Fas activation

Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. J Exp Med 12 — Conversion of membrane-bound Fas CD95 ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 8 — J Immunol 11 —9. Lack of proapoptotic activity of soluble CD95 ligand is due to its failure to induce CD95 oligomers.

J Interferon Cytokine Res 23 8 —7. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol 6 — Regulation of FAS exon definition and apoptosis by the Ewing sarcoma protein. Cell Rep 7 4 — Inhibition of death receptor signals by cellular FLIP.

Nature —5. EMBO J 14 22 — Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies. J Biol Chem 35 — J Immunol 9 — Molecular ordering of the initial signaling events of CD Mol Cell Biol 22 1 — CD95 signaling via ceramide-rich membrane rafts. J Biol Chem 23 — Molecular mechanisms of ceramide-mediated CD95 clustering.

Biochem Biophys Res Commun 4 — Exp Eye Res 75 1 :1—8. Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death. Ligand-independent redistribution of Fas CD95 into lipid rafts mediates clonotypic T cell death. Nat Immunol 5 2 —9.

Ceramide enables fas to cap and kill. J Biol Chem 26 — Activation interferes with the APO-1 pathway in mature human T cells. Int Immunol 5 6 — Signaling and transcriptional control of Fas ligand gene expression. Fas modulation of apoptosis during negative selection of thymocytes. Immunity 5 6 — Nat Rev Immunol 9 7 —9. Activation-induced cell death in T cells. Immunol Rev — Nature —4. Requirement of Fas expression in B cells for tolerance induction. Eur J Immunol 32 1 — FAS inactivation releases unconventional germinal center B cells that escape antigen control and drive IgE and autoantibody production.

Immunity 42 5 — J Exp Med 1 — Eur J Immunol 24 4 — CD95 engagement induces disseminated endothelial cell apoptosis in vivo: immunopathologic implications. Blood 99 8 —7. Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL.

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Immunity 14 5 — CNS immune privilege: hiding in plain sight. Nagata S, Suda T. Fas and Fas ligand: lpr and gld mutations. Immunol Today 16 1 — Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. N Engl J Med 22 —9. Clin Immunol 1 :1—6. Adv Cancer Res — Fas ligand-dependent suppression of autoimmunity via recruitment and subsequent termination of activated T cells.

Clin Immunol 1 — Death induced by CD95 or CD95 ligand elimination. Cell Rep 7 1 — The role of CD95 and CD95 ligand in cancer.

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J Neurol Sci 1 :1— Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J Exp Med 6 — Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis. Eur J Immunol 27 12 — Fas and Fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system.

J Immunol 7 —9. Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis. J Immunol 7 —3. Finally, treatment of T hybridoma cells with 9-cis retinoic acid or glucocorticoids, which are known to prevent activation-induced T cell apoptosis, inhibited the up-regulation of FasL. We conclude that up-regulated expression of FasL and its subsequent interaction with Fas accounts for the apoptotic response of T cell hybridomas to activation, and that retinoic acid and corticosteroids inhibit activation-induced apoptosis by preventing up-regulation of FasL.

Publication types Research Support, Non-U. Gov't Research Support, U. In response to injury, cytokines and chemokines trigger astrocyte proliferation and migration into the lesioned area where astrocytes contribute to the formation of the glial scar that inhibits axonal regeneration in the CNS [ 1 ]. Characteristic features of reactive astrocytes are morphological changes with cell body hypertrophy and increased expression of a number of proteins absent or weakly expressed in their resting state.

TIMP-1 is a 31 kDa multifunctional secreted glycoprotein that possesses, in addition to its MMP inhibitor activity, growth promoting activities in a number of non neural cells [ 2 - 4 ]. We first demonstrated that TIMP-1 is massively and sequentially upregulated in cortical areas of rat brain after kainate-induced seizures, first in resistant neurons and subsequently in reactive astrocytes [ 5 ].

