Accumulation and Activation-Induced Release of Preformed Fas (CD95) Ligand During the Pathogenesis of Experimental Graft-Versus-Host Disease

Abstract

Fas (CD95/APO-1) ligand (FasL)-mediated cytotoxicity has been implicated in tissue destruction in a variety of diseases, including acute graft-vs-host disease (GVHD). In this study, we have analyzed FasL expression and regulation during the course of experimental murine acute GVHD. Although activation-induced FasL-mediated cytotoxicity in control T cells was sensitive to the immunosuppressant cyclosporin A, we observed that functional FasL expression of GVHD T cells became increasingly cyclosporin A unresponsive. This was found to be the result of a massive in vivo accumulation and intracellular storage of FasL protein and its release in a transcription- and protein synthesis-independent manner. Immunohistochemistry analysis of FasL expression in situ revealed accumulation of FasL-expressing cells in the spleen, the liver, and small intestine, with a typical cytoplasmic and granular expression pattern. Thus, we conclude that the release of preformed FasL by infiltrating donor T cells may contribute to recipient tissue damage during the pathogenesis of acute GVHD.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells

“…Culture of activated murine T cells in IL-2-containing medium for 7 days leads to expression and accumulation of FasL in intracellular stores. Immunohistochemical detection of FasL revealed that T cell blasts store FasL in granule-like structures (Figure 1a), confirming previous observation by us 3 and others. 7 A similar pattern was observed when cells were stained for the granule marker CD107a/Lamp-1 (Figure 1b).…”

“…To assess the contribution of de novo synthesized versus preformed and granule-stored FasL in this process, activation-induced FasL transcription was blocked by cyclosporin A (CsA). 3 Figure 2c shows anti-CD3-induced FasL-mediated killing was only partially blocked by CsA, indicating that a substantial proportion of FasL killing activity was mediated by preformed and degranulated FasL.…”

“…3,9 The activation-induced movement of secretory lysosomes towards the cell surface and into the immunological synapse proceeds along the microtubules, whereas the last step to plasma membrane appears to be actin filament-dependent. 10 However, the exact signaling events initiated by the T cell receptor leading to the degranulation and cell surface expression of FasL are largely unknown.…”

“…6 Furthermore, it was shown that FasL is accumulated in activated T cells in granule-like structures and can be rapidly released in a protein synthesis-independent manner upon restimulation. 3,7,8 These FasL-containing granule-like structures likely represent so-called secretory lysosomes with important functions in degranulation-induced cytotoxicity in T cells. Interestingly, Griffiths and co-workers 7,8 have recently shown that intracellular FasL co-localizes with other markers of secretory lysosomes, such as CD107a/Lamp-1, CD63, perforin and granzymes.…”

“…1,2 FasL-mediated target cell apoptosis and tissue destruction has also been associated with various forms of immunopathology, including acute graft-versus-host disease and hepatitis. 1,3 As many tissue cells express high levels of Fas and are exquisitely sensitive to FasL-induced cell death, FasL expression must be tightly regulated. A crucial element in the control of activation-induced FasL expression and function is its transcriptional regulation by various transcription factors.…”