species

Mechanisms Underlying Interferon-γ-Induced Priming of Microglial Reactive Oxygen Species Production.

Microglial priming and enhanced reactivity to secondary insults cause substantial neuronal damage and are hallmarks of brain aging, traumatic brain injury and neurodegenerative diseases. It is, thus, of particular interest to identify mechanisms involved in microglial priming. Here, we demonstrate that priming of microglia with interferon-γ (IFN γ) substantially enhanced production of reactive oxygen species (ROS) following stimulation of microglia with ATP. Priming of microglial ROS production was substantially reduced by inhibition of p38 MAPK activity with SB203580, by increases in intracellular glutathione levels with N-Acetyl-L-cysteine, by blockade of NADPH oxidase subunit NOX2 activity with gp91ds-tat or by inhibition of nitric oxide production with L-NAME. Together, our data indicate that priming of microglial ROS production involves reduction of intracellular glutathione levels, upregulation of NADPH oxidase subunit NOX2 and increases in nitric oxide production, and suggest that these simultaneously occurring processes result in enhanced production of neurotoxic peroxynitrite. Furthermore, IFNγ-induced priming of microglial ROS production was reduced upon blockade of Kir2.1 inward rectifier K+ channels with ML133. Inhibitory effects of ML133 on microglial priming were mediated via regulation of intracellular glutathione levels and nitric oxide production. These data suggest that microglial Kir2.1 channels may represent novel therapeutic targets to inhibit excessive ROS production by primed microglia in brain pathology

Activation of adherent vascular neutrophils in the lung during acute endotoxemia

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells

COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord

BACKGROUND: While multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, there is increasing evidence for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids ameliorate progression of clinical disease in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three key molecules known to be expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord. METHODS: Frozen human post mortem spinal cord specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were available for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In addition, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a marker of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified by computerized image and optical density analysis respectively. RESULTS: In control spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, mostly bipolar with short processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, by image analysis. Inflammatory cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-2 by activated microglial cells/macrophages. An expected 70-kDa band was seen by Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-2 immunostaining and optical density of the COX-2 70-kDa band in the MS group (r = 0.89, P = 0.0011, n = 10). MS and ALS specimens also had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions. CONCLUSION: It is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in turn induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-2 and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS

Selective P2X7 receptor antagonists for chronic inflammation and pain

ATP, acting on P2X7 receptors, stimulates changes in intracellular calcium concentrations, maturation, and release of interleukin-1β (IL-1β), and following prolonged agonist exposure, cell death. The functional effects of P2X7 receptor activation facilitate several proinflammatory processes associated with arthritis. Within the nervous system, these proinflammatory processes may also contribute to the development and maintenance of chronic pain. Emerging data from genetic knockout studies have indicated specific roles for P2X7 receptors in inflammatory and neuropathic pain states. The discovery of multiple distinct chemical series of potent and highly selective P2X7 receptor antagonists have enhanced our understanding of P2X7 receptor pharmacology and the diverse array of P2X7 receptor signaling mechanisms. These antagonists have provided mechanistic insight into the role(s) P2X7 receptors play under pathophysiological conditions. In this review, we integrate the recent discoveries of novel P2X7 receptor-selective antagonists with a brief update on P2X7 receptor pharmacology and its therapeutic potential

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X1,2,4,7 and P2Y1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects