Unraveling the Bone Tissue Microenvironment in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western countries. Although characterized by the progressive expansion and accumulation of leukemic B cells in peripheral blood, CLL cells develop in protective niches mainly located within lymph nodes and bone marrow. Multiple interactions between CLL and microenvironmental cells may favor the expansion of a B cell clone, further driving immune cells toward an immunosuppressive phenotype. Here, we summarize the current understanding of bone tissue alterations in CLL patients, further addressing and suggesting how the multiple interactions between CLL cells and osteoblasts/osteoclasts can be involved in these processes. Recent findings proposing the disruption of the endosteal niche by the expansion of a leukemic B cell clone appear to be a novel field of research to be deeply investigated and potentially relevant to provide new therapeutic approaches.

A High Percentage of CD16+ Monocytes Correlates with the Extent of Bone Erosion in Chronic Lymphocytic Leukemia Patients: The Impact of Leukemic B Cells in Monocyte Differentiation and Osteoclast Maturation.

Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.

Establishment and Characterization of a Novel Fibroblastic Cell Line (SCI13D) Derived from the Broncho-Alveolar Lavage of a Patient with Fibrotic Hypersensitivity Pneumonitis.

Hypersensitivity pneumonitis (HP) is a diffuse interstitial lung disease (ILD) caused by the inhalation of a variety of antigens in susceptible individuals. Patients with fibrotic HP (fHP) may show histopathological and radiological manifestations similar to patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia-like pattern of fibrosis) that are associated with a worse prognosis. We describe here the establishment and characterization of a fibroblastic cell line derived from the broncho-alveolar lavage (BAL) of a patient with fHP, a 53 year old man who presented at our Pneumology Unit with cough and dyspnea. The fHP diagnosis was based on international criteria and multidisciplinary discussion. Primary fibroblasts were expanded in vitro until passage 36. These fibroblasts displayed morpho/phenotypical features of myofibroblasts, showing high positivity for α-smooth muscle actin, type I collagen, and fibronectin as determined by quantitative RT-PCR and cyto-fluorographic analysis. Cytogenetic analyses further evidenced trisomy of chromosome 10, which interestingly harbors the FGF2R gene. To our knowledge, this is the first fibroblastic cell line derived from an fHP patient and might, therefore, represent a suitable tool to model the disease in vitro. We preliminarily assessed here the activity of pirfenidone, further demonstrating a consistent inhibition of cells growth by this antifibrotic drug.

The HGF/c-MET axis as a potential target to overcome survival signals and improve therapeutic efficacy in multiple myeloma.

Multiple myeloma (MM) accounts for about 10% of hematologic malignancies, and it is the second most frequent hematologic neoplasm after lymphomas. The exact etiology of MM is still unknown and, despite the introduction of more effective and safe drugs in recent years, MM remains an incurable disease. Intrinsic and acquired resistance of malignant B cells to pharmacological treatments still represents an obstacle for survival improvement. Activation of the hepatocyte growth factor/c-MET axis has been reported as involved in MM pathogenesis: hepatocyte growth factor (HGF) levels are in fact higher in sera from MM patients than in healthy controls, the HGF/c-MET pathway may be activated in an autocrine or paracrine manner, and it is interesting to note that a higher c-MET phosphorylation is associated with disease progression. Several studies have further demonstrated the over-activation of c-MET either in resistant cell lines or in primary malignant plasma cells purified from bone marrow of patients resistant to chemotherapy. For this reason, c-MET has been proposed as a potential marker of multidrug resistance in the disease. Here, we first summarize the potential role of HGF/c-MET interaction in disease evolution and then describe novel approaches targeting this axis which could be conceptually utilized, alone or in combination with standard therapies, to treat MM and possibly overcome drug resistance.

Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rß1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rß1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

Potentiation of cisplatin-induced antiproliferative and apoptotic activities by the antiarrhythmic drug procainamide hydrochloride.

