Takashi Suda

Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.

The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.

Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVEnTo investigate the expression and function of Fas ligand (FasL),which can be in a membrane-bound or soluble form, in the joints of patients with rheumatoid arthritis (RA) and osteoarthritis (OA).nnnMETHODSnThe concentration of soluble FasL (sFasL) in serum and synovial fluid (SF) from 24 OA and 38 RA patients was measured using an enzyme-linked immunosorbent assay. The expression of FasL on SF lymphocytes (SFL) and peripheral blood lymphocytes (PBL) was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. A cytotoxic killing assay of membrane-bound FasL and purified sFasL against cultured synovial cells was also performed.nnnRESULTSnSoluble FasL was detected in the SF of patients with RA and OA, but not in their serum. The concentration of SF sFasL was remarkably higher in patients with severe RA than in patients with mild RA or with OA. RT-PCR showed that SFL, but not PBL, from RA patients expressed messenger RNA for FasL. Membrane-bound FasL induced apoptosis in cultured synovial cells from the RA and OA patients, but naturally processed human sFasL did not.nnnCONCLUSIONnSFL from RA patients expressed FasL, and cleaved sFasL accumulated in the SF of inflamed joints. The different killing activity of membrane-bound FasL and sFasL against synovial cells may regulate Fas-mediated apoptosis in synovial cells.

The membrane-bound but not the soluble form of human Fas ligand is responsible for its inflammatory activity

The ectopic expression of Fas ligand (FasL/CD95L) in tissues or tumors induces neutrophil infiltration and the destruction of the tissues or the rejection of tumors. It has been suggested thatthe infiltrated neutrophils are responsible for the latter phenomena. FasL is synthesized as a type II transmembrane protein, and soluble FasL is produced by a proteolytic mechanism from the membrane‐bound form. We previously demonstrated that uncleavable membrane‐bound FasL of mice induces IL‐1β release from inflammatory cells, and suggested that the IL‐1β enhances neutrophil infiltration. However, recent papers reported that human soluble FasL is directly chemoattractive to neutrophils in vitro and proposed that the soluble form of FasL is responsible for its inflammatory activity. Therefore, in this report, we investigated which form is responsible for the inflammatory activities of human FasL. We produced tumor cell lines expressing one or both forms of human FasL. Cells expressing both forms or only the membrane‐bound form of FasL induced neutrophil infiltration when transplanted into the peritoneal cavity of syngeneic mice, while cells expressing only the soluble form did not. Purified soluble FasL failed to induce neutrophil infiltration in vivo. IL‐1β release from inflammatory peritoneal exudate and acceleration of tumor rejection were also mediated by membrane‐bound but not soluble FasL. These results indicate that the membrane‐bound form of FasL is primarily responsible for its inflammatory activity.

Separation of the Tumor Rejection Antigen of Rous Sarcoma Virus-induced Murine Fibrosarcoma

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)‐induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freunds adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)‐solubilized fraction after the SDS‐extraction of CSA1M membranes. This CSA1M SDS‐solubilized preparation gave protection against syngeneic RSV‐induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV‐unrelated tumor cells. The membrane or SDS‐solubilized fraction from RSV‐unrelated tumor cells was unable to generate anti‐CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS‐solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS‐solubilized preparation was applied to a Sephacryl S‐300 superfine column, TRA activity was recovered in the range of molecular weight of 50‐90 kD. Further fractionation of this TRA‐positive fraction by SDS‐polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56–68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV‐induced tumor‐specific in vivo protective immunity.

The Complementary Registration Method for Vocal Tract of Sibilant /s/

We analyzed the sibilant /s/, an unvoiced consonant, by considering turbulence and vortex sound. The morphology of an oral tract is required when doing numerical analyses, and because magnetic resonance imaging (MRI) and cone-beam computed tomography (CBCT) are complementary, we use them as the registration technique in our proposed method. Calculating the approximate function to represent the morphological features of these geometries enabled us to associate the MRI and CBCT datasets with each other by referring to the functions in our proposal method. We applied the method to an actual person’s 4-dimensional MRI and CBCT datasets, and as a result were able to determine the appropriate registration result. The result was validated in a special configuration in which the result image was compared with the CBCT image of an actual person’s oral tract while pronouncing sibilant /s/. The validation demonstrates how our proposed method accurately registers the MRI and CBCT datasets and can thereby be used to perform numerical analyses in speech science research.

