In vitro and in vivo models to study chemokine regulation of angiogenesis.
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Allantois↗
Biomedical subjects
Angela Santoni
Publications and source records attributed to Angela Santoni.
Evaluation of Rho family small G-protein activity induced by integrin ligation on human leukocytes.
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Antibodies↗
The cyclopentenone-type prostaglandin 15-deoxy-delta 12,14-prostaglandin J2 inhibits CD95 ligand gene expression in T lymphocytes: interference with promoter activation via peroxisome proliferator-activated receptor-gamma-independent mechanisms.
15-Deoxy-delta(12,14)-PGJ(2) (15d-PGJ(2)) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. 15d-PGJ(2) is a natural ligand of the peroxisome proliferator-activated receptor (PPAR)-gamma nuclear receptor, but relevant PPARgamma-independent actions mediated by this prostanoid have been described. Fas (APO-1/CD95) and its ligand (Fas-L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death and has been implicated in diseases in which lymphocyte homeostasis is compromised. Moreover, several studies have described the pathogenic functions of Fas and Fas-L in vivo, particularly in the induction-progression of organ-specific autoimmune diseases. In this study we describe the effect of 15d-PGJ(2) on the activation of the fas-L gene in T lymphocytes. We show that 15d-PGJ(2) inhibits fas-L mRNA expression, activation-induced cell death, and fas-L promoter activity by mechanisms independent of PPARgamma and mediated by its chemically reactive cyclopentenone moiety. Our data indicate that 15d-PGJ(2) may repress fas-L activation by interfering with the expression and/or transcriptional activity of different transcription factors (early growth response types 3 and 1, NF-kappaB, AP-1, c-Myc, Nur77) whose altered balancing and transactivation may contribute for overall repression of this gene. In addition, the activation/expression of the heat shock response genes HSF-1 and HSP70 is not directly involved in the repression, and the electrophilic molecule cyclopentenone (2-cyclopenten-1-one) may reproduce the effects mediated by 15d-PGJ(2). These results suggest that modulation of Fas-L by 15d-PGJ(2) in T cells may represent an additional tool to consider for treatment of specific autoimmune and inflammatory disorders.
Animals↗
Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration.
Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.
Cell Adhesion↗
Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells.
Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), pre-stimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5-9 days, induced CD3-/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover, we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies.
Antibodies, Monoclonal↗
Analysis of the role of chemokines in angiogenesis.
Chemokines, a large family of inflammatory cytokines, have been shown to play a critical role in the regulation of angiogenesis during several pathophysiologic processes, such as tumor growth, wound healing and ischemia. Semiquantitative or quantitative angiogenesis assays are commonly utilized to screen the angiogenic or angiostatic activity of chemokines. These include in vitro endothelial cell activation assays and ex vivo or in vivo models of neovascularization. Chemokines may exert their regulatory activity on angiogenesis directly or as a consequence of leukocyte infiltration and/or the induction of growth factor expression. The effect of chemokines on endothelium can be assessed by performing in vitro assays on purified endothelial cell populations or by in vivo assays. Nevertheless, each model used to evaluate the angiogenic or angiostatic activity of a discrete factor has advantages and limitations. Thus, in order to avoid under- or overestimating the regulatory effect of chemokines on angiogenesis and to evaluate all aspects of the angiogenic process, multiple assays are usually performed. This review summarizes past and recent studies on chemokines as modulators of angiogenesis with particular emphasis on the methods currently used for the assessment of chemokine-mediated angiogenic or angiostatic responses.
Animals↗
Defective expression of the T-cell receptor-CD3 zeta chain in T-cell acute lymphoblastic leukaemia.
This study analysed the T-cell receptor (TCR)-CD3 zeta complex and the signal transduction apparatus of T-acute lymphoblastic leukaemia (T-ALL) blasts, and investigated the function of the ubiquitin-proteasome system. In all nine T-ALL samples studied, the leukaemic cells showed a marked reduction in the expression of the zeta chain, while a variety of tyrosine kinases (p56lck, ZAP70 and SYK) were normally present. There was no expression of the FcepsilonRIgamma chain. To confirm that this aberration was specific to immature T-ALL blasts, we investigated two patients with lymphoproliferative disorders of granular lymphocytes (LDGL), characterized by the expansion of mature T lymphocytes and found normal zeta chain expression. The reduction of the zeta chain protein was not reversible after 72 h stimulation with the anti-CD3 monoclonal antibody and interleukin 2, either alone or in combination. Northern blot analysis indicated that the reduced protein expression did not correspond to a defect at the mRNA level, nor were mutations in the coding region of the zeta chain found. We, therefore, hypothesized that the observed reduction of protein expression in T-ALL blasts could be secondary to an increased degradation at the proteasome level. Following selective inhibition of the proteasome, a marked increase of the zeta chain expression was observed. Moreover, an increase in the surface expression of CD3 was also documented. Taken together, these results indicate that the expression of the zeta subunit of the TCR-CD3 complex is consistently reduced in T-ALL blasts and that degradation of the protein is mediated by the proteasome system.
