Isabella Cascino, Giuliana Papoff, Adriana Eramo, and Giovina Ruberti

Department of Immunobiology, Institute of Cell Biology, National Research Council, Rome, Italy.

Received 12/01/95; Accepted 01/11/96; On-line 1/1/96

As yet, there is no a definitive answer to this question, but a number of considerations suggest that this is at least a likely possibility. Evidence for a biological effect of sFas derives from two sets of data.

A) In vivo observations of quantitative variations of sFas under physiological and pathological conditions.

1. Intrahepatic T lymphocytes in the mouse have been shown to possess different sensitivity to apoptosis according to the mRNA expression of a soluble Fas isoform (Fas ß) (44).

2. Sera of patients with systemic lupus erithematosus (28) and sera of patients with different high- and low-grade malignant B- and T-cell leukemias and lymphomas (45) have been reported to have an increased level of FasTMDel (FasExo6Del).

The results in the mouse system appear clear-cut, except for the fact that they are limited to RNA expression and lack data on Fas proteins. The data concerning sera of autoimmune patients are more open to criticism because the cells producing sFas have not been characterized. Direct proof of a role of sFas in those diseases remains to be established. Moreover, the ELISA used in the above studies detected only FasTMDel (FasExo6Del) but not the other variants with deletions in the extracytoplasmic regions. This problem can now be addressed by an ELISA allowing the detection all Fas isoforms so far identified (30). A correlation between the expression pattern of the different sFas in human T cells and sensitivity to apoptosis remains to be determined. Preliminary data from our group show marked qualitative and quantitative variations in the contribution of each variant to the "soluble Fas-pool" in different cell lines and in PBMC from different individuals.

It will be interesting to test whether these variations are associated with a differential regulation of apoptosis.

B) In vitro apoptosis inhibition studies.

Antibody to Fas and recombinant soluble FasL (rFasL) induce apoptosis in some tumor cell lines (Reviewed in Ref. 15). Early observations of an apoptosis-inhibition function of sFas molecules were based on Fas antibody- induced apoptosis that is far from being a physiological mechanism (28, 29). However several isoforms also block Fas activation due to interaction with its natural ligand suggesting a more likely potential regulatory function (30).

The mechanism of the inhibitory effect is still unknown. Following Fas/FasL interaction, the signal is transmitted to the death domain and0 from this to the apoptosis machinery. A necessary requirement for this process is supposed to be the trimerization of the Fas molecules, as suggested by the homology to TNFalpha and TNFß (46-50), the fact that IgM anti-Fas antibodies have agonistic activities (8) and the report that FasL forms homotrimers (51). Based on these data, two models can be postulated, called the "competition" model and the "inactive heterotrimers" model. These are shown schematically in Fig. 2.

The "competition" model assumes that sFas molecules compete with Fas for binding. This possibility is unlikely for Ab-induced apoptosis since sFas isoforms, with the exception of FasExo6Del, are not recognized by the CH-11 agonistic IgM Ab. However, this possibility may be valid for FasL-induced apoptosis. In this case, this would imply different mechanisms for apoptosis inhibition in the two in vitro systems. In the "inactive heterotrimer" model, we propose that the sFas forms are still able to trimerize with Fas but that they are not able to form active trimers because of the lack of other domains including the death domain. As a consequence, the signal provided by FasL or by Fas antibody is prevented from reaching the inside of the cell and results in inhibition of apoptosis. The fact that all variants exhibit a marked inhibition points toward the N-terminal domain as being responsible for this effect. The "inactive heterotrimer" model, even though more appealing is, at the present, only a working hypothesis that may help to organize further experimentation. A possible analogy with sFas apoptosis inhibition can be found in the inhibition of tyrosine kinase activity of the epidermal growth factor receptor (EGFR). This growth factor receptor was found to be regulated by a truncated receptor not by simple competition for available EGF but by specific association with the EGFR (52).

In conclusion, alternative splicing may be an important event in the regulation of Fas/FasL interaction and thus in the regulation of immune responses by these receptor-ligand pairs. However many important questions remain to be answered. Interestingly, other apoptosis genes like Ich-1, an Ice/ced3 related gene and bclx, a bcl2 related gene, can be expressed as splicing variants that either prevent or cause cell death depending on how the mRNA is processed (53, 54).