Leukocyte chemotaxis: from lysosomes to motility

Chemoattractants direct the extravasation of leukocytes to the site of immune response. New data highlight the role of synaptotagmins and Rab proteins in leukocyte chemotaxis

Finding the shortest path with PesCa: A tool for network reconstruction

The growing dimension and complexity of the available experimental data generating biological networks have increased the need for tools that help in categorizing nodes by their topological relevance. Here we present CentiScaPe, a Cytoscape app specifically designed to calculate centrality indexes used for the identification of the most important nodes in a network. CentiScaPe is a comprehensive suite of algorithms dedicated to network nodes centrality analysis, computing several centralities for undirected, directed and weighted networks. The results of the topological analysis can be integrated with data set from lab experiments, like expression or phosphorylation levels for each protein represented in the network. Our app opens new perspectives in the analysis of biological networks, since the integration of topological analysis with lab experimental data enhance the predictive power of the bioinformatics analysis

A deadly migration

CD95 has long been viewed as a death receptor regulating apoptosis. In this issue of Immunity, Letellier et al. (2010) tell us a different story, about the capability of CD95L to regulate leukocyte recruitment to sites of inflammation

Aberrant BLM cytoplasmic expressionassociates with DNA damage stress and hypersensitivity to DNA-damaging agents in colorectal cancer

Background Bloom syndrome is a rare and recessive disorder characterized by loss-of-function mutations of the BLM gene, which encodes a RecQ 30–50 DNA helicase. Despite its putative tumor suppressor function, the contribution of BLM to human sporadic colorectal cancer (CRC) remains poorly understood. Methods The transcriptional regulation mechanism underlying BLM and related DNA damage response regulation in independent CRC subsets and a panel of derived cell lines was investigated by bioinformatics analysis, the transcriptomic profile, a CpG island promoter methylation assay, Western blot, and an immunolocalization assay. Results In silico analysis of gene expression data sets revealed that BLM is overexpressed in poorly differentiated CRC and exhibits a close connection with shorter relapsefree survival even after adjustment for prognostic factors and pathways that respond to DNA damage response through ataxia telangiectasia mutated (ATM) signaling. Functional characterization demonstrated that CpG island promoter hypomethylation increases BLM expression and associates with cytoplasmic BLM mislocalization and increased DNA damage response both in clinical CRC samples and in derived cancer cell lines. The DNA-damaging agent S-adenosylmethionine suppresses BLM expression, leading to the inhibition of cell growth following accumulation of DNA damage. In tumor specimens, cytoplasmic accumulation of BLM correlates with DNA damage and cH2AX and phosphorylated ATM foci and predicts long-term progression-free survival in metastatic patients treated with irinotecan. Conclusions Taken together, the findings of this study provide the first evidence that cancer-linked DNA hypomethylation and cytosolic BLM mislocalization might reflect compromised levels of DNA-repair activity and enhanced hypersensitivity to DNA-damaging agents in CRC patients

Evidence of ζ Protein Kinase C Involvement in Polymorphonuclear Neutrophil Integrin-dependent Adhesion and Chemotaxis

Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation

Analysing omics data sets with weighted nodes networks (WNNets)

Current trends in biomedical research indicate data integration as a fundamental step towards precision medicine. In this context, network models allow representing and analysing complex biological processes. However, although effective in unveiling network properties, these models fail in considering the individual, biochemical variations occurring at molecular level. As a consequence, the analysis of these models partially loses its predictive power. To overcome these limitations, Weighted Nodes Networks (WNNets) were developed. WNNets allow to easily and effectively weigh nodes using experimental information from multiple conditions. In this study, the characteristics of WNNets were described and a proteomics data set was modelled and analysed. Results suggested that degree, an established centrality index, may offer a novel perspective about the functional role of nodes in WNNets. Indeed, degree allowed retrieving significant differences between experimental conditions, highlighting relevant proteins, and provided a novel interpretation for degree itself, opening new perspectives in experimental data modelling and analysis. Overall, WNNets may be used to model any high-throughput experimental data set requiring weighted nodes. Finally, improving the power of the analysis by using centralities such as betweenness may provide further biological insights and unveil novel, interesting characteristics of WNNets

RhoA and ζ PKC Control Distinct Modalities of LFA-1 Activation by Chemokines

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Molecular Mechanisms Involved in Lymphocyte Recruitment in Inflamed Brain Microvessels: Critical Roles for P-Selectin Glycoprotein Ligand-1 and Heterotrimeric Gi-Linked Receptors

AbstractLymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. We developed a novel intravital microscopy model to directly analyze through the skull the interactions between lymphocytes and the endothelium in cerebral venules of mice. No adhesive interactions were observed between lymphocytes and the nonactivated endothelium in the cerebral microcirculation. When brain venules were activated by pretreating mice with TNF-α or LPS, proteolipid protein 139–151 autoreactive T lymphocytes rolled and arrested; notably, only a few peripheral lymph node cells rolled and firmly adhered. Abs anti-P-selectin glycoprotein ligand-1 and anti-E- and P-selectin blocked tethering and rolling of autoreactive lymphocytes, suggesting that P-selectin glycoprotein ligand-1/endothelial selectins are critical in the recruitment of lymphocytes in inflamed brain venules. E- and P-selectin were expressed on cerebral vessels upon in vivo activation and had a patchy distribution during the preclinical phase of active and passive experimental autoimmune encephalomyelitis. LFA-1/ICAM-1 and α4 integrins/VCAM-1 supported rolling, but were not relevant to rolling velocity. Firm arrest was mainly mediated by LFA-1 and ICAM-1. Pretreatment of autoreactive lymphocytes with pertussis toxin blocked integrin-dependent arrest, implicating a requirement for Gi protein-dependent signaling in vessels from nonlymphoid districts. In conclusion, our data unveils the molecular mechanisms controlling the recruitment of autoreactive lymphocytes in inflamed cerebral vessels and suggest new insights into the pathogenesis of autoimmune inflammatory diseases of the CNS

Concurrency in leukocyte vascular recognition: developing the tools for a predictive computer model

Leukocyte recruitment has a crucial role in inflammation and immunity. An interplay between adhesion molecules and pro-adhesive agonists generates a complex molecular network controlling tissue-specific and inflammation-dependent leukocyte vascular recognition. Recent findings highlight the importance of quantitative parameters in controlling the specificity of leukocyte vascular recognition. Introduction of quantitative parameters demonstrates the non-linear behavior of the process and suggests the necessity for a revision of the traditional model. We propose a formalization of the original multi-step model of leukocyte vascular recognition by introducing the notion of concurrency that explains how the quantitative variation of pro-adhesive parameters might control the specificity and the sensitivity of this process. Moreover, we discuss how concurrency, by integrating quantitative parameters, constitutes a central concept for the implementation of a predictive computer modeling of leukocyte vascular recognition

Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule.

Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function