Adult↗
Memory T-cell competition for bone marrow seeding.
The presence in the bone marrow of memory CD8 T cells is well recognized. However, it is still largely unclear how T-cell migration from the lymphoid periphery to the bone marrow is regulated. In the present report, we show that antigen-specific CD4 T cells, as well as antigen-specific CD8 T cells, localize to the bone marrow of immunized mice, and are sustained there over long periods of time. To investigate the rules governing T-cell migration to the bone marrow, we generated chimeric mice in which the lymphoid periphery contained two genetically or phenotypically distinct groups of T cells, one of which was identical to the host. We then examined whether a distinct type of T cell had an advantage over the others in the colonization of bone marrow. Our results show that whereas ICAM1 and CD18 molecules are both involved in homing to lymph nodes, neither is crucial for T-cell bone marrow colonization. We also observed that memory-phenotype CD44high T cells, but not virgin-type CD44-/low T cells, preferentially home to the bone marrow upon adoptive transfer to normal young mice, but not to thymectomized old recipients where an existing memory T-cell pool precludes their free access. Thus, T-cell colonization of the bone marrow uses distinct molecules from those implicated in lymph node homing, and is regulated both by the properties of the T cell and by the competitive efficacy of other T cells inhabiting the same, saturable niche. This implies that the homing potential of an individual lymphocyte is not merely an intrinsic property of the cell, but rather a property of the lymphoid system taken as a whole.
Adoptive Transfer↗
The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway.
Several chemokines have been shown to regulate cellular apoptosis following discrete stimuli. It was previously demonstrated that the CC chemokine CCL1 (I-309) rescues thymic lymphoma cells from apoptosis by unknown mechanisms. The aim of our study was to characterize the role of the CC chemokine receptor 8 (CCR8), the only described receptor for CCL1, in the rescue of murine thymic lymphoma cells and murine thymocytes from dexamethasone (dex)-induced apoptosis. We show here that the CCR8-restricted agonist Kaposi sarcoma-associated herpesvirus-encoded chemokine viral macrophage-inflammatory protein-1 (vMIP-1) rescues thymic lymphoma cells from dex-induced apoptosis, similar to CCL1, and that such rescue is extracellular-regulated kinase-dependent. Although it has been hypothesized that the rescuing effect of CCL1 from apoptosis could be CCR8-mediated, here, we formally demonstrate the role of such receptor as its selective antagonist encoded by the MC148 gene of molluscum contagiosum virus MC148/vMCC-I inhibits v-MIP-1- and CCL1-induced rescue activity. In addition, CCR8 ligands inhibit dex-induced apoptosis of murine thymocytes with potential implications for thymic selection.
Animals↗
SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells.
Membrane recruitment of the SH2-containing 5' inositol phosphatase 1 (SHIP-1) is responsible for the inhibitory signals that modulate phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. Here we have investigated the molecular mechanisms underlying SHIP-1 activation and its role in CD16-mediated cytotoxicity. We initially demonstrated that a substantial fraction of SHIP-1-mediated 5' inositol phosphatase activity associates with CD16 zeta chain after receptor cross-linking. Moreover, CD16 stimulation on human primary natural killer (NK) cells induces the rapid and transient translocation of SHIP-1 in the lipid-enriched plasma membrane microdomains, termed rafts, where it associates with tyrosine-phosphorylated zeta chain and shc adaptor protein. As evaluated by confocal microscopy, CD16 engagement by reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly induces SHIP-1 redistribution toward the area of NK cell contact with target cells and its codistribution with aggregated rafts where CD16 receptor also colocalizes. The functional role of SHIP-1 in the modulation of CD16-induced cytotoxicity was explored in NK cells infected with recombinant vaccinia viruses encoding wild-type or catalytic domain-deleted mutant SHIP-1. We found a significant SHIP-1-mediated decrease of CD16-induced cytotoxicity that is strictly dependent on its catalytic activity. These data demonstrate that CD16 engagement on NK cells induces membrane targeting and activation of SHIP-1, which acts as negative regulator of ADCC function.
Antibody-Dependent Cell Cytotoxicity↗
Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells.
Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.
Animals↗
Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells.
CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.
Animals↗
Negative regulation of CD95 ligand gene expression by vitamin D3 in T lymphocytes.
Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)(2)D(3) is a secosteroid hormone that activates the nuclear receptor vitamin D(3) receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-gamma and IL-12 genes by 1,25(OH)(2)D(3) in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)(2)D(3) on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)(2)D(3) inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)(2)D(3)-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)(2)D(3), and confirm the interest for a possible pharmacological use of this molecule and its derivatives.
Animals↗
Production of MCP-1 and RANTES in bladder cancer patients after bacillus Calmette-Guerin immunotherapy.
