Giovanni Bernardini

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.

Identification of the CC chemokines TARC and macrophage inflammatory protein-1β as novel functional ligands for the CCR8 receptor

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I‐309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation‐regulated cytokine (TARC) and macrophage inflammatory protein‐1β (MIP‐1β) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.

CCL3 and CXCL12 regulate trafficking of mouse bone marrow NK cell subsets

Herein we have analyzed chemokine involvement in the trafficking of developing and mature mouse natural killer (NK) cells in the bone marrow (BM). We observed drastic changes of CCR1, CXCR3, and CXCR4 expression and function during progression from precursor NK (pNK) cells to immature DX5- NK (iNK) and mature DX5+ NK (mNK) cells. pNK and mNK cells expressed the 3 receptors, while only CXCR4 was detected on iNK cells. Correspondingly, mNK cells migrated to CXCL12, CXCL10, and CCL3, and pNK and iNK cells to CXCL12, whereas pNK cells migrated to CCL3 and CXCL10 only after CXCL12 stimulation. Comparison of BM, peripheral blood, and spleen mNK cell populations revealed that CXCL12, CXCL10, and CCL3 preferentially affected BM mNK cell migration. Administration of the CXCR4 antagonist, AMD-3100, to C57BL/6 mice induced strong reduction of mNK and iNK cells in the BM and increased their number in blood and spleen. Conversely, CCL3 administration selectively mobilized mNK cells from the BM and this effect correlated with its ability to inhibit CXCL12-mediated mNK cell responses in vitro. Our results suggest that the combined action of chemokines selectively regulates localization of NK cell subsets in the BM and direct their maturation and migration to the periphery.

Chemokines and glioma: invasion and more.

Increasing pieces of evidence indicate that the chemokine system influences several aspects of brain physiology and pathology. A deregulated chemokine expression pattern is observed during neurological diseases, including multiple sclerosis and brain tumor. Gliomas are the most common primary tumors affecting human central nervous system (CNS). Chemokines expressed by stromal cells or endogenously produced by glioma cells may influence tumor cell migration, invasion, proliferation, angiogenesis and immune cell infiltration in the tumor mass. Herein we focus on chemokines and chemokine receptors expressed by glioma cells and their role in the regulation of glioma cell functional behaviour, including its ability to attract immune cells.

Functional role of α4β1 and α5β1 integrin fibronectin receptors expressed on adriamycin-resistant MCF-7 human mammary carcinoma cells

Cytofluorimetric and reverse‐transcription polymerase chain reaction (RT‐PCR) analysis showed that adriamycin‐resistant (ADRR), but not sensitive (WT), MCF‐7 human mammary carcinoma cell lines express α4β1 and α5β1 integrins. ADRR cells adhere to fibronectin (FN), and only α5β1 is involved in cell adhesion to this glycoprotein, while α4β1 mediates cell binding to the cellular counter‐receptor VCAM‐1. Proliferation assays showed that FN, but not VCAM‐1, delivers a mitogenic signal to quiescent ADRR MCF‐7 cells. The activating signal is mediated by α5β1, since cell proliferation is inhibited in the presence of RGD peptide or specific antibody. Cell cycle analysis demonstrated that cell/FN interaction induces the re‐entry of ADRR MCF‐7 into S phase, and prevents them from undergoing serum deprivation‐induced apoptosis. Our data suggest that the presence of α5β1 on the resistant cells enables them to draw advantage from FN for both cell growth and survival. Int. J. Cancer 72:133–141, 1997.

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 apoptois 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.

CX3CR1 expression defines 2 KLRG1+ mouse NK-cell subsets with distinct functional properties and positioning in the bone marrow

During development in the bone marrow (BM), NK-cell positioning within specific niches can be influenced by expression of chemokine or adhesion receptors. We previously demonstrated that the maintenance in the BM of selected NK-cell subsets is regulated by the CXCR4/CXCL12 axis. In the present study, we showed that CX3CR1 is prevalently expressed on KLRG1(+) NK cells, a subset considered terminally differentiated. Two KLRG1(+) NK-cell populations endowed with distinct homing and functional features were defined according to CX3CR1 expression. In the BM, KLRG1(+)/CX3CR1(-) NK cells were mainly positioned into parenchyma, while KLRG1(+)/CX3CR1(+) NK cells exhibited reduced CXCR4 expression and were preferentially localized in the sinusoids. We also showed that α(4) integrin plays a pivotal role in the maintenance of NK cells in the BM sinusoids and that α(4) neutralization leads to strong reduction of BM KLRG1(+)/CX3CR1(+) NK cells. Moreover, we found that KLRG1(+)/CX3CR1(+) cells originate from KLRG1(+)/CX3CR1(-) NK-cell population and display impaired capability to produce IFN-γ and to lyse YAC-1 target cells on cytokine stimulation. Altogether, our findings show that CX3CR1 represents a marker of a KLRG1(+) NK-cell population with unique properties that can irreversibly differentiate from the KLRG1(+)/CX3CR1(-) NK cells during steady state conditions.

Chemokines and NK cells: Regulators of development, trafficking and functions

n Abstractn n NK cells are innate lymphocytes capable of killing malignant or infected cells and to produce a wide array of cytokines and chemokines following activation.n Chemokines, play critical roles in the regulation of NK cell tissue distribution in normal conditions as well as their rapid recruitment to the parenchyma of injured organs during inflammation, which is critical for NK cell ability to promote protective responses. In this regard, differences in chemokine receptor expression have been reported on specialized NK cell subsets with distinct effector functions and tissue distribution.n Besides their role in the regulation of NK cell trafficking, chemotactic molecules can also affect NK cell effector functions by regulating their priming and their ability to kill and secrete cytokines.n n

Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice

Mice exposed to standard (SE) or enriched environment (EE) were transplanted with murine or human glioma cells and differences in tumour development were evaluated. We report that EE exposure affects: (i) tumour size, increasing mice survival; (ii) glioma establishment, proliferation and invasion; (iii) microglia/macrophage (M/Mφ) activation; (iv) natural killer (NK) cell infiltration and activation; and (v) cerebral levels of IL-15 and BDNF. Direct infusion of IL-15 or BDNF in the brain of mice transplanted with glioma significantly reduces tumour growth. We demonstrate that brain infusion of IL-15 increases the frequency of NK cell infiltrating the tumour and that NK cell depletion reduces the efficacy of EE and IL-15 on tumour size and of EE on mice survival. BDNF infusion reduces M/Mφ infiltration and CD68 immunoreactivity in tumour mass and reduces glioma migration inhibiting the small G protein RhoA through the truncated TrkB.T1 receptor. These results suggest alternative approaches for glioma treatment.

NK cells and interferons

The role of Natural Killer cells in host defense against infections as well as in tumour surveillance has been widely appreciated for a number of years. Upon recognition of altered cells, NK cells release the content of cytolytic granules, leading to the death of target cells. Moreover, NK cells are powerful producers of chemokines and cytokines, particularly Interferon-γ (IFN-γ), of which they are the earliest source upon a variety of infections. Despite being armed to fight against pathogens, NK cells become fully functional upon an initial phase of activation that requires the action of several cytokines, including type I IFNs. Type I IFNs are now recognized as key players in antiviral defense and immune regulation, and evidences from both mouse models of disease and in vitro studies support the existence of an alliance between type I IFNs and NK cells to ensure effective protection against viral infections. This review will focus on the role of type I IFNs in regulating NK cell functions to elicit antiviral response and on NK cell-produced IFN-γ beneficial and pathological effects.