Induction of Fas Antigen Expression, and Apotopsis of Human B Cell by CD40 and Tumor Necrosis Factor Receptor Ligation

Chang, Dae Kook; Yoo, Dae Hyun; Kim, Seong Yoon

Abstract

OBJECTIVE
CD40 and the TNFR belong to the NGF/TNFR supergene family. Ligation of CD40 on B cells induces activation ignals leading to proliferation, Ig isotype class switching, germinal center formation but also induces Fas antigen expression.In addition,CD40 ligation induces pro-inflammatory cytokines including TNF-alpha and LT-alpha gene transcription by human B cell.TNF-alpha is a pleiotropic cytokine and also induces Fas antigen expression on various cells. Lately it has been known that TNF-alpha plays an important role in the pathogenesis of chronic inflammatory diseases,including rheumatoid arthritis,or chronic inflammatory bowel diseases.However there have been occurrence of autoantibodies,or autoimmune disease such as lupus after use of anti TNF-alpha blocking agents. In this report,we tested the relationship and biological significance of CD40 ligation and TNFR signaling with respect to Fas antigen expression on human B cells.
METHODS
Ramos Burkitt's lymphoma B cell was used as a prototype of ger-minal center B lymphocyte,and R2G6 cell was utilized as a model of activated germinal center B cell.CD40 lgation was performed by the coculture with CD40 ligand bearing L-293 cells,or anti-CD40 monoclonal antibody,whereas control was obtained with CD-8-L-293 cells or control antibody.Expression of Fas antigen was determined with flow cytometer.Apoptosis assay was conducted by two ways.Alamar blue reduction assay after sIgM cross linking or anti-Fas anti-body,in the presence or absence of CD40 ligation or TNF-alpha .In addition,DNA content assay was utilized to make sure the proportion of apoptotic Ramos B cells by various treatments.
RESULTS
1)CD40 and TNF-alpha induced Fas antigen expression on Ramos B cell line cells and rendered them susceptible to Fas-mediated apoptosis.2)CD40 and TNFR signaling upregulate Fas antigen independently.3)Both TNFR and CD40 signaling rescue sIgM crosslink induced apoptosis of Ramos B cell line cells,only CD40,but not TNFR,signaling rescues Ramos cells from Fas-mediated apoptosis.
CONCLUSION
Taken together,these results demonstrate that B cell signaling via two distinct members of the NGF/TNFR superfamily,CD40 and TNFR, independently engage the Fas pathway and provide mechanisms for eliminating B cells.Acting alone,both signals will ready B cells for Fas-mediated apoptosis. In concert with sIg signaling,the rescue effect provided uniquely by CD40 ligation assures the selective survival of only those B cells which have bound antigen and presented it to antigen-specific T(h) cells .