Search

Supplementary Table 1 from Comparative Analysis of Normal versus CLL B-Lymphocytes Reveals Patient-Specific Variability in Signaling Mechanisms Controlling LFA-1 Activation by Chemokines

Activation of lymphocyte function–associated antigen-1 (LFA-1) by chemokines is fine-tuned by inside-out signaling mechanisms responsible for integrin-mediated adhesion modulation. In the present study, we investigated the possibility of qualitative variability of signaling mechanisms controlling LFA-1 activation in chronic lymphocytic leukemia (CLL) cells. We pursued a multiplexed comparative analysis of the role of the recently described chemokine-triggered rho-signaling module in human normal versus CLL B-lymphocytes. We found that the rho-module of LFA-1 affinity triggering is functionally conserved in normal B-lymphocytes. In contrast, in malignant B-lymphocytes isolated from patients with B-CLL, the role of the rho-module was not maintained, showing remarkable differences and variability. Specifically, RhoA and phospholipase D1 were crucially involved in LFA-1 affinity triggering by CXCL12 in all analyzed patients. In contrast, Rac1 and CDC42 involvement displayed a consistent patient-by-patient variability, with a group of patients showing LFA-1 affinity modulation totally independent of Rac1 and CDC42 signaling activity. Finally, phosphatidylinositol-4-phosphate 5-kinase isoform 1γ (PIP5KC) was found without any regulatory role in all patients. The data imply that the neoplastic progression may completely bypass the regulatory role of Rac1, CDC42, and PIP5KC, and show a profound divergence in the signaling mechanisms controlling integrin activation in normal versus neoplastic lymphocytes, suggesting that patients with CLL can be more accurately evaluated on the basis of the analysis of signaling mechanisms controlling integrin activation. Our findings could potentially affect the diagnosis, prognosis, and therapy of CLL disorders. [Cancer Res 2009;69(24):9281–90]

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β 2 -AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton’s tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7–mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton’s tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

JAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1–mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking.

Ibrutinib is increasingly adopted for treating lymphoid malignancies. While growing amounts of data pile up about Ibrutinib mechanism of action on neoplastic B cells, little is known about its impact on other immune cells. Here we investigated the effect of Ibrutinib on monocyte/macrophage functions. (1) Ibrutinib treatment of purified human monocytes affected both chemoattractant-triggered inside-out as well as integrin-mediated outside-in signaling events, thus provoking defective adhesion and spreading on purified integrin ligands, respectively. (2) In in vitro cell-culture experiments, Ibrutinib promoted a differentiation shift of monocytes to fibrocyte-like cells, characterized by the acquisition of a typical elongated cell morphology. Importantly, this clear-cut shape transition also occurred upon culturing monocytes with sera derived from Ibrutinib-treated patients, thus clearly suggesting that the drug concentrations achievable in vivo can generate the phenotypic shift. (3) Ibrutinib-induced fibrocyte-like cells showed adhesion deficiency, altered phagocytic properties, and, with respect to macrophages, they acquired the capability of generating larger amounts of reactive oxygen species, possibly displaying different metabolic activities. Taken together, our results indicate that Ibrutinib has profound effects on the monocyte/macrophage immunobiology. They may finally shed some light about the biological ground of several Ibrutinib-related toxicities.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

