Oligomerization of soluble Fas antigen induces its cytotoxicity.

Soluble Fas antigen can protect cells against Fas-mediated apoptosis. High level soluble Fas antigen characteristic for blood of patients with autoimmune disease or cancer is believed to prevent the elimination of autoimmune lymphocytes or tumor cells. Here we first report that human recombinant FasDeltaTM, i.e. soluble Fas generated by alternative splicing of the intact exon 6, is capable of inducing death of transformed cells by "reverse" apoptotic signaling via transmembrane Fas ligand. FasDeltaTM, as well as transmembrane Fas antigen, can be either monomeric or oligomeric, and both its forms are efficient in blocking Fas-mediated apoptosis, although the cytotoxic activity is exhibited solely by the latter. An in vivo analysis of soluble Fas antigen showed that unlike in healthy controls, nearly the total FasDeltaTM present in sera of rheumatoid arthritis patients was oligomeric. This resulted in suppression of cell proliferation in the experimental sera and in its promotion in controls. Thus, oligomerization/depolymerization of soluble Fas antigen can regulate its activity and contribute to the pathogenesis of autoimmune diseases and cancer.

Receptors and their ligands from the families of TNF 1 receptors and TNF ligands are crucial for cell proliferation, differentiation, and death. Some of these functions are realized through ligand-induced activation of transmembrane receptors with subsequent signal transduction to the receptor-expressing cell. The molecular mechanisms of this kind of transduction are well studied and described in many reviews (1)(2)(3). Other functions performed by the TNF receptor-ligand system are realized through the so-called "reverse signaling", with transmembrane ligand acting as a receptor, i.e. a molecule that receives and delivers the signal, and the receptor (soluble or transmembrane) serving as a ligand, i.e. the signaling molecule. The reverse signaling has been shown for many members of the TNF ligand family (4 -11). However, the activation modes and pathways of the reverse signal are virtually unexplored and rarely reported on (11)(12)(13)(14)(15)(16)(17).
Fas antigen (Fas) and Fas ligand (FasL) play a key role in maintaining homeostasis of the immune system. FasL-induced activation of transmembrane Fas resulting in death of Fasbearing cells underlies selection of T-and B-lymphocytes and target elimination by T-and natural killers (3,18). FasLmediated reverse signaling was first revealed independently by two research teams (9,10) and shown to participate in growth control of CD8ϩ and CD4ϩ T-lymphocytes. Suzuki and Fink (9,19,20) reported that cross-linked FasIgG enhanced proliferation of CD8ϩ T-lymphocytes. Desbarats et al. (10) showed that activation of transmembrane Fas ligand on CD4ϩ T-lymphocytes inhibited expression of IL-2, thereby suppressing cell proliferation and causing cell death. Presumably, signal transduction via Fas ligand involves kinases Erk 1 and 2, phospholipase A2, and some proteins containing SH3 and WW domains, specifically, Fyn kinase (14 -16).
The current study deals with the possibility of occurrence of FasL-mediated reverse signaling in transformed cells of various origin. It was shown that human recombinant soluble Fas (Fas⌬TM) is capable of inducing cell death by apoptosis with an efficiency dependent on the level of FasL expression on the target cells. Anti-Fas antibodies blocked Fas⌬TM-induced cell death. The cytotoxic activity of recombinant soluble Fas proved to result from Fas oligomerization. Moreover, soluble Fas in sera of rheumatoid arthritis patients was shown to be oligomeric. This finding suggests that in blood serum, soluble Fas antigen activity is regulated not only by the level of Fas expression but also by its oligomarization/depolymerization.

EXPERIMENTAL PROCEDURES
Cell Cultures and Reagents-The following cell cultures were used: human histiocytic lymphoma cell line U-937, human cervical adenocarcinoma HeLa cells, and human Burkitt's lymphoma Raji cells. All cell lines were from Specialist collection of continuous cell lines of vertebrates (Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia). Antibodies to Fas antigen and Fas ligand were from Bioarsenal (Moscow), to caspase-3 and poly(ADP-ribose) polymerase from Pharmingen BD. Fas ligand exhibiting cytotoxic activity and inactive FasL-FLAG were a gift from Prof. D. Wallach.