BACKGROUND: We describe the potentiation of antiproliferative and apoptotic activities triggered by cis-diamminedichloroplatinum(II) (DDP), and obtained in vitro by the co-administration of procainamide hydrochloride (PdHCl) in murine P388, and human A2780 and A549 cells. METHODS: We determined the antiproliferative and apoptotic activities of DDP and PdHCl combinations by different techniques. Moreover, cell cycle analysis, restriction enzyme inhibition followed by agarose gel electrophoresis, and TUNEL analysis of tumour cells in vivo were also used to strengthen our hypothesis. RESULTS: Our results show that PdHCl may significantly increase the inhibition of cell proliferation and apoptosis. Experiments in vivo showed that the co-administration of DDP and PdHCl increased the percentage of apoptotic cells compared to DDP alone treatment, both in subcutaneous (sc) and intraperitoneal (ip) P388 tumours. We finally demonstrated that the co-administration of PdHCl prevents DNA digestion accounting for a restriction enzyme inhibition that in some cases was greater than that obtained by DDP alone. Moreover, when PdHCl was mixed with the reaction products (RP) of DDP (RP-PdHCl) we obtained a restriction enzyme inhibition greater for some enzymes (Bsp1407I, Hin1II, and Psp1406I) than that obtained by the DDP-PdHCl solution. CONCLUSIONS: On the whole our data demonstrate that the class I antiarrhythmic drug PdHCl may increase the antiproliferative activity of DDP by improving its triggering of apoptosis, and that this phenomenon may be likely linked to the formation of a new Pt compound.

Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages.

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.

ABCB1 Structural Models, Molecular Docking, and Synthesis of New Oxadiazolothiazin-3-one Inhibitors.

Docking methods are powerful tools for in silico screening and drug lead generation and optimization. Here, we describe the synthesis of new inhibitors of ABCB1 whose design was based on construction and preliminary confirmation of a model for this membrane transporter of the ATP-binding cassette family. We chose the strategy to build our three-dimensional model of the ABCB1 transporter by homology. Atomic coordinates were then assayed for their reliability using the measured activity of some oxadiazolothiazin-3-one compounds. Once established their performance by docking analysis, we synthesized new compounds whose forecasted activity was tested by MTT and cytofluorimetric assays. Our docking model of MDR1, MONBD1, seems to reliably satisfy our need to design and forecast, on the basis of their LTCC blockers ability, the inhibitory activity of new molecules on the ABCB1 transporter.

An interaction between hepatocyte growth factor and its receptor (c-MET) prolongs the survival of chronic lymphocytic leukemic cells through STAT3 phosphorylation: a potential role of mesenchymal cells in the disease.

BACKGROUND: Chronic lymphocytic leukemia cells are characterized by an apparent longevity in vivo which is lost when they are cultured in vitro. Cellular interactions and factors provided by the microenvironment appear essential to cell survival and may protect leukemic cells from the cytotoxicity of conventional therapies. Understanding the cross-talk between leukemic cells and stroma is of interest for identifying signals supporting disease progression and for developing novel therapeutic strategies. DESIGN AND METHODS: Different cell types, sharing a common mesenchymal origin and representative of various bone marrow components, were used to challenge the viability of leukemic cells in co-cultures and in contact-free culture systems. Using a bioinformatic approach we searched for genes shared by lineages prolonging leukemic cell survival and further analyzed their biological role in signal transduction experiments. RESULTS: Human bone marrow stromal cells, fibroblasts, trabecular bone-derived cells and an osteoblast-like cell line strongly enhanced survival of leukemic cells, while endothelial cells and chondrocytes did not. Gene expression profile analysis indicated two soluble factors, hepatocyte growth factor and CXCL12, as potentially involved. We demonstrated that hepatocyte growth factor and CXCL12 are produced only by mesenchymal lineages that sustain the survival of leukemic cells. Indeed chronic lymphocytic leukemic cells express a functional hepatocyte growth factor receptor (c-MET) and hepatocyte growth factor enhanced the viability of these cells through STAT3 phosphorylation, which was blocked by a c-MET tyrosine kinase inhibitor. The role of hepatocyte growth factor was confirmed by its short interfering RNA-mediated knock-down in mesenchymal cells. CONCLUSIONS: The finding that hepatocyte growth factor prolongs the survival of chronic lymphocytic leukemic cells is novel and we suggest that the interaction between hepatocyte growth factor-producing mesenchymal and neoplastic cells contributes to maintenance of the leukemic clone.