// Antonella Rigo 1, 2 , Isacco Ferrarini 1, 2 , Angela Bonalumi 1 , Cristina Tecchio 1 , Alessio Montresor 3 , Carlo Laudanna 3 and Fabrizio Vinante 1, 2 1 Section of Hematology, Department of Medicine, University of Verona, Verona, Italy 2 Cancer Research and Cell Biology Laboratory, Department of Medicine, University of Verona, Verona, Italy 3 Section of General Pathology, Department of Medicine, University of Verona, Verona, Italy Correspondence to: Fabrizio Vinante, email: fabrizio.vinante@univr.it Keywords: apoptosis; autophagy; chronic lymphocytic leukemia; treatment; α-bisabolol Received: March 01, 2018 Accepted: April 28, 2018 Published: May 25, 2018 ABSTRACT The sesquiterpene α-bisabolol (α-BSB) is a cytotoxic agent against acute leukemia and chronic myeloid leukemia cells. Here the profile of α-BSB citotoxicity was evaluated ex vivo in primary mononuclear blood cells isolated from 45 untreated B-chronic lymphocytic leukemia (B-CLL) patients. We studied the effects of α-BSB by flow cytometric and western blotting techniques with the following findings: (1) α-BSB was an effective proapoptotic agent against B-CLL cells (IC 50 42 ± 15 μM). It was also active, but to a lesser extent, on normal residual B cells and monocytes (IC 50 68 ± 34 and 74 ± 28 μM, respectively; p < 0.01), while T-cells, though not achieving IC 50 , were nevertheless decreased. (2) Lipid raft content positively correlated with α-BSB cell sensitivity, while neither the phenotype of B-CLL cells nor the disease clinical stage did affect the sensitivity to α-BSB. (3) Flow cytometry analysis evidenced the induction of pores in mitochondrial and lysosomal membrane after 3- to 5-hour exposure of B-CLL cells to α-BSB, leading to apoptosis; in contrast, western blotting analysis showed inhibition of the autophagic flux. Therefore, according to cellular selectivity, α-BSB is a cytotoxic agent preferentially active against leukemic cells, while its lower activity on normal B cells, monocytes and T cells may account for an additive anti-inflammatory effect targeting the leukemia-associated pro-inflammatory microenvironment. Consistent with the observed effects on intracellular processes, α-BSB should be regarded as a dual agent, both activating mitochondrial-based apoptosis and inhibiting autophagy by disrupting lysosomes.

Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.

Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of protein tyrosine phosphatases in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type γ (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced β2 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphorylation on the critical 1007–1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Item does not contain fulltext BACKGROUND: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in CF patients, and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. METHODS: Ex vivo adhesion and chemotaxis, flow cytometry, immunofluorescence, GTPases activity assays. RESULTS: We found that chemoattractant-induced activation of beta1 and beta2 integrins and of chemotaxis is defective in mononuclear cells isolated from CF patients. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of beta1 and beta2 integrins was restored by treatment of CF monocytes with the CFTR correcting drugs VX325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR inh-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that in CF monocytes chemoattractant-triggered activation of RhoA and CDC42 rho small GTPases, controlling integrin activation and chemotaxis respectively, was strongly deficient. CONCLUSION: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell-type selective, leukocyte adhesion deficiency disease providing new insights on CF pathogenesis.

Inflammation is a pathological hallmark of Alzheimer's disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimer's disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-β (Aβ) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aβ 42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimer's disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimer's disease-like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimer's disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimer's disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimer's disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimer's disease.

Methods: Confocal microscopy studies were performed to evaluate inflammation mechanisms. Intra-vital microscopy studies using two-photon microscopy were performed to visualize and analyze the movement of neutrophils inside brain vessels and in the parenchyma. In vitro rapid adhesion assays were performed on integrin ligands whereas and integrin affinity was measured using Image Stream technology. Neuropathological studies, fear conditioning and Y maze tests were performed to analyze the effect of inflammation mechanism inhibition on disease.

The increasing availability of large network datasets derived from high-throughput experiments requires the development of tools to extract relevant information from biological networks, and the development of computational methods capable of detecting qualitative and quantitative changes in the topological properties of biological networks is of critical relevance. We introduce the notions of node interference and robustness as measures of the reciprocal influence between nodes within a network. We examine the theoretical significance of these new, centrality-based, measures by characterizing the topological relationships between nodes and groups of nodes. Node interference analysis allows topologically determining the context of functional influence of single nodes. Conversely, the node robustness analysis allows topologically identifying the nodes having the highest functional influence on a specific node. A new Cytoscape plug-in calculating these measures was developed and applied to a protein-protein interaction network specifically regulating integrin activation in human primary leukocytes. Notably, the functional effects of compounds inhibiting important protein kinases, such as SRC, HCK, FGR and JAK2, are predicted by the interference and robustness analysis, are in agreement with previous studies and are confirmed by laboratory experiments. The interference and robustness notions can be applied to a variety of different contexts, including, for instance, the identification of potential side effects of drugs or the characterization of the consequences of genes deletion, duplication or of proteins degradation, opening new perspectives in biological network analysis.

