Cell-mediated cytotoxicity in perforin-less mice.

We have used a perforin-less (PO) mouse to explore alternate CTL-mediated lytic pathways. PO mice are unable to overcome an infection with LCMV in vivo. Nevertheless, splenocytes from infected mice show vigorous, antigen-specific cytotoxicity that requires the presence of the Fas antigen on target cells. The Fas lytic pathway is virtually indistinguishable, in terms of kinetics and magnitude of cytotoxicity, from perforin/granzyme-mediated lysis. It is rapidly induced in CTL upon occupation of the TcR, and requires protein synthesis for full expression. Upon removal of the activating signal, the capacity for fas-mediated lysis rapidly disappears. PO mice infected with LCMV also undergo what appears to be a CD8-mediated immunopathology, and rarely live beyond one month. The precise basis of this pathology is unknown at present. Given the widespread distribution of Fas in mice, particularly on inflamed tissues, the complete failure to clear virus from any tissue or organ is surprising.

The role of the Fas lytic pathway in a perforin-less CTL hybridoma.

The murine CTL hybridoma PMMI has been shown by the most sensitive techniques to be devoid of perforin. We thus used PMMI activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CTLs in the absence of perforin. We found that PMMI is equipped with membrane TNF-alpha as a potential lytic mechanism, but TNF-alpha is unlikely to be involved in acute (4 h) lytic reactions. On the other hand, PMMI readily lyses target cells expressing the gene for the Fas Ag, but does not lyse target cells expressing fas antisense DNA. The generation of fas-dependent lysis required protein synthesis in PMMI, but target cell protein synthesis was not required for lysis. Lysis of Fas-positive target cells by PMMI was accompanied by DNA fragmentation, and both lysis and DNA fragmentation were blocked by inhibition of protein synthesis in the effector cell. We find the relative extent and kinetics of fas-dependent lysis and DNA fragmentation indistinguishable from that seen in "classical" CTL lytic assays. Both fas- and perforin-dependent lysis were blocked by inhibitors of poly(ADP) ribosylation. We found very little difference in the sequence of events in target cells lysed by the fas pathway compared with the classical (probably perforin) lytic pathway. Given the widespread distribution of fas, particularly in hematopoietic target cells, caution may be required in interpreting the relationship between parameters such as DNA fragmentation and 51Cr-release solely on the basis of the granule exocytosis model.

Discrete signaling regions in the lymphotoxin-beta receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-kappaB pathways.

Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR.

Fas (CD95)-dependent cell-mediated immunity to Listeria monocytogenes.

Two distinct and complementary pathways, one mediated by perforin and the other dependent upon CD95 (Fas), effect cell-mediated cytotoxicity. We examined the relative roles of these pathways in host defenses against the intracellular bacterial pathogen Listeria monocytogenes by using murine listeriosis as a model system. Mice which lacked both perforin and Fas (P0L0) were generated, and their responses to primary and secondary listeriosis were compared to those of wild-type (WT), Fas-deficient (L0), and perforin knockout (P0) mice. Relative to WT mice during primary listeriosis, P0 mice exhibited a reduced capacity to clear the infection from their spleens but not their livers whereas L0 mice had elevated bacterial titers in their livers and a modestly increased titer in their spleens. In contrast, bacterial titers in P0L0 mice were increased approximately 50- to 560-fold in their spleens and 230- to 1, 000-fold in their livers; eventual clearance of listeriae from both organs was significantly delayed. Furthermore, the resistance of P0L0 mice to secondary listeriosis was significantly reduced in their spleens and livers compared to that of WT, P0, or L0 mice. In vitro experiments indicated that immune cytotoxic T lymphocytes (CTL) lysed L. monocytogenes-infected hepatocytes primarily via a Fas-dependent, perforin-independent mechanism. The absence of Fas severely abrogated the lysis of infected hepatocytes by immune CD8(+) CTL. Taken together, these results provide the first evidence for Fas-dependent CTL-mediated lysis of L. monocytogenes-infected hepatocytes and demonstrate complementary roles for Fas and perforin in host defenses against an intracellular bacterial pathogen.

Interferon production by the preimplantation sheep embryo.

Ovine trophoblast protein-1 (oTP-1), the major product secreted by the trophectoderm of the sheep conceptus between days 13 and 21 of pregnancy, is considered to mediate maternal recognition of pregnancy by maintaining the function of the corpus luteum. Its amino acid sequence has 40-55% identity with various mammalian interferons-alpha (IFN-alpha), and it has been shown to have antiviral activity. The present results confirm that oTP-1, which at days 15-17 of pregnancy is produced by a single embryo at more than 100 micrograms (greater than 1 million antiviral units) per day, is a functional IFN. A preparation of purified oTP-1 was made. Its amino-terminal sequence suggested that it consisted of a single homogeneous protein, so that its antiviral activity probably was not due to a contaminant. In a cytopathic effect inhibition assay with GBK-2 bovine cells challenged with vesicular stomatitis, its specific activity was 1.3 X 10(7) end point units/mg protein. It also protected GBK-2 cells against four other viruses, and A549 human cells against encephalomyocarditis virus. The antiviral activity was neutralized by an antiserum to human leukocyte IFN. Like human IFN-alpha, oTP-1 at concentrations as low as 10(-9) M inhibited the growth of GBK cells in culture and suppressed mitogen-stimulated incorporation of [3H]thymidine into ovine lymphocytes. Possible roles for oTP-1, functioning as an IFN-alpha during early pregnancy, are discussed.

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.

Efficient surface expression of platelet GPIIb-IIIa requires both subunits.

Platelet membrane GPIIb-IIIa is a member of the integrin family of heterodimeric adhesion receptors. Processing and export of certain leukocyte and melanoma integrins is disrupted in cells lacking one subunit. We found that surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits. In contrast, surface expression was not detected when the subunits were transfected individually. Immunoprecipitation of metabolically labeled transfected cells confirmed the presence of comparable levels of intracellular protein in all cases. When both subunits were transfected, post-translational cleavage of Pro-GPIIb to yield GPIIb heavy chain was also seen, while transfection with GPIIb alone resulted in coprecipitation of Pro-GPIIb with a second band that may be an endogenous beta subunit. Pro-GPIIb in these transfectants was not processed to yield GPIIb heavy chain. When transfected into COS cells alone, transiently expressed GPIIIa remained intracellular and did not appear to complex with any endogenous proteins. Thus, surface expression of processed GPIIb-IIIa depends on the presence of both subunits; the coordinate reduction of both subunits observed in some cases of Glanzmann's thrombasthenia may result from mutation affecting only one.

The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells.

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.