Biomedical subjects

Uwe-Karsten Hanisch

Publications and source records attributed to Uwe-Karsten Hanisch.

13 recordsLinked to original sources

PubMed2006

Microglial phenotype: is the commitment reversible?

Microglia, the standby cells for immune defense in the CNS, have a reputation for exacerbating the neural damage that occurs in neurodegenerative diseases. However, research over the past few years has established that microglia do not constitute a single, uniform cell population, but rather comprise a family of cells with diverse phenotypes--some that are beneficial and others that the CNS can barely tolerate and that are therefore destructive. This finding raised several questions. What instructs microglia to acquire a particular phenotype, and how do these phenotypes differ? How committed are microglia to a specific phenotype? Can destructive microglia become protective, and can protective microglia retain their beneficial phenotype even when they encounter a destructive environment? Here, we address these questions, and the background of research that elicited them.

Animals↗

PubMed2006

Activation of microglial cells by thrombin: past, present, and future.

In addition to its role in the coagulation cascade, the serine proteinase thrombin (factor IIa) activates cell surface proteinase-activated receptors (PARs) both within and outside the vascular system. PARs are expressed in the central nervous system and mediate thrombin-induced cellular responses in a variety of neural cell types, including microglial cells. Microglial activation by thrombin was reported to induce proliferation, cytokine release, and intracellular calcium signaling. Recently, additional experiments questioned whether these effects are mediated either by thrombin's proteolytic activity or by thrombin itself. Analysis of commercially available plasma-derived thrombin frequently used in the earlier studies showed that cyto/chemokine release-activating properties were not residing with thrombin but were with high molecular weight contaminant(s). In the absence of such contamination, no microglial activation was seen. We compared commercial-grade plasma-derived thrombin to pharmaceutical-grade recombinant thrombin devoid of any measurable contamination. The pharmaceutical-grade thrombin displayed a much more limited profile of microglia-activating properties, triggering only intracellular calcium signals and small changes in surface antigen expression. The signals induced by the pharmaceutical-grade thrombin were completely abolished by proteolytic inhibition, indicating that they are proteolysis-dependent, are most likely PAR mediated, and reflect thrombin's true microglia-activating potential. Prior reports using nonpharmaceutical-grade thrombin need to be reinterpreted critically given these new findings.

Animals↗

PubMed2005

c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia.

The activation and function of c-Jun N-terminal kinases (JNKs) were investigated in primary microglia cultures from neonatal rat brain, which express all three JNK isoforms. Lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and thrombin preparations induced a rapid and lasting activation of JNKs in the cytoplasm. In the nucleus, the activation patterns were rather complex. In untreated microglia, the small pool of nuclear JNKs was strongly activated, while the high-affinity JNK substrate c-Jun was only weakly phosphorylated. Stimulation with LPS increased the total amount of nuclear JNKs and the phosphorylation of the transcription factor c-Jun. Levels of activated JNKs in the nucleus, however, rapidly decreased. Analysis of the nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased, and the weakly expressed JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of c-Jun in this context. Upstream of JNKs, LPS induced a lasting activation of the constitutively present JNK kinase MKK4. The function of JNKs in LPS-triggered cellular reactions was investigated using SP600125 (0.5-5 microM), a direct inhibitor of JNKs. Inhibition of JNKs reduced the LPS-induced metabolic activity and induction of the AP-1 target genes cyclooxygenase-2 (Cox-2), TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in response to LPS, while ERK1/2 and p38 alpha had a more pronounced effect on LPS-induced cellular enlargement than JNKs. In summary, JNKs are essential mediators of relevant pro-inflammatory functions in microglia with different contributions of the JNK isoforms.

Animals↗

PubMed2005

Unraveling thrombin's true microglia-activating potential: markedly disparate profiles of pharmaceutical-grade and commercial-grade thrombin preparations.

Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-alpha release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.

Amino Acid Chloromethyl Ketones↗

PubMed2004

The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines.

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.

Amino Acid Sequence↗

PubMed2004

Microglia.