Bacillus Calmette-Guerin (BCG) therapy induces a local immunological response mediated by cellular immune and inflammatory reactions that enhance its anti-tumor efficacy in bladder cancer. Monocyte chemotactic protein-1 (MCP-1) and the "regulated on activation normal T expressed and secreted" chemokine (RANTES) are potent chemotactic molecules that attract monocytes and memory T cells. MCP-1 and RANTES levels in patients with superficial bladder cancer treated with intravesical instillations of BCG are significantly higher than in untreated cancer patients and controls. In the present study, the subjects were divided into three groups: (1) control subjects; (2) bladder cancer patients who did not receive BCG treatment; (3) bladder cancer patients who received intravesical administration of BCG. No differences in the basal production and expression of MCP-1 and RANTES mRNA were observed between BCG-treated and untreated patients. BCG treatment influenced the monocyte response to phytohemagglutinin (PHA) and BCG stimulation. After 24-h incubation, monocytes from BCG-treated bladder cancer patients released more MCP-1 and RANTES than those from untreated bladder cancer patients and controls. The anti-tumor effects of BCG observed in superficial bladder cancer therapy may depend on stimulation of the investigated chemokines, which attract monocytes/macrophages and memory T cells.
BCG Vaccine↗
Bone marrow CD8 T cells are in a different activation state than those in lymphoid periphery.
In addition to its well-established use for hematopoiesis reconstitution, bone marrow is considered with increasing interest as a possible source of mature cells for adoptive therapies, in particular for the immunotherapy of cancer. Nevertheless, the peculiarities of bone marrow T cells in comparison with those in lymphoid periphery are still largely unknown. In this report, we show for the first time that bone marrow CD8 T cells are in a different activation state than those in peripheral lymphoid organs. Firstly, we observed that mouse bone marrow contains a significantly higher percentage of blasts within the CD8 T cells than either spleen or lymph nodes, yet such enrichment is not due to recent antigenic stimulation. Secondly, when we challenged bone marrow CD8 T cells from immunized mice with their antigen in vitro, they displayed a faster response than those from the spleen. Thus, we suggest that the bone marrow could be a preferential source of CD8 T cells for adoptive therapies in those cases in which highly active effectors are required.
Animals↗
Preliminary in vivo experimental evidence on intratumoral morphine uptake. Possible clinical implications in cancer pain and opioid responsiveness.
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Analgesics, Opioid↗
Expansion of cytotoxic effectors with lytic activity against autologous blasts from acute myeloid leukaemia patients in complete haematological remission.
New therapeutic approaches are needed to improve the cure rates in acute myeloid leukaemia (AML). The present study was designed to investigate whether: (1) cytotoxic lymphocytes could be expanded from AML patients in complete remission; (2) their signal transduction machinery was preserved; (3) these cells were capable of producing cytokines involved in the cytolytic process; and (4) these cells showed cytotoxic activity against allogeneic and autologous blasts. By co-culturing blood mononuclear cells with feeder cells, we obtained an average 5.3-fold increase in the total cell number and a 35-fold increase in natural killer (NK) cells. Expression of the zeta chain and of tyrosine kinases of the Src and Syk-ZAP families involved in the triggering of NK functions was analysed on expanded cells. The results demonstrated a signal transduction apparatus preserved and quantitatively similar to that of normal donors. After phorbol myristate acetate and ionomicin stimulation, the ability of expanded cells to produce interferon gamma and tumour necrosis factor alpha was documented. Patients' expanded cells showed a cytotoxic activity against target lines and allogeneic blasts which was similar to that of normal donors. Purification experiments indicated that the NK cell fraction was responsible for most of the lytic effect. More significantly, these cells also exerted a lytic effect against autologous blasts that could be further enhanced following incubation with low-dose interleukin 2. These findings document the possibility of expanding cytotoxic effectors with preserved signal transduction machinery and autologous killing capacity from AML patients in remission, and suggest a new potential immunotherapeutic strategy for the management of early disease recurrence or of residual disease.
Acute Disease↗
Polyclonal Antibodies Against gp185HER2 Peptides: Their Putative Role in the Identification of a Particular HER2 Status in Patients With Breast Cancer.
SUMMARY: The HER2 oncogene and its relative oncoprotein, gp185HER2, a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, are overexpressed in a wide range of solid tumors including breast and ovarian cancer. In patients with breast cancer, both humoral and cell-mediated HER2 immune responses have been found as well as in some patients with gp185HER2 nonoverexpressing tumors. To establish whether peptide sequences identified as HLA-A2-restricted T-cell epitopes are expressed in breast tumor cell lines and tissues, we produced and characterized by different methodologic approaches polyclonal antibodies raised against four gp185HER2 peptides. Two of the antibodies recognized peptides eluted from the HLA-A2 groove of the mDAmB231 breast cancer cell line expressing a basal level of gp185HER2. Paraffin-embedded primary and metastatic breast tumors were specifically immunostained by all four reagents, thereby showing an overlapping reactivity. When this immunoreactivity was compared with that obtained using two different monoclonal antibodies, in 105 breast primary tumors and 36 corresponding lymph node metastases, we identified a subset of tumors that were negative with anti-gp185HER2 monoclonal antibodies and positive with the four antipeptide antibodies. Our novel observations provide in vivo evidence of the complexity involved in evaluating HER2 expression, and open a new path for understanding the biologic significance of HER2 status in breast tumors.
Journal Article↗