Inhibition of MDR1 activity and induction of apoptosis by analogues of nifedipine and diltiazem: an in vitro analysis.

We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4',6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC(50) reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.

Heterogeneous expression and function of IL-21R and susceptibility to IL-21-mediated apoptosis in follicular lymphoma cells.

OBJECTIVE: Interleukin (IL)-21, a member of the IL-2 family, has antitumor activity and is now being tested in non-Hodgkin's lymphoma in combination with anti-CD20 antibodies. IL-21 may either induce apoptosis or promote growth in different lymphoid malignancies. We therefore investigated the IL-21/IL-21R system in follicular lymphoma (FL) cells. MATERIALS AND METHODS: IL-21R expression was studied by reverse transcription polymerase chain reaction, immunofluorescence, and Western blot analyses. Apoptosis was measured by Annexin-V-propidium iodide staining. Signaling via IL-21R was studied using antibodies specific for phosphorylated Janus-activating kinase and signal transducers and activators of transcription proteins by Western Blot. RESULTS: IL-21R was found on primary FL cells in 15 of 15 cases at diagnosis and IL-21 increased apoptosis in 10 of 10 FL samples. However, cells from areas of diffuse growth in FL and from two diffuse lymphomas evolved from previous FL, showed low IL-21R expression. The latter were also resistant to IL-21-mediated apoptosis. Among lymphoma cell lines bearing the t(14;18) translocation, only 1 of 7 showed increased apoptosis in response to IL-21 stimulation. This cell line was IL-21R-positive, whereas five of six nonresponsive cell lines showed very low IL-21R expression. Intriguingly, one of the IL-21-resistant cell lines (DOHH2) expressed high levels of IL-21R. Treatment with IL-21 or IL-4 upregulated suppressor of cytokine signaling 3 gene expression in the IL-21-responsive cell line, but not in DOHH2 cells, which showed defective Janus-activating kinase/signal transducers and activators of transcription signaling in response to IL-21, in relationship to the lack of Janus-activating kinase 3 gene expression. CONCLUSION: These data indicate that low IL-21R expression or defective signal transduction downstream IL-21R may cause refractoriness to IL-21-mediated effects in some FL cells.

Two-step in vivo tumor targeting by biotin-conjugated antibodies and superparamagnetic nanoparticles assessed by magnetic resonance imaging at 1.5 T.

PURPOSE: The purpose of this study was to assess two-step in vivo tumor targeting by specific biotin-conjugated antibodies and ultrasmall superparamagnetic iron oxide (USPIO)-anti-biotin nanoparticles as contrast agents for magnetic resonance imaging (MRI) at 1.5 T. PROCEDURES: D430B human lymphoma cells, expressing the CD70 surface antigen, were injected either s.c. or i.v. to induce pseudo-metastases in NOD/SCID mice. Thirty micrograms of biotin-conjugated monoclonal anti-CD70 was injected i.v., followed 4 h later by 8 micromol Fe/Kg USPIO-anti-biotin. After 24 h, MRI was performed on T2* and b-FFE sequences. Signal intensity (SI) was calculated before and after USPIO-anti-biotin administration. RESULTS: Subcutaneous xenografts showed a dishomogeneous 30% decrease in SI on T2* with anti-CD70 + USPIO-anti-biotin treatment. Pseudo-metastatic xenografts showed a slight reduction in SI on T2*, but a 60% decrease in SI on b-FFE-weighted sequences. Prussian blue staining confirmed the presence of iron nanoparticles in the excised tumors. CONCLUSION: MRI at 1.5 T can detect tumors by a two-step in vivo biotin-based protocol, which may allow the targeting of any cell surface antigen.

The administration of demethyl fruticulin A from Salvia corrugata to mammalian cells lines induces "anoikis", a special form of apoptosis.

