Jerome J. A. Hendriks

Flavonoids Influence Monocytic GTPase Activity and Are Protective in Experimental Allergic Encephalitis

In the chronic disabling disease multiple sclerosis (MS), migration of monocytes across the blood-brain barrier is a crucial step in the formation of new lesions in the central nervous system (CNS). Infiltrating monocyte-derived macrophages secrete inflammatory mediators such as oxygen radicals, which contribute to axonal demyelination and damage, resulting in neurological deficits. Flavonoids are compounds occurring naturally in food, which scavenge oxygen radicals and have antiinflammatory properties. To investigate whether they might suppress clinical symptoms in MS, we treated rats sensitized for acute and chronic experimental allergic encephalomyelitis, an experimental model of MS, with flavonoids. We demonstrated that the flavonoid luteolin substantially suppressed clinical symptoms and prevented relapse when administered either before or after disease onset. Luteolin treatment resulted in reduced inflammation and axonal damage in the CNS by preventing monocyte migration across the brain endothelium. Luteolin influenced migration by modulating the activity of Rho GTPases, signal transducers involved in transendothelial migration. Oral administration of luteolin also significantly reduced clinical symptoms.

Signal-Regulatory Protein α-CD47 Interactions Are Required for the Transmigration of Monocytes Across Cerebral Endothelium

Monocyte infiltration into inflamed tissue requires their initial arrest onto the endothelial cells (ECs), followed by firm adhesion and subsequent transmigration. Although several pairs of adhesion molecules have been shown to play a role in the initial adhesion of monocytes to ECs, the mechanism of transendothelial migration is poorly defined. In this study, we have investigated the role of signal-regulatory protein (SIRP)α-CD47 interactions in monocyte transmigration across brain ECs. CD47 expression was observed in vivo on cerebral endothelium of both control animals and animals suffering from experimental allergic encephalomyelitis. To investigate whether SIRPα-CD47 interactions are instrumental in the trafficking of monocytes across cerebral EC monolayers, in vitro assays were conducted in which the migration of monocytes, but not adhesion, was found to be effectively diminished by blocking SIRPα and CD47 on monocytes and ECs, respectively. In this process, SIRPα was found to interact solely with its counterligand CD47 on ECs. Overexpression of the CD47 molecule on brain ECs significantly enhanced monocytic transmigration, but did not affect adhesion. SIRPα-CD47-mediated transendothelial migration involved Gi protein activity, a known signaling component of CD47. Finally, cross-linking of CD47 on brain ECs induced cytoskeletal reorganization of the endothelium, a process that was Gi protein independent. These data provide the first evidence that the interaction of CD47 with its monocytic counterligand SIRPα is of importance in the final step of monocyte trafficking into the brain, a critical event in the development of neuroinflammatory diseases.

Macrophage subsets and microglia in multiple sclerosis

Along with microglia and monocyte-derived macrophages, macrophages in the perivascular space, choroid plexus, and meninges are the principal effector cells in neuroinflammatory and neurodegenerative disorders. These phagocytes are highly heterogeneous cells displaying spatial- and temporal-dependent identities in the healthy, injured, and inflamed CNS. In the last decade, researchers have debated on whether phagocytes subtypes and phenotypes are pathogenic or protective in CNS pathologies. In the context of this dichotomy, we summarize and discuss the current knowledge on the spatiotemporal physiology of macrophage subsets and microglia in the healthy and diseased CNS, and elaborate on factors regulating their behavior. In addition, the impact of macrophages present in lymphoid organs on CNS pathologies is defined. The prime focus of this review is on multiple sclerosis (MS), which is characterized by inflammation, demyelination, neurodegeneration, and CNS repair, and in which microglia and macrophages have been extensively scrutinized. On one hand, microglia and macrophages promote neuroinflammatory and neurodegenerative events in MS by releasing inflammatory mediators and stimulating leukocyte activity and infiltration into the CNS. On the other hand, microglia and macrophages assist in CNS repair through the production of neurotrophic factors and clearance of inhibitory myelin debris. Finally, we define how microglia and macrophage physiology can be harnessed for new therapeutics aimed at suppressing neuroinflammatory and cytodegenerative events, as well as promoting CNS repair. We conclude that microglia and macrophages are highly dynamic cells displaying disease stage and location-specific fates in neurological disorders. Changing the physiology of divergent phagocyte subsets at particular disease stages holds promise for future therapeutics for CNS pathologies.