Generation of rhFas⌬TM and FasFc-The cDNA fragment Fas⌬TM was a product of reverse transcription followed by PCR in the presence of specific primers. mRNA isolated from human HeLa cells was used as a template. The amplified cDNA Fas⌬TM was cloned into vector pET30b(ϩ). Expression and purification of the protein from Escherichia coli BL21(DE3) were performed using the Talon metal affinity resin (Clontech) in accordance with the manufacturer's instructions.
The cDNA fragment Fas corresponding to amino acids 1-156 was amplified with specific primers and cloned into the vector Signal pIgplus (Ingenius). COS-1 cells were transfected with the obtained vector, the FasFc-containing incubation medium was collected, and the protein was purified according to the manufacturer's instructions (Ingenius).
Western Blot Analysis-Protein expression was studied using cell extracts yielded by cell lysis in buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml solutions of leupeptin, pepstatin, and aprotinin. The protein concentration was aligned according to Bradford (21). After electrophoresis in 12% PAGE in the presence of sodium dodecyl sulfate (22), the samples were transferred onto nitrocellulose membrane. For immunoblotting, murine monoclonal antibodies were used as primary antibodies, and anti-mouse antibodies conjugated with peroxidase were used as secondary antibodies. The antigen-antibody complex staining was performed using the ECLplus kit or diaminobezidine in the presence of hydrogen peroxide.
PCR products and DNA fragments of apoptotic cells were analyzed by 1% agarose or 8% acrylamide electrophoresis Serum Analysis-Blood was drawn from 28 patients with rheumatoid arthritis and from 11 healthy controls at the Moscow Medical Academy Hospital. Sera were harvested by centrifugation at 1500 ϫ g and thereafter stored at Ϫ70°C until further processing. A commercially available ELISA kit (Bioarsenal, Moscow, Russia) recognizing recombinant and natural sFas was used for quantification of sFas according to the manufacturer's instructions. Briefly, serum samples were diluted 10-fold and thereafter applied to a polystyrene multiplate precoated with murine monoclonal anti-Fas antibodies 2F3. After 2 h of incubation, plates were washed and thereafter incubated with monoclonal anti-Fas antibodies 10F10 conjugated to horseradish peroxidase. Staining by hydrogen peroxide and tetramethylbenzidine was performed for detection of bound antibodies and their subsequent quantifying with a microtiter plate reader at 490 nm. Standard curves were constructed using serial dilutions of recombinant sFas. Each serum sample was tested in triplicate.
Gel Filtration of sFas-Samples of Fas⌬TM or serum were applied to Toyopearl HW-55 or Superose 12 gel filtration columns using fast protein liquid chromatography equipment. The columns were equilibrated with Tris-buffered saline, pH 7.4, at a flow rate of 0.2 ml/min. The absorption of the eluent was monitored at 280 nm. Fractions were collected, and the amount of sFas was determined by sandwich-ELISA (see above). Molecular masses were determined by comparison with a standard curve of cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and dextran blue.

RESULTS
Cytotoxic Activity of Soluble Fas Antigen-In the cell, there are transmembrane and soluble forms of Fas (28 -30). Among the latter, the highest expression is characteristic of Fas⌬TM (generated by alternative splicing of the intact exon 6) that encodes the Fas transmembrane domain. For the purpose of cloning, Fas⌬TM mRNA isolated from HeLa cells was used. The recombinant protein was expressed and purified as described under "Experimental Procedures" and in accordance with the instructions by equipment manufacturers.
The molecular mass of Fas⌬TM isolated by SDS-PAGE electrophoresis was about 40 kDa, and it could be recognized by specific anti-Fas antibodies (Fig. 1A).
When added to various human cell cultures, Fas⌬TM exhibited the cytotoxic activity (Fig. 1B) to which the cells were sensitive to a variable degree. Antibodies to the extracellular, but not intracellular, portion of Fas blocked the Fas⌬TM-dependent apoptosis (Fig. 1C), thereby demonstrating specificity of Fas⌬TM as a cell killer.
