Heat-induced chaperone activity of HSP90.

The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 degrees C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. alpha-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.

Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes.

Apoptosis appears to play a major role in the differentiation and selection of T and B lymphocytes, but the mechanisms of clonal deletion of T cells in thymus are not well understood. We have prepared an anti-human Fas IgM mAb with associated apoptosis-inducing activity in Fas antigen-positive target cells including human T cells. We analyzed the expression of apoptosis antigen Fas on human thymocytes by cytofluorometry showing low, but significant amounts of Fas antigen on double-negative and double-positive undifferentiated thymocytes. On the contrary, most of the differentiated thymocytes (single-positive or CD3-brightest) expressed undetectable levels of Fas antigen. About 1-2% of thymocytes expressed high amounts of Fas antigen, and these cells, which were CD3-bright, were CD4-bright and CD8-low at the stage of late double-positive lineage. Immunohistological analysis shows these Fas-bright cells on the edge of the medulla. Stimulation through the TCR complex was shown to induce the expression of Fas antigen on thymocytes at the late double-positive stage and prolonged stimulation through the TCR complex rendered the Fas-bright thymocytes sensitive to apoptosis-inducing activity of anti-Fas. To show the involvement of the Fas system in the negative selection/clonal deletion of thymocytes, we organ-cultured human thymus in the presence of the superantigen, staphylococcus enterotoxin B (SEB), and new antagonistic anti-Fas mAb, which can inhibit the apoptosis-inducing activity of the original anti-Fas mAb. The SEB-reactive TCR complex on thymocytes was at first down-regulated by SEB, then the SEB-reactive clone was deleted by apoptosis, which was inhibited by an antagonistic anti-Fas mAb. Thus, Fas antigen is shown to be involved in the negative selection/clonal deletion of superantigen-reactive thymocytes.

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.

Anti-Fas monoclonal antibody is cytocidal to human immunodeficiency virus-infected cells without augmenting viral replication.

A cytotoxic monoclonal antibody (anti-Fas mAb) against the 200-kDa cell surface Fas antigen, which is associated with the tumor necrosis factor (TNF) receptor, was examined for its in vitro activity on human immunodeficiency virus (HIV)-infected cells. It was found that both TNF and anti-Fas mAb selectively killed the chronically HIV-infected cells. Uninfected cells were less sensitive to the antibody than those infected with HIV. When the cells were cultured in the presence of anti-Fas mAb immediately after the HIV infection, the spread of HIV-infected cells was suppressed by the antibody. TNF augmented both the synthesis of HIV-specific mRNA in HIV-infected cells and formation of multinucleated giant cells. In contrast, the anti-Fas mAb did not augment HIV replication or enhance the HIV-induced formation of syncytia. The results indicated that anti-Fas mAb mimicks the cytocidal action of TNF but does not augment HIV replication.

Identification of a cell surface 105 kd protein (Aic-2 antigen) which binds interleukin-3.

A mouse interleukin-3 (IL-3)-binding molecule that is an essential constituent of the mouse IL-3 receptor complex was identified as a cell surface protein of Mr 105 kd. A rat monoclonal antibody, anti-Aic-2 IgM, recognized and immunoprecipitated a cell surface 105 kd protein (Aic-2 antigen). The antigen (Aic-2) and IL-3 receptor were co-down-regulated upon incubation of IL-3-dependent mouse IC2 cells with either anti-Aic-2 IgM or IL-3 at 37 degrees C, whereas anti-Aic-2 did not inhibit the binding of IL-3 to IC2 cells at 15 degrees C. The Aic-2 antigen and IL-3 receptor were co-distributed on various cell lines and cell types. IL-3 was shown to bind specifically to the Aic-2 antigen (Mr 105 kd) in the immunoprecipitated complex with anti-Aic-2. Chemically cross-linking of IL-3 to surface molecules of IC2 cells produced three types of complexes with Mr 95, 140, and 200-340 kd in SDS-PAGE, and pre-incubation with anti-Aic-2 IgM at 37 degrees C reduced the intensity of the three bands to the same degree. Moreover, anti-Aic-2 immunoprecipitated these three IL-3-cross-linked complexes, whereas the same monoclonal antibody recognized a single 105 kd molecule. Anti-Aic-2 IgM enhanced IL-3-dependent growth of IC2 cells though it did not promote proliferation of IC2 cells.

A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor.

We have prepared an mAb specific for a human cell surface component (termed anti-Fas mAb). Anti-Fas shows cell-killing activity that is indistinguishable from the cytolytic activity of TNF. Fas antigen was characterized by western blotting, indicating that Fas antigen is a cell surface protein with a molecular weight of 200,000, which is different from the molecular weight of TNF-R. Fas antigen, however, is co-downregulated with the TNF-R when cells sensitive to the cytolytic activity of TNF are incubated with either TNF or anti-Fas. In contrast, Fas antigen on cells insensitive to TNF is not co-downregulated with the TNF-R. We suggest that the cell-killing activity of TNF is mediated by Fas antigen associated with the TNF-R.

The kinetics of interferon clearance in mice: comparison of mouse and human interferon.

Mice were injected with three kinds of interferon, i.e., mouse, human leucocyte and human fibroblast interferon, and the kinetics of their clearance from the blood was compared. After intravenous injection, mouse interferon was cleared very rapidly with an initial half life to two to three minutes, while both types of human interferon were cleared rather slowly with a corresponding value of about 20 minutes. When mouse interferon was injected intramuscularly or intraperitoneally, only very low levels of interferon activity were detected in the serum, and the recovery did not exceed 1% at any time. In contrast, both types of human interferon gave appreciable levels of activity in the serum upon injection by the same routes, and the levels remained stable during the observation period. The recovery of human interferon in the blood was about 10-fold that of mouse interferon. Mouse interferon was not inactivated in vitro by incubation with normal mouse serum. Our results suggest the validity of using homologous interferon for studies on the kinetics of interferon clearance in experimental animals.