Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation

BACKGROUND: In neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS. METHODS: Primary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton. RESULTS: Significant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology. CONCLUSIONS: Together our results indicate that the alternative activation status of macrophages promotes their migratory properties to chemoattractants relevant for neuroinflammatory diseases like MS. Conversely, classically activated, proinflammatory macrophages have reduced migratory properties. Based on our results, we postulate that the activation status of the macrophage influences the capacity of the macrophages to rearrange their cytoskeleton. This is the first step in understanding how modulation of macrophage activation affects macrophage migration in neuroinflammatory diseases like MS

Nuclear factor-{kappa}B is not essential for NADPH oxidase activity in neutrophils from anhidrotic ectodermal dysplasia patients.

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The Modjokerto Regency Area [East Java]

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcgamma receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcgamma receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response

The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, upregulation of host defense proteins through toll-like receptors and NFkappaB requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4-Myd88-and abl tyrosine kinase-dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an in vivo model of bacteremia, neutrophils crawled to and rapidly phagocytosed Escherichia coli sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens

Proliferating or Differentiating Stimuli Act on Different Lipid-dependent Signaling Pathways in Nuclei of Human Leukemia Cells

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-βII migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the α isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-βII occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-α to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease

Glass transition under confinement-what can be learned from calorimetry

Calorimetry is an effective analytical tool to characterize the glass transition and phase transitions under confinement. Calorimetry offers a broad dynamic range regarding heating and cooling rates, including isothermal and temperature modulated operation. Today 12 orders of magnitude in scanning rate can be covered by combining different types of calorimeters. The broad dynamic range, comparable to dielectric spectroscopy, is especially of interest for the study of kinetically controlled processes like crystallization or glass transition. Accuracy of calorimetric measurements is not very high. Commonly it does not reach 0.1% and often accuracy is only a few percent. Nevertheless, calorimetry can reach high sensitivity and reproducibility. Both are of particular interest for the study of confined systems. Low addenda heat capacity chip calorimeters are capable to measure the step in heat capacity at the glass transition in nanometer thin films. The good reproducibility is used for the study of glass forming materials confined by nanometer sized structures, like porous glasses, semicrystalline structures, nanocomposites, phase separated block copolymers, etc. Calorimetry allows also for the frequency dependent measurement of complex heat capacity in a frequency range covering several orders of magnitude. Here I exclusively consider calorimetry and its application to glass transition in confined materials. In most cases calorimetry reveals only a weak dependence of the glass transition temperature on confinement as long as the confining dimensions are above 10 nm. Why these findings contradict many other studies applying other techniques to similar systems is still an unsolved problem of glass transition in confinement