Microglia--the macrophage equivalent of the CNS--safeguards and supports neuronal functions. Threats to the CNS homeostasis can trigger a rapid transformation of these cells from a normally "resting" into alerted and "activated" states. Microglia primarily serves the tissue defence and protection when participating in mechanisms of innate and adaptive immunity. On the contrary, excessive acute or chronic microglial activation can provoke severe neuronal and glial damage by carrying or fuelling destructive cascades. Several factors and conditions have already been identified that maintain the resting phenotype or organize and control the activation process. Cells are thereby able to recognize a dangerous signal as well as to sense functional disturbance. Microglial activation is also proving a much more variable and adaptive process than previously noticed. Aiming at microglia as a therapeutic target, research may focus on intracellular pathways that are probably common to activation scenarios as triggered by various receptor systems. Certain signalling elements may have key roles in the cytosolic integration of sensory inputs and a conversion into programs of executive performance. As the integrative aspect of microglial activation becomes illuminated hope builds up also on strategies for selective interference with harmful outcomes in favour of the--phylogenetically approved--beneficial potential of these fascinating cells.

Animals↗

PubMed2004

The tyrosine kinase inhibitor AG126 restores receptor signaling and blocks release functions in activated microglia (brain macrophages) by preventing a chronic rise in the intracellular calcium level.

We recently reported that lasting activation of mouse microglial cells with bacterial lipopolysaccharide (LPS) chronically elevated the basal intracellular calcium concentration ([Ca2+]i). This correlated to an attenuated calcium signaling of complement (C5a) and purinergic (UTP) receptors as well as to the capacity for effective production of cytokines-chemokines. Here, we demonstrate that these adjustments in the [Ca2+]i regulation require a critical protein tyrosine kinase (PTK) function--even in varying stimulation scenarios. Changes in basal [Ca2+]i and calcium signaling are not restricted to Gram-negative bacterial confrontation. Pneumococcal cell wall (PCW) modelling Gram-positive infection causes virtually the same effects. Moreover, decreases in calcium signaling efficacy are neither associated with altered receptor expression, nor mediated by autocrine loops. Administration of microglial release products, transfer of conditioned supernatant or presence of a radical scavenger during LPS or PCW treatments have no consequence. However, both the elevation in basal [Ca2+]i as well as the suppression of C5a- and UTP-evoked calcium signals are selectively and dose-dependently reversed by tyrphostin AG126, a PTK inhibitor that, moreover, blocks inducible nitric oxide and cytokine-chemokine release. The findings suggest that the AG126-sensitive PTK critically controls both sensory and executive features of the microglial activation process via sustained up-regulation of basal [Ca2+]i.

Animals↗

PubMed2004

Tyrosine kinase inhibition reduces inflammation in the acute stage of experimental pneumococcal meningitis.

Bacterial meningitis is an acute inflammatory disease of the central nervous system with a mortality rate of up to 30%. Excessive stimulation of the host immune system by bacterial surface components contributes to this devastating outcome. In vitro studies have shown that protein tyrosine kinase inhibitors are highly effective in preventing the release of proinflammatory cytokines induced by pneumococcal cell walls in microglia. In a well-established rat model, intracisternal injection of purified pneumococcal cell walls induced meningitis characterized by increases in the regional cerebral blood flow and intracranial pressure, an influx of leukocytes, and high concentrations of tumor necrosis factor alpha (TNF-alpha) in the cerebrospinal fluid. Compared with the values at the beginning of the experiment, intraperitoneal injection of tyrphostin AG 126 reduced the increases in regional cerebral blood flow (at 6 h, 127% +/- 14% versus 222% +/- 51% of the baseline value; P < 0.05) and intracranial pressure (at 6 h, 0.8 +/- 2.4 versus 5.4 +/- 2.0 mm of Hg; P < 0.05), the influx of leukocytes (at 6 h, 1,336 +/- 737 versus 4,350 +/- 2,182 leukocytes/microl; P < 0.05), and the TNF-alpha concentration (at 6 h, 261 +/- 188 versus 873 +/- 135 pg/microl; P < 0.05). These results demonstrate that inhibition of AG 126-sensitive tyrosine kinase pathways may provide new approaches for preventing excessive inflammation and reducing the increases in blood flow and intracranial pressure in the acute phase of bacterial meningitis.