Recently demethyl fruticulin A was identified as the major diterpenoid component of the exudates produced by the trichomes of Salvia corrugata leafs. Given the documented apoptotic effects of some of the other known components of the exudates from Salvia species, we assessed if demethyl fruticulin A, once administered to mammalian cells, was involved in the onset of apoptosis and if its biological effects were exerted through the participation of a scavenger membrane receptor, CD36. Three model cell lines were chosen, one of which lacking CD36 expression. Functional availability of the receptor, or its transcriptional rate, were blocked/reduced with a specific antibody or by the administration of vitamin E. Immunodetection of cell cytoskeletal components and tunel analysis revealed that demethyl fruticulin A triggers the onset of anoikis, a special form of apoptosis induced by cell detachment from the substrate. Impairment of CD36 availability/transcription confirmed the receptor partial involvement in the intake of the substance and in anoikis, as also sustained by FACS analysis and by the downregulation of p95, a marker of anoikis, upon blockade of CD36 transcription. However, experiments with CD36-deficient cells suggested that alternate pathways, still to be determined, may take part in the biological effects exerted by demethyl fruticulin A.

Inhibition of MDR1 activity in vitro by a novel class of diltiazem analogues: toward new candidates.

The reversal of multidrug resistance by 22 molecules [8-aryl-8-hydroxy-5-R'-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (1a-i) and 8-aryl-8-alkoxy-5-methyl-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (2a-m)] related to myocardial-calcium-channel-modulator diltiazem was studied in multidrug resistant A2780/DX3 and their sensitive counterpart A2780 cells. MTT, cytofluorimetry assays, and fluorescence microscopy analyses were used to define activity and accumulation of doxorubicin with or without the diltiazem-like modulators. Of the 22 molecules, 1a, 2f, 2g, and 2m were able to overcome the established criteria for the selection in A2780/DX3 cells (IC(50) reduction > or = 25%), but only 2f, 2g, and 2m caused a significant increase of intracellular accumulation of doxorubicin. In conclusion, experiments lead to the identification of three diltiazem-like molecules able to increase the intracellular accumulation of doxorubicin by inhibiting the MDR1 function, thus potentiating its antiproliferative activity in multidrug resistant A2780/DX3 cells.

The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways.

The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common gamma chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and prevented apoptosis induced by surface IgM cross-linking. These effects were more evident in cells stimulated via surface CD40, which exhibited increased cell expression of IL-15Ralpha chain and, in some of the cases, also of IL-2Rbeta. IL-21 failed to induce CLL cell proliferation and instead promoted apoptosis. Following cell exposure to IL-15, phosphorylation of STAT5 was predominantly observed, whereas, following stimulation with IL-21, there was predominant STAT1 and STAT3 activation. Moreover, IL-15 but not IL-21 caused an increased phosphorylation of Shc and ERK1/2. Pharmacological inhibition of JAK3 or of MEK, which phosphorylates ERK1/2, efficiently blocked IL-15-induced CLL cell proliferation and the antiapoptotic effect of this cytokine. The knowledge of the signaling pathways regulating CLL cell survival and proliferation may provide new molecular targets for therapeutic intervention.

Role of IL-21 in immune-regulation and tumor immunotherapy.

IL-21, the most recently discovered member of the IL-2 cytokine family, is an attractive subject for research due to its involvement in experimental models of autoimmunity, its ability to down-regulate IgE production, and its anti-tumor properties. Its interest for cancer immunotherapy stems from its physiological immune-enhancing functions. These include regulation of T, B and NK cell proliferation, survival, differentiation, and effector functions. IL-21's functional activities partially overlap those of IL-2. Both cytokines display similar structural features and use the common gamma-chain receptor and its downstream signaling pathways. Besides its activities on normal lymphoid cells, IL-21 is an in vitro growth factor for myeloma and acute-T cell leukemia cells, whereas it induces the apoptosis of B-CLL (chronic lymphocytic leukemia) cells. These findings indicate that the IL-21/IL-21R system exerts opposite functions in different lymphoid neoplasias, and suggest its employment in B-CLL therapy. Since IL-2, but not IL-21, is specifically required for the development of regulatory T (Treg) cell immune-suppressive functions, IL-21 may be a new tool for cancer immunotherapy. It is, in fact, a powerful anti-tumor agent in a variety of murine experimental tumor models through its activation of specific or innate immune responses against neoplastic cells. The preliminary data from phase-I clinical studies suggest that the use of IL-21 is feasible and may result in immune-enhancing effects.

Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells.

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.