Flavonoids inhibit myelin phagocytosis by macrophages; a structure–activity relationship study

Demyelination is a characteristic hallmark of the neuro-inflammatory disease multiple sclerosis. During demyelination, macrophages phagocytose myelin and secrete inflammatory mediators that worsen the disease. Here, we investigated whether flavonoids, naturally occurring immunomodulating compounds, are able to influence myelin phagocytosis by macrophages in vitro. The flavonoids luteolin, quercetin and fisetin most significantly decreased the amount of myelin phagocytosed by a macrophage cell line without affecting its viability. IC(50) values for these compounds ranged from 20 to 80 microM. The flavonoid structure appeared to be essential for observed effects as flavonoids containing hydroxyl groups at the B-3 and B-4 positions in combination with a C-2,3 double bond were most effective. The capacity of the various flavonoids to inhibit phagocytosis correlated well with their potency as antioxidant, which is in line with the requirement of reactive oxygen species for the phagocytosis of myelin by macrophages. Our results implicate that flavonoids may be able to limit the demyelination process during multiple sclerosis.

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Quantitative real time polymerase chain reaction (qPCR) has become a widely used tool to examine gene expression levels. Reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple reference genes is becoming the standard, although the most suitable reference genes depend on the applied treatment as well as the tissue or cell type studied. In this study the stability of various reference genes was investigated in cultures of oligodendrocytes derived from either mature or neonatal rats, the latter also in the presence of the liver X receptor (LXR) agonist. The expression stability of ten commonly used reference genes (HPRT, GAPDH, 18S, ActB, CycA, Tbp, Rpl13A, YWHAZ, HMBS, Pgk1) was analyzed using geNorm and NormFinder. When comparing the different types of cell cultures, Rpl13A, CycA, Pgk1 and YWHAZ were identified as most stable genes. After LXR agonist treatment, CycA, Pgk1 and Rpl13A were found to be the most stable by both geNorm and NormFinder. HMBS and the commonly used housekeeping genes GAPDH and 18S turned out to be the most variable according to geNorm and NormFinder. In conclusion, the use of multiple reference genes, instead of only one, in qPCR experiments with rat oligodendrocytes is strongly advised and standard housekeeping genes such as GAPDH and 18S are not recommended as they appear to be relatively unstable under the experimental conditions used. Reference gene selection should always be performed for each individual experiment, since useful reference genes are very specific for every situation.

Leukemia inhibitory factor is produced by myelin-reactive T cells from multiple sclerosis patients and protects against tumor necrosis factor-α-induced oligodendrocyte apoptosis

In multiple sclerosis (MS), damage to oligodendrocytes is believed to be caused by an aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that these T cells are not exclusively detrimental but might also exert protective effects. We report for the first time that myelin‐reactive T‐cell clones from eight MS patients (6/19) and five healthy controls (4/11) produce leukemia inhibitory factor (LIF), a member of the neuropoietic family of neurotrophins. In addition, T‐cell clones specific for tetanus toxoid, CD4+ and CD8+ T cells, and monocytes, but not B cells, secreted LIF. LIF‐producing T lymphocytes and macrophages were also identified immunohistochemically in both active and chronic‐active MS lesions. We further demonstrated dose‐dependent protective effects of LIF on tumor necrosis factor‐α‐induced apoptosis of oligodendrocytes. In conclusion, our data demonstrate that peripheral and CNS‐infiltrating T cells from MS patients produce LIF, a protective factor for oligodendrocytes. This study emphasizes that secretion of LIF may contribute to the neuroprotective effects of autoreactive T cells.

