Carl F. Ware

Rapid colormetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colorimetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colorimetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH -5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10-50-fold more sensitive than the ESH -5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.

Lymphotoxin β, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface

The lymphokine tumor necrosis factor (TNF) has a well-defined role as an inducer of inflammatory responses; however, the function of the structurally related molecule lymphotoxin (LT alpha) is unknown. LT alpha is present on the surface of activated T, B, and LAK cells as a complex with a 33 kd glycoprotein, and cloning of the cDNA encoding the associated protein, called lymphotoxin beta (LT beta), revealed it to be a type II membrane protein with significant homology to TNF, LT alpha, and the ligand for the CD40 receptor. The gene for LT beta was found next to the TNF-LT locus in the major histocompatibility complex (MHC), a region of the MHC with possible linkage to autoimmune disease. These observations raise the possibility that a surface LT alpha-LT beta complex may have a specific role in immune regulation distinct from the functions ascribed to TNF.

APOPTOSIS MEDIATED BY THE TNF-RELATED CYTOKINE AND RECEPTOR FAMILIES

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)‐related family of membrane‐anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin β‐receptor (LTβR), among others. The MHC‐encoded cytokines, TNF and LTα, form homomeric trimers, whereas LTβ assembles into heterotrimers with LTα, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LTβR are involved in induction NFκB and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the “death domain” homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways.

Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis?

Fas/APO-1, a member of the NGF/TNF receptor superfamily expressed on the cell-surface of normal and malignant cells, is known to induce cell death by apoptosis. In the present study, we have investigated Fas/APO-1 gene defects in a human osteosarcoma cell line resistant to the apoptosis-inducing effects of anti-Fas. cDNA cloning and sequencing revealed that these cells contained both authentic and mutant Fas/APO-1 containing a 63 base pair in-frame deletion spanning the transmembrane domain, designated DFas/APO-1. Direct evidence for the existence of a soluble Fas/APO-1 protein was obtained by immunoprecipitation and Western blotting. Taken together with prior studies demonstrating a role for Fas/APO-1 and Fas ligand, respectively, in tumor target cell killing by cytotoxic T-lymphocytes, production of soluble Fas/APO-1 might have significant implications in malignant disease pathogenesis.

Production of lymphotoxin (LTα) and a soluble dimeric form of its receptor using the baculovirus expression system

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.

Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies following transplantation into mice with severe combined immunodificiency disease (SCID)

Mice with severe combined immunodeficiency disease (SCID) have been investigated for their ability to grow xenogenic hybridomas of mouse, rat and human origin. Two rat X mouse hybridoma lines (187.1.10 and 3B9) and 1 mouse X mouse hybridoma (2D9) grown in pristane-treated SCID mice as ascites tumors showed a 100-200-fold increase in monoclonal antibody levels over the amount produced in vitro with a total yield up to 0.5 g of antibody per animal. A human X human hybridoma, CLL-11-D1, exhibited a 1000-fold increase in human immunoglobulin levels in ascites (1.3 mg/ml) as compared to that obtained in tissue culture. Analyses of the antibody protein in the SCID ascites produced by these hybridomas using protein electrophoresis, SDS polyacrylamide gel electrophoresis and high resolution isoelectric focusing indicated the antibodies were monoclonal and free from any contaminating immunoglobulins. Yields of monoclonal antibodies of over 90% purity could be obtained from the ascites by a single ammonium sulfate precipitation step. This study indicates that SCID mice provide several significant advantages over other in vivo methods for the production of pure monoclonal antibodies of human, rat, or mouse origin.

A rat anti-mouse kappa chain specific monoclonal antibody, 187.1.10: purification, immunochemical properties and its utility as a general second-antibody reagent

A rat IgG1 monoclonal antibody, produced by hybridoma 187.1.10, exhibits specificity for mouse immunoglobulins containing kappa light chains (Yelton et al., 1981). The 187.1.10 hybridoma cell line secreted upwards of 200 micrograms/ml of monoclonal antibody in tissue culture and the secreted product was purified in a single step by antigen-immunoadsorbent affinity chromatography. The homogeneity of the purified 187.1.10 protein was determined by isoelectrofocusing and SDS gel electrophoresis. Equilibrium binding analyses of the radioiodinated 187.1.10 antibody indicated a strong interaction with its antigen of KA = 2 X 10(9) l/mole. The 187.1.10 antibody did not readily bind to Staph. aureus protein A unless it was complexed with antigen. The binding of immune complexes of 187.1.10 to protein A was shown to be dependent on the Fc region of the antigen. The utility of the 187.1.10 monoclonal antibody as a general second antibody reagent for studying mouse immunoglobulins was demonstrated in a rapid solid phase immunoprecipitation assay to detect and analyze radioiodinated membrane proteins of a human cytotoxic T cell line.