Selective TIMP-1 upregulation in astrocytes has also been reported after experimental autoimmune encephalomyelitis [ 6 ] or cerebral ischemia [ 7 ]. Interestingly, none of the aforementioned studies reported TIMP-1 expression in reactive microglial cells, highlighting the possibility of a specific role for TIMP-1 in astrocytes among glial cells. Nevertheless, the effects of TIMP-1 in astrocytes are still largely unknown. We thus investigated the influence of TIMP-1 null mutation [ 13 ] on the response of cultured astrocytes to two cytokines of the TNF superfamily know to be induced in similar physiopathological conditions than TIMP As shown in Fig.

We confirmed previous data [ 21 - 23 ] demonstrating the absence of anti-Fas toxicity in astrocytes. Characterisation of astrocyte cultures. Phase contrast photomicrographs of astrocyte cultures. No apparent morphological differences were seen between WT and KO astrocytes. Effect of treatments on cell viability.

In control cultures, gel zymography Fig. We also detected a conspicuous band at kDa. Phenanthroline and BB abolished gelatinase activity, indicating that it was generated by metalloproteinases not shown. Total gelatinase expression detected in gel zymography. The total gelatinase expression was higher in KO astrocytes as compared to WT astrocytes. Astrocytes are the most abundant cells of the brain and express cytokines and adhesion molecules involved in different phases of neuroinflammation, including leukocyte recruitment in the CNS.

Among these proteins, ICAM-1 and MCP-1 are strongly up-regulated during neuroinflammation, and preferentially in astrocytes [ 29 - 32 ]. Accordingly, both proteins are considered as markers of inflammatory response. Quantification of ICAM-1 expression. Since cell proliferation is a hallmark of glial response to neuroinflammatory challenge, we measured the incorporation of 3 H-thymidin, as an index of proliferation, into first passage synchronised astrocytes in response to cytokines and mrTIMP-1 Fig.

Even though the levels of 3 H-thymidin uptake in KO astrocytes treated with TIMP-1 were equivalent to those of WT astrocytes, statistical significance with respect to the untreated KO control was not reached, possibly because of a high level of constitutive 3 H-thymidin incorporation in the mutant astrocytes. MCP-1 is one of the main chemokines involved in the chemoattraction of monocytes and lymphocytes under inflammatory conditions [ 35 , 36 ]. The data Fig.

In contrast, no effect of anti-Fas antibody was observed in WT or KO astrocytes as compared to their controls. The effect of astrocytes on lymphocyte migration was assessed using Transwell chambers and was expressed by the total number of migrated cells A and as an index of migration B. Note that anti-Fas treatment alone without astrocytes in the lower compartment induced lymphocyte migration.

The present report shows that TIMP-1 null mutation attenuates the inflammatory response of astrocytes to Fas activation. The constitutive expression of gelatinases in general, and MMP-2 in particular, is higher in KO than in WT astrocytes, suggesting that in the absence of TIMP-1, the baseline regulation of one of its main targets is altered.

We can not rule out the possibility that the absence of cell death under TNF treatment in KO astrocytes may be actually related to a relatively higher baseline proliferative potential in these cultures. We also show that astrocytes are resistant to Fas-mediated cell death in keeping with previous findings in human [ 21 , 22 ] and mouse astrocyte cultures [ 23 ]. However, this is the first report demonstrating that the activation of Fas stimulates MMP-9 release by astrocytes.

Together, these data support the idea that the activation of receptors of the TNF superfamily triggers inflammatory rather than death signals in astrocytes, and that MMP-9 could act as an inflammatory factor downstream of Fas activation. It is likely that the induced expression of MMP-9 amplifies the inflammatory cascade initiated by cytokines through mechanisms involving the proteolytic activation of latent forms of cytokines.

The most striking finding of the present study was the reduced response displayed by TIMP-1 deficient astrocytes to anti-Fas antibody, in clear contrast with the response of their WT counterparts.