JAK tyrosine kinases promote integrin activation and integrin-meditated lymphocyte trafficking by bridging chemokine receptors to activation of Rho and Rap., Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1– and VLA-4–mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

Integrin-mediated adhesion is a general concept referring to a series of adhesive phenomena including tethering-rolling, affinity, valency, and binding stabilization altogether controlling cell avidity (adhesiveness) for the substrate. Arrest chemokines modulate each aspect of integrin activation, although integrin affinity regulation has been recognized as the prominent event in rapid leukocyte arrest induced by chemokines. A variety of inside-out and outside-in signaling mechanisms have been related to the process of integrin-mediated adhesion in different cellular models, but only few of them have been clearly contextualized to rapid integrin affinity modulation by arrest chemokines in primary leukocytes. Complex signaling processes triggered by arrest chemokines and controlling leukocyte integrin activation have been described for ras-related rap and for rho-related small GTPases. We summarize the role of rap and rho small GTPases in the regulation of rapid integrin affinity in primary leukocytes and provide a modular view of these pro-adhesive signaling events. A potential, albeit still speculative, mechanism of rho-mediated regulation of cytoskeletal proteins controlling the last step of integrin activation is also discussed. We also discuss data suggesting a functional integration between the rho- and rap-modules of integrin activation. Finally we examine the universality of signaling mechanisms regulating integrin triggering by arrest chemokines.

Activation of lymphocyte function–associated antigen-1 (LFA-1) by chemokines is fine-tuned by inside-out signaling mechanisms responsible for integrin-mediated adhesion modulation. In the present study, we investigated the possibility of qualitative variability of signaling mechanisms controlling LFA-1 activation in chronic lymphocytic leukemia (CLL) cells. We pursued a multiplexed comparative analysis of the role of the recently described chemokine-triggered rho-signaling module in human normal versus CLL B-lymphocytes. We found that the rho-module of LFA-1 affinity triggering is functionally conserved in normal B-lymphocytes. In contrast, in malignant B-lymphocytes isolated from patients with B-CLL, the role of the rho-module was not maintained, showing remarkable differences and variability. Specifically, RhoA and phospholipase D1 were crucially involved in LFA-1 affinity triggering by CXCL12 in all analyzed patients. In contrast, Rac1 and CDC42 involvement displayed a consistent patient-by-patient variability, with a group of patients showing LFA-1 affinity modulation totally independent of Rac1 and CDC42 signaling activity. Finally, phosphatidylinositol-4-phosphate 5-kinase isoform 1γ (PIP5KC) was found without any regulatory role in all patients. The data imply that the neoplastic progression may completely bypass the regulatory role of Rac1, CDC42, and PIP5KC, and show a profound divergence in the signaling mechanisms controlling integrin activation in normal versus neoplastic lymphocytes, suggesting that patients with CLL can be more accurately evaluated on the basis of the analysis of signaling mechanisms controlling integrin activation. Our findings could potentially affect the diagnosis, prognosis, and therapy of CLL disorders. [Cancer Res 2009;69(24):9281–90]

In this paper, evidence is provided that p75 neurotrophin receptor (p75NTR) exerts an opposite role on the cytotoxic function of beta-amyloid (Abeta) depending on the different state of the peptide, fibrillar or oligomeric soluble form. Previous work in our laboratory has shown that the expression of p75NTR is required for cell death in vitro by Abeta peptides in fibrillar form (G. Perini, V. Della-Bianca, V. Politi, G. Della Valle, I. Dal-Pra, F. Rossi, U. Armato. Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines. J. Exp. Med. 195 (2002) 907-918). In the present study, performed by using the same cell clones and procedures as in previous paper, we show that: (a) soluble oligomers of Abeta(1-42) exert a cytotoxic activity independent of p75NTR, (b) the expression of p75NTR exerts a protective role against the toxic activity of soluble oligomers, (c) this role is due to an active function of the juxtamembrane sequence of the cytoplasmic region of p75NTR and (d) the protective function is mediated by phosphatidylinositide 3-kinase (PI3K) activity.

SummaryEndothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.