No effect of Fas⌬TM on cell distribution over the cell cycle phases was observed (data not shown). As revealed by Western blotting and analysis of DNA from apoptotic cells, Fas⌬TM induced cell death by apoptosis ( Fig. 2A). Caspase-3 that was formed in the cells 4 h after treatment cleaved poly(ADPribose) polymerase to trigger internucleosome fragmentation of the DNA (31). Interestingly, prior to the DNA fragmentation in apoptotic cells, FasL expression decreased with increasing amount of Fas mRNA (Fig. 2B). Because inhibition of RNA synthesis by actinomycin D enhances Fas⌬TM-induced apoptosis (Fig. 1B), it can be believed that a change in expression of FasL and Fas mRNA is a protective response to the treatment by target cells.
A reason for variable degrees of Fas⌬TM sensitivity shown by cell cultures could lie in different levels of expression of acceptor molecules mediating the action of Fas⌬TM. Among potential Fas⌬TM targets, there could be FasL, Fas receptor, or probably other proteins (30,32,33). An analysis of expression of FasL and Fas receptor in the studied cell cultures showed that the level of expression of Fas antigen was nearly the same in all the samples, whereas FasL expression was higher in HeLa and U-937 cells as compared with Raji (Fig. 3A). However, HeLa and U-937 cells were more sensitive to Fas⌬TM cytotoxicity than Raji cells (Fig. 1B). To verify the correlation between the FasL expression level and efficiency of Fas⌬TMinduced cell death, Fas⌬TM cytotoxicity was assayed using HeLa cells transiently transfected with FasL cDNA-bearing vector in the antisense orientation. As found, inhibition of FasL expression led to an increased cell resistance against Fas⌬TM (Fig. 3B).
A direct evidence for FasL contribution to Fas⌬TM-induced cell death came from an analysis of the specificity of interactions between recombinant Fas⌬TM and Fas ligand. In the experiment, protein extracts of Fas⌬TM-sensitive cell cultures passed through a Fas⌬TM-containing column. After eluting, the Fas⌬TM-bound proteins were subjected to Western blotting with specific anti-FasL antibodies (Fig. 3C). FasL was detected in the protein fraction bound to Fas⌬TM. Hence, the obtained Fas⌬TM was able to bind FasL.
Taken together, these results suggest that it is FasL that serves as a target for Fas⌬TM.
Oligomerization of Soluble Fas Antigen-Further studies of the Fas⌬TM sample showed that after its separation by gel filtration, the protein was contained by the fraction corresponding to a molecular mass of about 150 kDa (Fig. 4). This demonstrated the ability of Fas⌬TM to be oligomeric, which was in good agreement with earlier findings by other researchers (32,33). It is noteworthy that only samples containing a high molecular fraction of Fas⌬TM displayed the Fas⌬TM cytotoxic activity.
To verify the suggestion that the cytotoxic activity of Fas⌬TM depends on its oligomerization, we used FasFc, a COS cell-derived combination of the extracellular portion of Fas and the Fc fragment of human IgG. When added to cell cultures, FasFc exhibited no notable cytotoxicity, although its crosslinking with the use of anti-Fc antibodies induced cell death (Fig. 5A). Note that both monomeric and cross-linked FasFc efficiently blocked Fas ligand-induced apoptosis (Fig. 5B). Thus, interactions of both cross-linked FasFc and recombinant oligomeric Fas⌬TM with transmembrane Fas ligand induce apoptosis of target cells. Binding of both oligomeric and monomeric Fas to soluble Fas ligand suppresses the cytotoxic activity of the latter.
Thus, cytotoxicity of Fas⌬TM comes from its oligomerization. The reasons for Fas⌬TM oligomerization are still unclear. It may be believed that spontaneous self-association of Fas⌬TM results from interactions between CRD1/PLAD regions of the extracellular portion of Fas and/or the "death domains" of the Fas intracellular region at high concentrations of Fas⌬TM (33)(34)(35).
Oligomeric Fas Antigen in the Sera of Patients-The fact of oligomerization of recombinant Fas⌬TM suggested existence of natural oligomeric soluble Fas. Specifically, a high concentration of soluble Fas was reported for blood drawn from patients with autoimmune disease (36,37). Because rheumatoid arthritis (RA) is known to be characterized by a higher concentration of soluble Fas in the blood of a patient, a question arises as to whether Fas is monomeric or oligomeric in sera from RA patients. For this purpose, the sera were separated by gel filtration, and the obtained fractions were assayed for the Fas presence using sandwich ELISA.