Acute Disease↗

PubMed2003

Expression of proteinase-activated receptors in mouse microglial cells.

Microglia are the resident immune cells of the CNS: they are activated rapidly by CNS damage and perform the function of tissue macrophages. The first steps during microglial activation are currently under intense study, and it is widely believed that substances released from damaged brain tissue can trigger this process. We recently reported that the blood coagulation factor thrombin, which enters the CNS during breakdown of the blood-brain barrier, activates microglial cells. The cellular effects of thrombin and trypsin-like proteases are mediated by proteinase-activated receptors (PARs). Based on our prior data we hypothesized that microglial cells express these receptors. Using RT-PCR and flow cytometry, we report that primary mouse microglial cells, as well as the murine microglial cell lines BV-2 and N9, indeed express PARs, albeit at different levels. Demonstrating multiple PARs on microglia may enhance the attractiveness of PARs as therapeutic targets in neuroinflammatory disorders.

Animals↗

PubMed2003

Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function.

Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia.

Animals↗

PubMed2003

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro.

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.

Adenosine Triphosphate↗

PubMed2002

Microglia as a source and target of cytokines.

Cytokines constitute a significant portion of the immuno- and neuromodulatory messengers that can be released by activated microglia. By virtue of potent effects on resident and invading cells, microglial cyto- and chemokines regulate innate defense mechanisms, help the initiation and influence the type of immune responses, participate in the recruitment of leukocytes to the CNS, and support attempts of tissue repair and recovery. Microglia can also receive cyto- and chemokine signals as part of auto- and paracrine communications with astrocytes, neurons, the endothelium, and leukocyte infiltrates. Strong responses and modulatory influences can be demonstrated, adding to the emerging view that microglial behavior is highly dependent on the (cytokine) environment and that reactions to a challenge may vary with the stimulation context. In principle, microglial activation aims at CNS protection. However, failed microglial engagement due to excessive or sustained activation could significantly contribute to acute and chronic neuropathologies. Dysregulation of microglial cytokine production could thereby promote harmful actions of the defense mechanisms, result in direct neurotoxicity, as well as disturb neural cell functions as they are sensitive to cytokine signaling.

Animals↗

PubMed2002

Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages.

During bacterial infections of the CNS, activated microglia could support leucocyte recruitment to the brain through the synthesis of cyto- and chemokines. In turn, invading leucocytes may feedback on microglial cells to influence their chemokine release pattern. Here, we analyzed the capacity of interferon-gamma (IFNgamma) to serve as such a leucocyte-to-microglia signal. Production of cyto- and chemokines was stimulated in mouse microglia cultures by treatments with lipopolysaccharide (LPS) from Gram-negative Escherichia coli or cell walls from Gram-positive Streptococcus pneumoniae (PCW). IFNgamma presence during the stimulation (0.1-100 ng/mL) modulated the patterns of LPS- and PCW-induced cyto- and chemokine release in a dose-dependent, potent and complex manner. While amounts of TNFalpha and IL-6 remained nearly unchanged, IFNgamma enhanced the production of IL-12, MCP-1 and RANTES, but attenuated that of KC, MIP-1alpha and MIP-2. Release modulation was obtained with IFNgamma preincubation (treatment of cells before LPS or PCW administration), coincubation and even delayed addition to an ongoing LPS or PCW stimulation. Together the changes observed for the microglial chemokine release under IFNgamma would shift the chemoattractive profile from favouring neutrophils to a preferential attraction of monocytes and T lymphocyte populations--as actually seen during the course of bacterial meningitis. The findings support the view of activated microglia as a major intrinsic source for an instant production of a variety of chemokines and suggest that leucocyte-derived IFNgamma could potentially regulate the microglial chemokine release pattern.

Animals↗