Notch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161⁺/CD56⁺ NK cells from these cultures contained a tumor necrosis factor alpha (TNF-α)⁺/granulocyte macrophage–colony-stimulating factor (GM-CSF)⁺ subset, an interferon gamma (IFN-γ)⁺ subset, mostly included within the former, and very few IFN-γ⁻/IL-13⁺ cells. Instead, most immature CD161⁺/CD56⁻ NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-α, and GM-CSF, but not IFN-γ, and contained an IL-5⁺ subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-γ. Part of these produced both IFN-γ and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161⁺ NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-γ.

Group B streptococci (GBS) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS–macrophage interaction in the absence of type‐specific antibodies, we examined the features of GBS survival in thioglycollate‐elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC)‐dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS‐Ia) and type III GBS strain COH 31r/s (GBS‐III), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon‐γ (IFN‐γ) and lipopolysaccharide from Escherichia coli (LPS) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN‐γ and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro‐organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12‐myristate 13‐acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBS‐infected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC‐dependent cell signalling was also demonstrated by the reduced c‐fos gene expression in GBS‐infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival.

A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8–mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.