CD40 triggering enhances fludarabine-induced apoptosis of chronic lymphocytic leukemia B-cells through autocrine release of tumor necrosis factor-alpha and interferon-gama and tumor necrosis factor receptor-I-II upregulation.
OBJECTIVE
In chronic lymphocytic leukemia (CLL) B-cells are refractory to activation signals and to apoptosis. CD40 triggering, however, rescues CLL B-cells from their anergic state and upregulates the FAS receptor. We therefore studied whether CD40 triggering enhances CLL B-cell sensitivity to fludarabine, and receptors or cytokines potentially involved in apoptosis.


METHODS
CD40-activation of CLL B-cells was carried out by co-culture with CD40L-transfected cells. After fludarabine treatment, apoptosis was evaluated by propidium iodide (PI), annexin-V/PI or DiOC6 staining and flow cytometry analysis. Modulation of Bcl-2, of tumor necrosis factor receptor (TNFRI/II) and release of tumor necrosis factor (TNF)alpha/interferon (IFN)gamma was also analyzed. Furthermore, addition of caspase-inhibitors or anti-TNFalpha/-IFNgamma monoclonal antibodies to fludarabine-treated cells allowed us to determine the mediators of apoptosis. Student's t tests or ANOVA variance statistical analysis were performed to evaluate whether any differences observed might be considered significant.


RESULTS
CD40 triggering enhanced fludarabine sensitivity of CLL B-cells, downmodulated Bcl-2 and upregulated TNFRI/II. Caspases 1 and 6 were the major caspases involved in fludarabine apoptosis induction in resting B cells, while only anti-TNFalpha/-IFNgamma monoclonal antibodies reduced apoptosis in activated cells. In agreement with this observation, autocrine production of TNFalpha and IFNgamma by CD40-activated CLL B cells was found.


CONCLUSIONS
B-cells from a considerable proportion of CLL cases studied (11/20) are more prone to fludarabine-induced apoptosis after CD40 triggering; accordingly Bcl-2 expression was lower in activated cells. Moreover, upregulation of TNFRI/II, release of TNFalpha and IFNgamma, and inhibition of apoptosis by anti-TNFalpha/-IFNgamma monoclonal antibodies in CD40-activated cells strongly suggest that these cytokines may play a role in sensitizing B-cells to fludarabine treatment.
de Totero
Daniela
D
Department of Internal Medicine, University of Genoa, Italy. detotero@yahoo.com
Tazzari
P L
PL
Capaia
Matteo
M
Montera
Maria Pina
MP
Clavio
Marino
M
Balleari
Enrico
E
Foa
Robin
R
Gobbi
Marco
M
D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D019898:Autocrine Communication / Q000187:drug effects*; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D019013:CD40 Antigens / Q000378:metabolism* / Q000502:physiology; D023201:CD40 Ligand / Q000378:metabolism; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D019013:CD40 Antigens; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D023201:CD40 Ligand; D007371:Interferon-gamma; D014740:Vidarabine; C024352:fludarabine