Lack of immunosuppressive effects of acute and subacute administration of malathion on murine cellular and humoral immune responses

Abstract Malathion has been previously shown to cause allergic responses and suppress the generation of a humoral immune response in vivo . In this study, the effect of in vivo administration of malathion on cellular, humoral and mitogenic responses was examined. Acute (50% LD 50 ) or subacute (10% LD 50 per day for 14 days) treatment with malathion in vivo did not affect the in vivo generation of specific antibody secreting cells to sheep red blood cells (SRBC) or cytotoxic T lymphocytes (CTL) to allogeneic tumor. However, 5 days following acute administration of malathion, there was a slight increase in humoral immune responsiveness. Acute treatment with 50% LD 50 purified malathion did not affect body weight, splenic cell number, or thymus size. However, mitogenic responses to Concanavalin A (Con A) and lipopolysaccharide (LPS) was significantly enhanced on all days tested following acute administration of malathion. In contrast, subacute treatment with malathion did not affect mitogenic response to Con A or LPS, but led to a significant decrease in thymic cell number.

Fas involvement in human NK cell apoptosis: lack of a requirement for CD16-mediated events.

Propriocidal regulation of T cells refers to apoptosis induced by interleukin‐2 (IL‐2) activation with subsequent antigen receptor stimulation. We previously reported that natural killer (NK) cells also exhibit propriocidal death. Cell death can be induced following occupancy of the FcγRm (CD16) receptor when NK cells were pretreated with IL‐2, IL‐12, or IL‐15. Here we show other triggering receptors on NK cells such as CD44, anti‐NK‐receptor antibodies, and pharmacological activation can result in the cell death signal. Requirement for cell interactions indicated that cell contact was required; however, unlike cell‐mediated lysis, extracellular calcium was not required. Like T cells, the process of cell death for NK cells was receptor‐induced apoptosis. Activation‐induced apoptosis of T cells is mediated by members of the tumor necrosis factor (TNF) cytokine superfamily. We examined the involvement of TNF receptor family members or Fas in this rapid cell death. Antibody directed against Fas, TNFR60, TNFR80, LTBR, and LTα failed to inhibit receptor‐induced death. Therefore, NK cells appear to demonstrate a rapid apoptotic episode when CD16 is cross‐linked, but the mechanism of this apoptosis is quite different than was observed in T cells with CD3. The direct examination of the Fas pathway on activated NK cells revealed that susceptibility required longer treatment times and IL‐2 activation. This susceptibility was paralleled by increased Fas‐ligand expression. Therefore, NK cells can demonstrate an apoptotic response to CD16, CD44, NK receptors, and Fas. The enumeration of ligands capable of eliciting NK cell death and the in vivo relevance of this observation require further study. J. Leuhoc. Biol. 61: 209–215; 1997.

Effects of acute administration of O,S,S-trimethyl phosphorodithioate on the generation of cellular and humoral immune responses following in vitro stimulation☆

The time course of immune modulation induced by acute treatment with O,S,S-trimethyl phosphorodithioate (OSS-TMP), an impurity in technical formulations of malathion, was examined in female C57BL/6 mice. The immune parameters studied included the generation of cytotoxic T lymphocytes (CTL) to alloantigen (H-2 incompatible) and antibody secreting cells to sheep red blood cells, proliferative response to the mitogens, and interleukin-2 (IL-2) production. Acute administration of the non-toxic doses of OSS-TMP, i.e. 20 or 40 mg/kg, led to an elevation in the generation of a CTL response on day 1 or 7, respectively. At 20 mg/kg OSS-TMP, the antibody response was elevated at day 3. However, at a dose of 40 mg/kg OSS-TMP, the antibody response was suppressed at day 1 following treatment. Following acute administration of 60 or 80 mg/kg OSS-TMP, the generation of an antibody and CTL responses was suppressed at all time points tested with 1 exception. One day following treatment at a dose of 60 mg/kg OSS-TMP, there was no change in the CTL response. At day 7 following treatment, the mitogenic responses to lipopolysaccharide and phytohemagglutinin were elevated at all doses of OSS-TMP administered. At this time point, however, the proliferative response to Concanavalin A was elevated in a dose dependent manner. IL-2 production was suppressed following acute administration of 60 or 80 mg/kg OSS-TMP at all time points tested and at all doses tested on day 5 following treatment. These data indicate that OSS-TMP, unlike its congener, O,O,S-trimethyl phosphorothioate, enhances the generation of humoral and cell mediated immune responses of C57BL/6 mice following administration of non-toxic doses.