Sara M. Mariani

Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage

TRAIL (APO‐2 ligand) and CD95L (CD95/APO‐1/Fas ligand) share the highest homology among the TNF family members and the ability to induce apoptosis. These similarities raise the issue of a potential functional redundancy between the two ligands. We have previously shown that CD95L‐resistant cells may be sensitive to TRAIL, even though apoptosis induced by both ligands is blocked by caspase inhibitors. Here we investigated TRAIL protein expression in cells of T and B origin and compared its regulation of expression with that of CD95L. A rabbit antibody (Ab) to a peptide sequence in the extracellular region of TRAIL identified recombinant TRAIL (rTRAIL) produced by Sf9 cells as a protein of approximately 32 – 33 kDa and soluble rTRAIL as a 19 – 20‐kDa protein. In human and mouse cells, the Ab identified a 33 – 34‐kDa and an additional 19 – 20‐kDa protein only in human cells. Both transformed cells of the T and B lymphocyte lineage were found to react with the anti‐TRAIL Ab by immunoblot analysis and surface staining. The majority of the cells analyzed co‐expressed TRAIL and CD95L. Two cell lines showed a mirror‐pattern, one being TRAILhigh CD95Llow and the other TRAILlow CD95Lhigh, thus suggesting the existence of a cell type‐specific regulation of expression of the two ligands. Differently from CD95L, surface TRAIL was not up‐regulated by any of the metalloprotease inhibitors tested, independently of the cell type analyzed. Conversely, reactivity with the anti‐TRAIL but not with the anti‐CD95L Ab was enhanced by cysteine protease inhibitors. An in vitro cleavage assay showed that generation of soluble rTRAIL was dependent on the functional activity of cysteine proteases, as it was blocked by leupeptin and E64 but not by the metalloprotease inhibitor 1,10‐phenanthroline. Thus, even though TRAIL and CD95L share structural and functional properties, they have unique properties as they differ in their regulatory pathways, i.  e. cell‐type‐dependent expression and sensitivity to protease inhibitors.

Surface expression of TRAIL/Apo‐2 ligand in activated mouse T and B cells

Like other members of the TNF family, TRAIL/Apo‐2 ligand induces apoptosis in sensitive target cells in a caspase‐dependent fashion. We recently found that TRAIL may be constitutively expressed on the surface of mouse and human tumor cells of T and B origin. To define the pattern of TRAIL expression in normal immune cells, freshly isolated splenocytes, Concanavalin A/IL‐2‐activated T cells and lipopolysaccharide‐activated B cells were analyzed by surface staining with or without secondary stimulation. Activated, but not resting, CD3+ cells expressed TRAIL in an activation‐dependent fashion. Conversely, freshly isolated B220+ cells displayed surface TRAIL and CD95L that were retained following activation. Restimulation with the protein kinase C activator phorbol 12‐myristate 13‐acetate and the calcium ionophore ionomycin or an agonistic anti‐CD3 monoclonal antibody induced significant up‐regulation of surface TRAIL and CD95L in CD3+ , TCRα β cells with CD4+ or CD8+ phenotype. Similarly to CD95L, TRAIL up‐regulation was protein synthesis dependent and cyclosporin A sensitive. These results indicate that both TRAIL and CD95L are displayed on the cell surface of activated immune cells and may thus represent complementary effector pathways in the regulatory functions of T and B cells.