The role of mast cells in neuroinflammation

Mast cells (MCs) are densely granulated perivascular resident cells of hematopoietic origin and well known for their pathogenetic role in allergic and anaphylactic reactions. In addition, they are also involved in processes of innate and adaptive immunity. MCs can be activated in response to a wide range of stimuli, resulting in the release of not only pro-inflammatory, but also anti-inflammatory mediators. The patterns of secreted mediators depend upon the given stimuli and microenvironmental conditions, accordingly MCs have the ability to promote or attenuate inflammatory processes. Their presence in the central nervous system (CNS) has been recognized for more than a century. Since then a participation of MCs in various pathological processes in the CNS has been well documented. They can aggravate CNS damage in models of brain ischemia and hemorrhage, namely through increased blood–brain barrier damage, brain edema and hemorrhage formation and promotion of inflammatory responses to such events. In contrast, recent evidence suggests that MCs may have a protective role following traumatic brain injury by degrading pro-inflammatory cytokines via specific proteases. In neuroinflammatory diseases such as multiple sclerosis, the role of MCs seems to be ambiguous. MCs have been shown to be damaging, neuroprotective, or even dispensable, depending on the experimental protocols used. The role of MCs in the formation and progression of CNS tumors such as gliomas is complex and both positive and negative relationships between MC activity and tumor progression have been reported. In summary, MCs and their secreted mediators modulate inflammatory processes in multiple CNS pathologies and can thereby either contribute to neurological damage or confer neuroprotection. This review intends to give a concise overview of the regulatory roles of MCs in brain disease.

Leukemia inhibitory factor modulates production of inflammatory mediators and myelin phagocytosis by macrophages.

Leukemia inhibitory factor (LIF) promotes survival of glial cells and neurons during autoimmune and injury responses in the central nervous system (CNS). While various studies indicate that LIF also modulates ongoing inflammatory responses, data on underlying events are lacking. In this study we demonstrate that LIF modulates macrophage function. LIF inhibits the production of oxygen radicals and TNFalpha and stimulates myelin uptake by macrophages. These effects of LIF are accompanied by activation of the JAK/STAT3 signalling pathway. Our findings demonstrate that LIF has anti-inflammatory properties and enhances myelin clearance, implicating that LIF may be an important factor in CNS inflammatory disease.

Leukemia inhibitory factor induces an antiapoptotic response in oligodendrocytes through Akt-phosphorylation and up-regulation of 14-3-3

Leukemia inhibitory factor (LIF) promotes the survival of oligodendrocytes (OLG) both in vitro and in an animal model of multiple sclerosis. Here, we show that LIF protects mature rat OLG cultures selectively against the combined insult of the proinflammatory cytokines interferon‐γ and tumor necrosis factor‐α, but it does not protect against oxidative stress nor against staurosporine induced apoptosis. We further demonstrate that LIF activates the janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and the phosphatidylinositol 3 kinase/Akt pathway in mature OLG. We show that LIF protection is independent of suppressors of cytokine signaling and Bcl‐2 mRNA expression levels. To gain further insight into the protective mechanism, a quantitative proteomic approach (DIGE) was applied to identify differentially expressed proteins in LIF‐treated OLG. Our results indicate that LIF induces a shift in the cellular machinery toward a prosurvival execution program, illustrated by an enhanced expression of isoforms of the antiapoptotic molecule 14‐3‐3. These data provide further insight into the mechanisms of LIF‐mediated protection of mature OLGs.

Selective Identification of Macrophages and Cancer Cells Based on Thermal Transport through Surface-Imprinted Polymer Layers

In this article, we describe a novel straightforward method for the specific identification of viable cells (macrophages and cancer cell lines MCF-7 and Jurkat) in a buffer solution. The detection of the various cell types is based on changes of the heat transfer resistance at the solid-liquid interface of a thermal sensor device induced by binding of the cells to a surface-imprinted polymer layer covering an aluminum chip. We observed that the binding of cells to the polymer layer results in a measurable increase of heat transfer resistance, meaning that the cells act as a thermally insulating layer. The detection limit was found to be on the order of 10(4) cells/mL, and mutual cross-selectivity effects between the cells and different types of imprints were carefully characterized. Finally, a rinsing method was applied, allowing for the specific detection of cancer cells with their respective imprints while the cross-selectivity toward peripheral blood mononuclear cells was negligible. The concept of the sensor platform is fast and low-cost while allowing also for repetitive measurements.