Stefania Morrone

Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed “cardiospheres”) from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

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.

CD69-triggered ERK activation and functions are negatively regulated by CD94/NKG2-A inhibitory receptor

CD69 represents a functional triggering molecule on activated NK and T cells, capable of inducing cytotoxic activity and costimulating cytokine production. It belongs to the C‐lectin type superfamily, and its gene maps in the NK gene complex, close to other genes coding for NK receptors. CD94 / NKG2‐A complex is the inhibitory receptor for the non classical MHC class I molecule HLA‐E on human NK cells. To investigate CD69‐initiated signal transduction pathways, and to evaluate CD94 / NKG2‐A interference on CD69 triggering ability, we have generated transfectants expressing both receptors in the RBL cell line. Here we report that CD69 engagement leads to the activation of extracellular signal‐regulated kinase (ERK) enzymes belonging to the MAPK family, and that this event is required for CD69‐mediated cell degranulation. Moreover, we show that the co‐engagement of CD94 / NKG2‐A inhibitory receptor effectively suppresses both CD69‐triggered cell degranulation in RBL transfectants, through the inhibition of ERK activation, and CD69‐induced cytotoxicity in human NK cells. Thus, here we provide new information on the molecular mechanisms initiated by CD69 activation receptor, and show that CD69‐initiated signaling pathways and functional activity are negatively regulated by CD94 / NKG2‐A inhibitory complex.

In vivo modulation of the distribution of thymocyte subsets : effects of estrogen on the expression of different T cell receptor Vβ gene families in CD4-, CD8- thymocytes

Estrogen treatment of mice has been shown to deplete CD4+, CD8+ double-positive (DP) thymocytes and to alter the relative proportion of CD4+ and CD8+ single-positive (SP) thymocytes. In this work, we have studied the effect of the steroid hormone 17 beta-estradiol (E2) on the different subsets of CD4-/CD8- double-negative (DN) thymocytes by analyzing the expression of CD5, CD3-epsilon and of several V beta gene family products of the T cell antigen receptor (TCR). After in vivo administration of E2 a significant decrease in the number and proportion of dull CD5+, CD3-, beta-TCR- DN thymocytes was observed. In contrast E2 treatment significantly increased the proportion of bright CD5+, CD3+, beta-TCR+ DN cells. The E2-induced increase in DN/TCR+ cells was observed for subsets expressing V beta 6, V beta 8, and V beta 11, but not V beta 3 gene products of the TCR. Thus, estrogen administration results in a selective inbalance of the DN thymocyte subsets by depleting an immature, dull CD5+, CD3-, TCR beta- DN subset, while enriching a mature, bright CD5+, CD3+, TCR beta+ DN subset of cells. In addition to TCR beta+ DN thymocytes, an increased proportion of CD4+ and CD8+ SP thymocytes expressing V beta 8, V beta 6, and V beta 11, but not V beta 3, TCR proteins was also observed after E2 administration. An involvement of intrathymic cytokine production in mediating the hormone action is suggested by the ability of estrogen to increase the levels of IL-1 alpha mRNA of intact thymus. Our data suggest that estrogen exerts its effects on a broad range of immature cells, including dull CD5+, CD3-, beta-TCR- DN and DP thymocytes.

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 Cγ2 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.

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways.

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high‐affinity‐specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin‐coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c‐Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: A new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (FcεRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, FcεRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of FcεRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating FcεRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the FcεRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of FcεRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

Dichotomic effects of IFN-γ on the development of systemic lupus erythematosus-like syndrome in MRL-lpr/lpr mice

Systemic lupus erythematosus (SLE)‐prone female MRL‐lpr / lpr (MRL‐lpr) mice were treated with mouse or rat IFN‐γ under different experimental conditions, both prophylactically in 6‐ to 8 week‐old animals and therapeutically in 12‐ to 18‐week‐old SLE‐affected mice. It was found that IFN‐γ heterogeneously modulated the course of the disease in MRL‐lpr mice. When administered prophylactically, IFN‐γ favorably modulated the histological, serological and clinical signs of the disease. Relative to untreated or PBS‐treated control animals, the MRL‐lpr mice which received IFN γ were virtually free of inflammatory infiltration of the kidneys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1, IgG2a and IgG3 and anti‐double strand (ds) and single strand (ss) DNA antibodies, milder skin vasculitis, significantly reduced enlargment of their lymph nodes and lower weight of the spleens. IFN‐γ also lowered the rate of mortality of MRL‐lpr mice. In contrast to these findings, therapeutically administered IFN‐γ worsened the course of the disease in MRL‐lpr mice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti‐ds and ‐ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS‐treated control mice. The dichotomic effect of IFN‐γ on disease manifestation in MRL‐lpr mice offers new insights into the complex role of this cytokine in the regulation of systemic autoimmunity such as SLE.

Inhibition of intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1 and interleukin-4 by nitric oxide expression in migraine patients

Abstract The mechanisms of the postulated ”sterile” inflammation in migraine were studied utilizing flow cytometry (intercellular adhesion molecule 1, ICAM-1; interleukin-1 receptor, IL-1R) and enzyme-linked immunosorbent assay (soluble intercellular adhesion molecule 1, sICAM-1; interleukin-4, IL-4). Twenty patients suffering from migraine without aura, 20 healthy subjects, and 10 patients suffering from episodic tension headache were selected. All of the migraine patients were studied during a migraine crisis experimentally induced by the administration of isosorbide dinitrate (a nitric oxide donor), and 10 out the 20 were also studied during a spontaneous migraine attack. A sharp decrease in the expression of ICAM-1 (F=5.09, p<0.001 and F=2.46, p<0.05, respectively), sICAM-1 1 (F=6.21, p<0.0001 and F=3.99, p<0.007, respectively) and serum IL-4 (F=6.23, p<0.001 and F=3.64, p<0.01, respectively) were observed in experimentally induced and spontaneous migraine attacks. There was no change with respect to IL-IR 1 receptor expression values. The two control groups, tested with the same experimental procedure, showed no changes in ICAM-1 and IL-1R or in in sICAM-1 and IL-4. Our data suggest that migraine patients are more sensitive to exogenous NO than controls. In addition, our results indicate that experimental migraine crisis, induced by an NO donor, is mediated by the inhibition of IL-4 and subsequently of ICAM-1. It is likely that the described ICAM-1 downregulation inhibits during a migraine attack the critical step of transendothelial migration into the cerebral tissues of activated leukocytes, as proposed in the ”sterile inflammation” hypothesis.