Tumor-specific T cell lines: capacity to proliferate and produce interleukin 2 in response to various forms of tumor antigens.

Anti‐tumor proliferative T cell lines were established from cultures of lymph node cells from BALB/c mice immunized to syngeneic CSAIM fibrosarcoma with the CSAIM tumor cell membrane. The cultures were maintained throughout in the absence of exogenous interleukin 2 (IL2) J. Cell surface phenotypes of all T cell lines established were Thy‐1+, Ig‐, L3T4+ and Lyt‐2‐. Their proliferation was induced in a tumor antigen dose‐dependent fashion and a tumor antigen‐specific way. Such proliferative responses were inhibited by the addition to cultures of anti‐class II H‐2d (anti‐I‐Ad) or anti‐L3T4 but not of anti‐class I H‐2d or anti‐Lyt‐2 monoclonal antibody. None of the T cell lines exhibited any cytotoxic T lymphocyte activity but they all produced IL2 upon stimulation with CSAIM tumor antigens, indicating that they represent helper‐type T cell (Th) lines. The activation of these tumor‐specific Th lines was induced with either CSAIM tumor cells themselves, or their membrane or detergent‐solubilized fraction depending on the presence of antigen‐presenting cells (APC). Most importantly, activation was also inducible by membranous tumor antigen‐pulsed APC, which were capable of producing potent anti‐tumor protective immunity when administered in vivo into syngeneic BALB/c mice. These results indicate that the tumor‐specific Th lines established here can be activated with various forms of tumor antigens for their expression of helper function. Since Th lines of this type have not been described previously, our Th lines provide an intriguing tool for investigating the cellular and molecular mechanisms by which tumor‐specific Th recognize tumor antigens.

The Tumor Rejection Antigen Separated from Rous Sarcoma Virus‐induced Murine Fibrosarcoma Exhibits a Molecular Weight of Approximately 60 kD but Differs from Functional pp60src

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was obtained in a solubilized form by sodium dodecyl sulfate (SDS) extraction of plasma membrane fraction from Rous sarcoma virus (RSV)‐induced CSA1M fibrosarcoma cells (BALB/c origin). Analyses by Sephacryl S‐300 gel filtration and SDS‐polyacrylamide gel electrophoresis revealed that TRA activity was recovered in the fraction with a molecular weight of approximately 60 kD. Unfractionated crude SDS‐solubilized preparation contained gp70 as detected by rabbit anti‐gp70 antiserum, whereas such reactivity was lost in the fraction exhibiting the molecular weight of about 60 kD. Since this fraction retained pp60src activity, the relation of TRA to pp60src was further investigated. pp60v‐src was also obtained from the lysate of v‐src‐expressing yeast transformant. Immunization of BALB/c mice with such pp60v‐src‐containing lysate failed to induce any significant tumor protection. The above 60 kD fraction of CSA1M solubilized antigens was allowed to bind to Sepharose beads coupled with anti‐pp60src monoclonal antibody and separated into the bead‐bound and bead‐unbound fractions. The bead‐bound fraction that was recovered from pp60src‐binding beads (pp60src‐positive fraction) did not exhibit the TRA activity. In contrast, immunization with the fraction depleted of pp60src activity (bead‐unbound fraction) resulted in potent tumor protection. These results indicate that the solubilized membranous component(s) of CSA1M with a molecular weight of approximately 60 kD, which is distinct from functional pp60src, functions as the TRA against RSV‐induced CSA1M tumor cells.

Application of T Cell—T Cell Interaction to Enhanced Tumor-Specific Immunity Capable of Eradicating Tumor Cells in Vivo

Investigations have attempted to delineate the consequences of malignant transformation of cells by the appearance of new cell surface structures [tumor-associated antigens (TAA) or tumor-associated transplantation antigens (TATA)] that could be identified by specific antiserum or by their ability to induce a specific cellular immune response. Considerable efforts have been undertaken to establish the significance of these tumor cell surface structures by correlating their cell surface expression with changes that take place during the course of neoplastic disease. The most compelling evidence for the existence of TATA comes from the study of chemically induced tumors of inbred rodents. These tumors express neoantigens capable of immunizing syngeneic or autochthonous hosts against subsequent challenge with the same tumor.(1–4)