Fleur Bossi

CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38(+)CD49d(+) versus CD38(-)CD49d(-) CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38(+)CD49d(+) but not CD38(-)CD49d(-) cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68(+) macrophage infiltration was particularly high in BMB from CD38(+)CD49d(+) CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor alpha overproduction. These effects were apparent in BMB from CD38(+)CD49d(+) CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1(+) stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38(+)CD49d(+) CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells.

In vivo Targeting of Human Neutralizing Antibodies against CD55 and CD59 to Lymphoma Cells Increases the Antitumor Activity of Rituximab

An in vivo model of human CD20+ B-lymphoma was established in severe combined immunodeficiency mice to test the ability of human neutralizing miniantibodies to CD55 and CD59 (MB55 and MB59) to enhance the therapeutic effect of rituximab. The miniantibodies contained single-chain fragment variables and the hinge-CH2-CH3 domains of human IgG(1). LCL2 cells were selected for the in vivo study among six B-lymphoma cell lines for their high susceptibility to rituximab-dependent complement-mediated killing enhanced by MB55 and MB59. The cells injected i.p. primarily colonized the liver and spleen, leading to the death of the animals within 30 to 40 days. Thirty percent of mice receiving biotin-labeled rituximab (25 microg) i.p. on days 4 and 11 after cell injection survived to 120 days. Administration of biotin-labeled rituximab, followed by avidin (40 microg) and biotin-labeled MB55-MB59 (100 microg) at 4-h intervals after each injection resulted in the survival of 70% of mice. Surprisingly, 40% of mice survived after the sole injection of avidin and biotin-labeled MB55-MB59, an observation consistent with the in vitro data showing that the miniantibodies induced killing of approximately 25% cells through antibody-dependent cell cytotoxicity. In conclusion, MB55 and MB59 targeted to tumor cells represent a valuable tool to enhance the therapeutic effect of rituximab and other complement-fixing antitumor antibodies.

Platelet-Activating Factor and Kinin-Dependent Vascular Leakage as a Novel Functional Activity of the Soluble Terminal Complement Complex

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.

The Neutrophil-Activating Protein of Helicobacter pylori Crosses Endothelia to Promote Neutrophil Adhesion In Vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.

An Alternative Role of C1q in Cell Migration and Tissue Remodeling: Contribution to Trophoblast Invasion and Placental Development

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α4β1 integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q−/− mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

Novel pathogenic mechanism and therapeutic approaches to angioedema associated with C1 inhibitor deficiency.

BACKGROUND Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. OBJECTIVE Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. METHODS The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. RESULTS We observed that the attack phase plasma from C1 inhibitor-deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor-high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1beta and was markedly reduced by brefeldin A. CONCLUSION Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium

This study was prompted by the observation that decidual endothelial cells (DECs), unlike endothelial cells (ECs) of blood vessels in normal skin, kidney glomeruli and brain, express surface-bound C1q in physiologic pregnancy. This finding was unexpected, because deposits of C1q are usually observed in pathologic conditions and are associated with complement activation. In the case of DECs, we failed to detect immunoglobulins and C4 co-localized with C1q on the cell surface. Surprisingly, DECs expressed mRNA for the three chains of C1q and secreted detectable level of this component in serum-free medium. The ability to synthesize C1q is acquired by DECs during pregnancy and is not shared by ECs obtained from endometrium and from other sources. Cell-associated C1q has a molecular weight similar to that of secreted C1q and is released from DECs following treatment with heparinase or incubation at low pH. This suggests that C1q binds to DECs and it is not constitutively expressed on the cell surface. C1q is localized at contact sites between endovascular trophoblast and DECs and acts as an intercellular molecular bridge because adhesion of endovascular trophoblast to DECs was inhibited by antibodies to C1q and to a receptor recognizing its globular portion expressed on trophoblast.

Bilirubin inhibits the TNFα-related induction of three endothelial adhesion molecules

Since an increased serum unconjugated bilirubin (UCB) level has been proposed as an independent protective factor against atherosclerotic disease, we investigated the molecular events at the basis of this effect. HUVEC and H5V cells were treated with TNFalpha and UCB and the effects assessed on E-selectin, VCAM-1 and ICAM-1. In HUVEC cells, UCB blunted the TNFalpha-induced gene upregulation of E-selectin VCAM-1 and ICAM-1. The same pattern was observed in H5V cells except for ICAM-1. UCB also inhibited the PMN endothelial adhesion in HUVEC H5V cells. Western blot and FACS analysis confirmed that UCB prevented TNFalpha-induced over-expression of adhesion molecules proteins in H5V cells. These data contribute to further explain the protective effect of bilirubin against development of atherosclerosis.

Mast cells are critically involved in serum‐mediated vascular leakage in chronic urticaria beyond high‐affinity IgE receptor stimulation

To cite this article: Bossi F, Frossi B, Radillo O, Cugno M, Tedeschi A, Riboldi P, Asero R, Tedesco F, Pucillo C. Mast cells are critically involved in serum‐mediated vascular leakage in chronic urticaria beyond high‐affinity IgE receptor stimulation. Allergy 2011; 66: 1538–1545.

C1q as a unique player in angiogenesis with therapeutic implication in wound healing

Significance C1q is a well-known initiator of the complement classical pathway and interacts with several immune and nonimmune cells inducing complement activation-independent functions. Endothelial cells represent one of the potential targets of C1q that binds to cell surface-expressed receptors and stimulates inflammation. Here we report a unique and hitherto unrecognized function of C1q to promote angiogenesis acting through the globular heads. The angiogenic activity of C1q was supported by its ability to induce new vessel formation in in vitro and in vivo models of wound healing. These findings have important implications for the treatment of clinical diseases associated with impaired angiogenesis such as chronic skin ulcers in diabetic patients. We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa−/− mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and the healed wound area were significantly increased in C1q-treated rats. On the basis of these results we suggest that C1q may represent a valuable therapeutic agent that can be used to treat chronic ulcers or other pathological conditions in which angiogenesis is impaired, such as myocardial ischemia.