Autocrine VEGF mediates the antiapoptotic effect of CD154 on CLL cells M Farahani 1 , AT Treweeke 1 , CH Toh 1 , KJ Till 1 , RJ Harris 1 , JC Cawley 1 , M Zuzel 1 and H Chen 1 1 Department of Haematology, University of Liverpool, Royal Liverpool University Hospital, Liverpool, UK CD154 is an important regulator of chronic lymphocytic leukaemia (CLL)-cell survival. In CLL, high serum levels of VEGF are a feature of advanced disease, and we and others have previously shown that CLL cells produce and secrete this growth factor. Since CD154 stimulates VEGF production in other cell types, and VEGF is known to promote cell survival, we examined whether the cytoprotection of CLL cells by CD154 involves VEGF. We report for the first time that treatment of CLL cells with CD154 results in increased VEGF production and demonstrate involvement of NF-jB in this process. Moreover, we show that CD154-induced CLL-cell survival is reduced by anti-VEGF-neutralising antibody and by inhibiting VEGF recep- tor (VEGFR) signalling with SU5416. However, addition of exogenous VEGF alone or blocking secreted autocrine VEGF had little or no effect on CLL-cell survival. We therefore conclude that CLL-cell cytoprotection in the presence of CD154 requires combined signalling by both CD40 and VEGFR. This combined signalling and resulting cytoprotection were shown to involve NF-jB activation and increased survivin production. In conclusion, our findings identify autocrine VEGF as an important mediator of the antiapoptotic effect of CD40 ligation, and thus provide new insights into CLL-cell rescue by CD154 in lymphoreticular tissues. Leukemia (2005) 19, 524–530. doi:10.1038/sj.leu.2403631 Published online 27 January 2005 Keywords: CD154; VEGF; CLL Introduction Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of long-lived, mature neoplastic B-lymphocytes in blood and tissues. 1,2 It is believed that prolonged in vivo survival of malignant cells contributes to their clonal expansion. 3,4 The fact that the malignant cells, despite progressively accumulating in vivo, rapidly undergo apoptosis when cultured in vitro implies that microenvironmental factors are likely to play a prominent role in prolonging the lifespan of CLL cells. VEGF is one factor that could potentially be important for the pathogenesis and prognosis of CLL. CLL cells themselves produce VEGF, 5,6 and high levels of this growth factor in serum, 7 as well as high expression of VEGF receptor (VEGFR)-2 on the malignant cells, 8 correlate with shortened patient survival. Moreover, in a previous study, 5 we observed increased vascularisation in enlarged lymph nodes of CLL patients and proposed that this was caused by paracrine stimulation of endothelial cells by CLL-cell-derived VEGF. Increased micro- vessel density has also been reported in the bone marrow of patients with CLL. 9 We suggested that increased angiogenesis might play an important role in the survival of tissue-phase CLL cells by providing metabolic support. 5 In addition to its role in angiogenesis, VEGF has been found to be an autocrine survival factor for some cell types including solid tumour and endothelial cells, 10–12 as well as CLL cells. 13 VEGF, like many other growth factors, often achieves its effects in cooperation with other stimuli. 14 An important microenvir- onmental factor affecting CLL-cell survival is CD40 ligand (CD154) expressed on activated T cells, monocytes, macro- phages, dendritic cells and platelets. 15,16 Its receptor CD40 is expressed by mature B-lymphocytes including CLL cells, and also by some nonhaemic cell types. 17 Stimulation of CD40 on endothelial cells, monocytes, fibroblasts and multiple myeloma cells upregulates production of VEGF by these cells. 18–20 Furthermore, neutralisation of autocrine VEGF by anti-VEGF antibody (Ab) completely abolished the mitogenic effect of CD154 on endothelial cells. 18 In CLL cells, ligation of CD40 by CD154 is known to inhibit spontaneous and drug-induced cell death. 21–24 However, whether this effect also requires cell stimulation by the secreted autocrine VEGF is not known. In the present study, we show that the previously observed promotion of CLL-cell survival by CD154 does indeed depend on autocrine VEGF production, and that both VEGFR and CD40 engagement are essential for this effect. We also show that VEGF provides a significant contribution to CD154-induced NF-kB activation and upregulation of survivin, one of the NF-kB target genes. Materials and methods Patients All patients had typical CLL with high numbers of circulating malignant cells as defined morphologically (prolymphocytes o10%) and immunophenotypically (CD19 þ , CD5 þ , CD23 þ and weak light-chain-restricted surface immunoglobulin). Patients’ samples were obtained after informed consent and with the approval of the Liverpool Research Ethics Committee. Relevant clinical and laboratory data of the patients studied are included as Supplementary information accompanying the paper. Materials Human VEGF Quantikine Colorimetric Sandwich ELISA (en- zyme-linked immunosorbent assays) kit, anti-human VEGF- neutralising monoclonal antibody (mAb) (MAB293), recombi- nant human VEGF165 and PLGF were all from R & D Systems (Abingdon, UK). Anti-human p65 mAb was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The CD154 kit (ALX-850- 064) containing recombinant human soluble CD154 trimeric protein (rhsCD154) and enhancer was from Alexis Biochemicals (San Diego, CA, USA). TransAM NF-kB p65 assay kit and nuclear extract kit were from Active Motif North America (Carlsbad, CA, USA). The NF-kB translocation inhibitor SN50, its control peptide (SN50M) and the VEGFR tyrosine kinase inhibitor SU5416 were from Calbiochem (Nottingham, UK). Recombinant human VEGF-E was from RELIA Tech GmbH (Braunschweig, Germany). Human Total Survivin ELISA kit was Received 8 September 2004; accepted 12 November 2004; Published online 27 January 2005 Correspondence: Dr M Farahani, Department of Haematology, Royal Liverpool University Hospital, Daulby Street, Liverpool L69 3GA, UK; Fax: þ 44 151 706 4311; E-mail: Mosavar@liv.ac.uk Leukemia (2005) 19, 524–530 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu SPOTLIGHT