Some of the mechanisms underlying these actions are related with the ability of TIMPs to prevent proteolytic processing of membrane receptors, such as death receptors of the TNF superfamily. It has been suggested that the stabilisation of Fas by endogenous TIMP-1 and TIMP-3 and synthetic MMP inhibitors is responsible for increased sensitivity to apoptosis of various cell types, including Dev neural precursors cells [ 45 ] or cancer cells [ 46 ]. Recently, MMP-7 has been identified as a sheddase of Fas that induces apoptosis resistance in tumour cells [ 47 ].

Considering that astrocytes express high constitutive levels of Fas [ 23 ] and that astrocytes deficient for TIMP-1 likely have an increased proteolytic activity, we hypothesise that constitutive cleavage of Fas may indeed reduce the levels of Fas at the membrane, and consequently hamper astrocyte reactivity to anti-Fas antibody treatment.

It is tempting to speculate that elevated levels of MMP-2 contribute directly or indirectly to Fas cleavage in normal conditions. The exposure of cells to a Fas-L-like factor would favour the downregulation of MMP-2 in an attempt to re-establish the levels of the receptor. Nevertheless, the role of MMP-2 as a Fas sheddase has not been demonstrated yet. Astrocyte proliferation is a hallmark of reactivity in inflammatory settings.

Our data support the idea that TIMP-1 exerts a control on cell growth, as previously suggested by other authors in different cell types [ 48 , 4 ]. Moreover, it has been shown that TIMP-1 accumulates in the nucleus of some cell types [ 49 ] and it has been suggested that it could regulate the cell cycle via a MMP-independent mechanism. This idea is reinforced by the finding in our laboratory that mrTIMP-1 increases by 2-fold the number of astrocytes in a mixed neuronal-astrocyte culture, and that this effect is not mimicked by broad spectrum MMP inhibitors unpublished results.

Perhaps, for this reason, a pro-inflammatory stimulus with cytokines or TIMP-1 did not induce further significant increases in KO astrocyte proliferation.

It is possible that a low constitutive expression of TIMP-1 maintains astrocytes in a resting "low proliferative" state that reacts promptly to a pro-inflammatory stimulus. On the contrary, the chronic absence of TIMP-1 may cause a state of permanent mild "reactivity" or "inflammatory status" that increases the response threshold for new exogenous pro-inflammatory stimuli. From the above, it follows that TIMP-1 might actually act as a homeostatic factor for astrocytes in the resting state, and as a pro-inflammatory factor in stressful conditions, notably in seizing and ischemic brains, where early and massive induction of TIMP-1 in neurons [ 5 , 7 ] precede astrocyte reactivity.

Pro-inflammatory effects of TIMP-1 have also been suggested in in vivo models of rheumatoid arthritis [ 50 ]. We expected that mutant astrocytes resistant to Fas-mediated induction of MCP-1 would attract lymphocytes less efficiently than WT astrocytes displaying a strong upregulation of MCP-1 in the same treatment conditions. These data are coherent with previous reports demonstrating that soluble FasL is chemotactic for neutrophils and that an anti-Fas antibody mimics this effect [ 51 ].

In addition, it has been shown that Fas activation stimulates motility in the absence of cytotoxicity [ 52 ].

This finding suggests that MCP-1 alone is not sufficient to critically influence the chemoattraction of lymphocytes. The activity of MCPs is regulated by MMP-mediated proteolytic processing, resulting in new bioactive truncated proteins with antagonist effects on leukocyte migration. In this scenario, MMPs would indirectly act as anti-inflammatory agents [ 34 ].

We are just starting to unveil the importance that the proteolytic modification of bioactive factors may have in the control of cell behaviour and intercellular communication. All experimental procedures were performed in compliance with our institutional guidelines after obtaining the authorisation of the Laboratory Animal Committee of the Medical Faculty.

Genetically modified KO and WT mice have an identical genetic background. Astrocytes were obtained from the brains of 2-day-old mice. After removal of the meninges, the brains were dissociated into a single-cell suspension by trypsinisation and mechanical disruption.