Novel role of CD40 in Fas-dependent apoptosis of cultured salivary epithelial cells from patients with Sjögren's syndrome.
OBJECTIVE
To determine the role of Fas and CD40 in the molecular mechanism of salivary epithelial cell death in Sjogren's syndrome (SS).


METHODS
The expression of Fas and CD40 in SS salivary epithelial cells was analyzed by flow cytometry. Induction of apoptosis with anti-Fas and/or anti-CD40 monoclonal antibodies (mAb) was examined by morphologic analysis, DNA fragmentation, and TUNEL assay. Expression of c-FLIP, Fas-associated phosphatase 1, FADD, Bcl-2, Bcl-x(L), and Bcl-x(S) was determined by Western blot analysis.


RESULTS
Expression of Fas and CD40 was significantly higher in SS salivary epithelial cells than in normal cells after interferon-gamma (IFNgamma) stimulation (P < 0.001 for both Fas and CD40). Although neither anti-Fas (CH11) nor anti-CD40 mAb alone could induce typical apoptosis, the two together and preincubation with IFNgamma efficiently induced apoptosis in SS salivary epithelial cells. This apoptosis was almost completely blocked by neutralizing anti-Fas mAb (ZB4), whereas an antagonistic mAb to CD40 (ch5D12) partially inhibited anti-Fas/anti-CD40-induced apoptosis. Also, c-FLIP, an important inhibitory molecule in the Fas death pathway, was strongly expressed in SS salivary epithelial cells, but its expression was down-regulated at the protein level by anti-CD40 mAb.


CONCLUSIONS
CD40 signals promote Fas-dependent death of SS salivary epithelial cells by down-regulating c-FLIP expression. The presence of c-FLIP in these cells may explain their resistance to undergoing apoptosis in response to either anti-Fas or anti-CD40 mAb, despite their surface expression of both proteins. These findings suggest that SS salivary epithelial cell death requires the cooperation of both Fas and CD40.
Ping
Li
L
Kanazawa Medical University, Kahoku-gun, Ishikawa-ken, Japan.
Ogawa
Noriyoshi
N
Sugai
Susumu
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D019013:CD40 Antigens / Q000032:analysis / Q000276:immunology / Q000502:physiology*; D002478:Cells, Cultured; D053938:DNA Fragmentation; D015536:Down-Regulation; D004847:Epithelial Cells / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D008297:Male; D008875:Middle Aged; D012469:Salivary Glands / Q000737:chemistry / Q000473:pathology*; D012859:Sjogren's Syndrome / Q000473:pathology / Q000503:physiopathology*; D019014:fas Receptor / Q000032:analysis / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D019013:CD40 Antigens; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D019014:fas Receptor; D007371:Interferon-gamma