In RA sera with 3-5-fold Fas expression (up to 10 ng/ml) as compared with controls, the major part of found to belong to high molecular fractions with a molecular mass of 150 -200 kDa (Fig. 6A). Confirmation of the presence of Fas antigen in these fractions came from their immunoprecipitation with anti-Fas antibodies (Fig. 6B). In healthy controls, no soluble Fas was detected in high molecular fractions, and no other Fas but monomeric was observed (data not shown). However, it cannot be ruled out that this was a result of a low concentration of the receptor.
Since oligomeric recombinant Fas exhibited the cytotoxic activity, we assayed some sera containing oligomeric soluble Fas at high concentrations (6 -10 ng/ml) for such an activity. As was expected, addition of 20% healthy control to HeLa cell culture resulted in a higher proliferation of the cells. Note that non-cytotoxic FasL-FLAG capable of interacting with Fas produced no effect on the proliferation-stimulating activity of normal sera (Fig. 6C). Unlike cultivation with controls, that with RA sera containing soluble Fas at a high concentration resulted in a suppressed proliferation of HeLa cells, and the cytotoxicity was notably down-regulated by FasL-FLAG. Hence, cytotoxicity of RA sera is determined, at least partially, by oligomeric soluble Fas antigen. Besides, the suppression of proliferation of transformed cells by sera from autoimmune disease patients suggests that Fas is a key signaling molecule providing a link between cancero-and autoimmune genesis. DISCUSSION Here we report that (a) Fas⌬TM is capable of inducing death of transformed cells; (b) in vivo there exists oligomeric soluble Fas antigen, e.g. in RA sera; and (c) cytotoxicity of soluble Fas antigen results from its oligomerization.
We believe that Fas⌬TM cytotoxicity is indicative of a novel functional role of soluble Fas antigen. Taken together, the literature data and our results suggest that functionally, soluble Fas antigen not only inhibits Fas ligand cytotoxicity and hence, Fas-mediated apoptosis (28), but also triggers cell death through reverse signaling via transmembrane Fas ligand. This requires more attention to the cases of higher expression of soluble Fas with distinction as to its apoptosis-inhibiting and apoptosis-stimulating activities. Specifically, inhibition of apoptosis of peripheral blood lymphocytes by soluble FasL in hepatocellular carcinoma patients described by Nakamoto et al. (38) may be a result of neutralization of cytotoxic soluble Fas antigen whose high level of expression in hepatocellular carcinoma samples has been reliably documented (39,40). Sup- pressed cytotoxic soluble Fas may also underlie the therapeutic effect of antibodies to Fas antigen reported in a number of papers (41)(42)(43). Besides, the effects of metalloprotease (that cleaves membrane-bound FasL) and its inhibitors can also be considered from the viewpoint of reverse signaling as events inhibiting or promoting signal transduction via transmembrane FasL (44 -46).
The cytotoxicity-oligomerization relationship of soluble Fas seems to offer novel interpretations of previous findings. First, the illusory contradiction concerning the FasFc effect upon lymphocytes reported by Suzuki and Fink (f) on the one hand and by Desbarats et al. (10) on the other could arise from the difference between FasFc samples used. Probably, the former research team used monomeric non-cytotoxic FasFc, while the latter dealt with dimeric FasFc capable of inducing death of T-lymphocytes (9, 10). Next, it can be believed that certain mutations of Fas antigen that are likely to underlie autoimmune diseases (47)(48)(49) affect not only cytotoxic signal transduction via membrane Fas but also oligomerization of soluble Fas and, hence, realization of reverse signaling as well. Biologically, Fas⌬TM oligomerization can shed light on the reason for the existence of several forms of soluble Fas antigen (29,30), which probably lies in synthesis of molecules that interact with Fas ligand but are unable to form homooligomers and, hence, to trigger the cytotoxic reverse signaling. Moreover, the ability of soluble Fas antigen to make high molecular associations presupposes rigid regulatory mechanisms of its oligomerization/ depolymerization.
Finally, one can leave room for possible associations of soluble Fas with heterological proteins that bear a CRD1-like motif and provide a signal exchange between the Fas-Fas ligand system and cytokines of other families.
Thus, the cytotoxic properties of oligomeric soluble Fas antigen reported here contribute to our knowledge of functioning of the Fas-Fas ligand system and open up new opportunities in the treatment of tumors and autoimmune diseases.