CD95 Ligand (CD95L) in Normal Human Lymphoid Tissues: A Subset of Plasma Cells Are Prominent Producers of CD95L

CD95(Fas/APO-1)-ligand (CD95L) mediates apoptosis by trimerization of the CD95 receptor on the surface of sensitive cells. In vitro studies have shown CD95L expression mainly by activated T cells and suggested a role for CD95L in the regulation of immune responses. Little is known, however, about the cellular distribution of CD95L in situ in the normal human immune system. We investigated CD95L expression in tissue sections of the thymus, lymph node, spleen, tonsil, and gastrointestinal tract using in situ hybridization and two monoclonal antibodies. In all these organs, cells expressing CD95L message and protein were scarce and comprised scattered lymphocytes, rare nonlymphoid cells, and a subset of epithelioid endothelial cells. Surprisingly, a subset of plasma cells turned out to be the most prominent producers of CD95L, matching the reports on CD95L in myeloma cells. CD95L+ plasma cells were most numerous in the mucosa-associated lymphoid tissue. This also applied to acquired mucosa-associated lymphoid tissue in chronic gastritis in which CD95L+ plasma cells were found scattered in the lamina propria. Our data suggest that plasma cells as yet may be neglected modulators of immune responses.

Expression of biologically active mouse and human CD95/APO-1/Fas ligand in the baculovirus system.

CD95L (CD95/APO-1/Fas ligand) is a type II transmembrane glycoprotein that induces apoptosis in sensitive target cells. CD95L can be proteolytically cleaved from the membrane by a metalloprotease and occurs in a soluble form. Thus CD95L may act as a cytotoxic effector molecule at a distance from the producer cell. In order to develop an expression system yielding large quantities of CD95L, we expressed mouse and human CD95L tagged with a FLAG sequence in insect cells (Sf9) infected with recombinant baculovirus. CD95L expressed by Sf9 cells was detected with rabbit antibodies directed against the carboxy-terminal region of CD95L (which is highly conserved between mouse and human CD95L) and with an anti-FLAG monoclonal antibody. Immunoblotting showed that recombinant mouse and human CD95L expression was associated with the presence of 40 kDa and 32-33 kDa proteins. CD95L released into the supernatant of infected Sf9 cells specifically induced apoptosis in sensitive target cells, thus indicating that recombinant mouse and human CD95L were functional. The presence of the amino-terminal FLAG sequence did not modify this biological activity. Infection of Sf9 cells with recombinant baculoviruses may thus provide an efficient system for the expression of biologically active recombinant CD95L.

P53 escape from P19ARF-trapped MDM2

Maintenance of steady-state levels of the tumor suppressor p53 is very dependent on its interaction with Mdm2. This multifunctional protein antagonizes p53 activity by inhibition of p53-dependent transcription, as well as by enforcement of p53 nuclear transport and cytoplasmic degradation. But while Mdm2 antagonizes p53, what controls Mdm2? Jason D. Weber and colleagues from the St. Judes Childrens Research Hospital and SUNY now report in the May issue of Nature Cell Biology that the nucleolar protein Arf, a product of the Ink4 locus, sequesters Mdm2 in the nucleoli after activation of the oncoprotein Myc. Similarly, while Arf and Mdm2 linger in the nucleoli, p53 takes action in the nuclei of senescent mouse fibroblasts. Thus, Arf takes the stage as an essential element in the control of p53 activation, raising questions about the in vivo tumor-promoting effect of tumor-associated Arf mutants with defective intracellular co-localization.

Detection of active infection of Sf9 insect cells by recombinant baculoviruses

Production of different recombinant proteins in baculovirus AcMNPV (BV)-infected cells may be facilitated by the availability of immunoassays to monitor active infection of Sf9 insect cells. To this end, two hybridomas secreting mouse monoclonal antibodies (mAbs) were established to different BV-related products. The proteins recognized by mAb SM22 and SM62 were easily detectable by immunoblotting and immunostaining in Sf9 cells infected with recombinant BV (rBV), but not in non-infected cells. Their production paralleled that of the recombinant proteins analyzed but was independent of the type of recombinant protein expressed. Thus, immunoassays with these mAbs allow: (1) daily monitoring of the infection occurring in small and large scale cultures of Sf9 cells using a defined rBV; (2) preliminary assessment of active rBV infection in the absence of a specific reagent for the recombinant protein and (3) single-reagent comparison of the infection achieved in Sf9 cells exposed to rBVs expressing different recombinant proteins.