Soluble Cytotoxin Research Articles

Tritrichomonas foetus: characterization of isolates and partial purification of a secreted cytotoxin

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro. Index Descriptors and Abbreviations: Trichomoniasis; Tritrichomonas foetus; cytotoxins; American Type Culture Collection (ATCC); concentrated parasite-conditioned medium (CPCM); cytotoxic unit (CU 50); hemagglutinating unit (HA 100); Dulbecco’s modified Eagle’s medium (DMEM); gel filtration chromatography (GF); hydrophobic interaction chromatography (HIC); ion exchange chromatography (IEC); optical density (OD); phosphate-buffered saline (PBS); serum-free RPMI-1640 medium (RPMI).

Toxicity of pneumolysin to pulmonary endothelial cells in vitro.

Pneumococcal pneumonia and bacteremia are associated with appreciable mortality, most of which occurs very early in the course of infection. An initial step in the pathogenesis of pneumococcal pneumonia may include disruption of the pulmonary endothelial barrier with subsequent alveolar hemorrhage. We sought to determine whether soluble factors from Streptococcus pneumoniae can directly injure pulmonary endothelial cells in vitro and to identify pneumococcal toxins that may be involved in endothelial cell injury. Suspensions of S. pneumoniae (10(8) organisms per ml) caused significant injury to cultured bovine pulmonary artery endothelial cells in a time-dependent manner. The degree of endothelial cell cytotoxicity differed among S. pneumoniae strains; among the strains tested, a type 14 strain was the most cytotoxic and a type 3 strain was the least cytotoxic. During autolysis, type 14 S. pneumoniae released a soluble endothelial cell cytotoxin that was distinct from S. pneumoniae capsular and cell wall polysaccharides. The soluble cytotoxin was further characterized as a thiol-activated, heat-sensitive protein that coeluted with purified pneumolysin during gel filtration. The identity of the S. pneumoniae endothelial cell cytotoxin as pneumolysin was further supported by the ability of purified pneumolysin and the inability of S. pneumoniae mutants which lack pneumolysin to injure endothelial cells, as well as by the inhibition of the soluble S. pneumoniae cytotoxin by a neutralizing antibody to pneumolysin. Pneumolysin appears to be the major S. pneumoniae soluble cytotoxin for pulmonary endothelial cells in vitro and may be an important factor in the pathogenesis of alveolar hemorrhages in S. pneumoniae infections.

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. II. A novel CTL-produced cytotoxin that is antigenically distinct from tumor necrosis factor and alpha-lymphotoxin.

We have cloned lines of IL 2-dependent human T cells derived from alloantigen, soluble antigen (tetanus toxoid), mitogen, or IL 2-stimulated peripheral blood lymphocytes and characterized their surface marker expression and cytolytic activity. The surface phenotype and cytolytic function was compared with the ability of these T cell clones to release cytotoxic lymphokines in response to mitogenic lectins. The cytotoxins released by these CTL clones were detected on the murine L929 target cells in a 16-hr assay. All of the T cell clones, whether stimulated by HLA alloantigens, tetanus toxoid, or mitogens, exhibited killer cell activity and the capacity to secrete a soluble cytotoxin(s). Specific polyclonal antisera to recombinant human tumor necrosis factor (rTNF) and human alpha-lymphotoxin (alpha LT) were unable to neutralize the cytotoxic activity released by most of these CTL clones. These results indicate that human CTL produce a novel antigenic form(s) of cytotoxin that we have termed CTL-toxin. Supernatants from several CTL clones yielded a cytotoxic activity that was partially neutralized (10 to 40%) by saturating levels of anti-TNF (but not anti-alpha LT) indicating that human CTL may be capable of producing a TNF-like molecule. Only two out of 60 CTL clones studied thus far produced a cytotoxic activity that was partially neutralized by anti-alpha LT (20 to 40%). Collectively, these results suggest that although both the CD4 and the CD8 subpopulations of human cytotoxic T cells may be capable of releasing several types of cytotoxins in response to mitogenic signals, the predominant cytotoxin is distinct from alpha LT and TNF.

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins.

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.

Production of α-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (T G+) or IgM (T M+), or the absence of both IgG and IgM receptors (T G−M−). When restimulated with alloantigen of the same derivation, T G+, T M+, and T G−M− cells yielded almost equal amounts of cytotoxin. Anti-α-lymphotoxin serum neutralized most of this cytotoxic activity indicating that α-lymphotoxin (α-LT) constituted most of this activity. Although T G−M− cells function as effectors in allogeneic cytotoxicity, T G+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by α-LT. Whether T M+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, T G+, T M+, and T G−M− cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-α-LT. The T G+ cells incorporated less thymidine than the T M+ or T G−M− cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in T M+ cells, where positive and negative results have been reported.

Differences in Cytotoxic Effects of Activated Murine Peritoneal Macrophages and J774 Monocytic Cells on Metastatic Variants of B16 Melanoma<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

The cytotoxic effects of activated peritoneal macrophages and the J774 reticulum cell sarcoma cell line on B16 melanoma cells of differing metastatic potential were investigated in vitro. The melanoma target cells were sublines of low (B16-F1) or high (B16-F10) lung colonization potential as well as a subline of high (B16-B14b) brain colonization ability. Thioglycolate-elicited peritoneal macrophages from syngeneic C57BL/6 mice and J774 cells were activated in vitro with polyinosinic-polycytidylic acid (poly I:C) and used as effector cells. Macrophage-mediated cytolysis was determined by means of 24- to 72-hour radioactivity release assays with [125I]5-iodo-2'-deoxyuridine-labeled melanoma cells; the results indicated that the more metastatic sublines B16-F10 and B16-B14b were less susceptible to cytolysis by activated macrophages and J774 cells than was the poorly metastatic B16-F1 subline. The poly I:C-activated effector cells also released soluble cytotoxin(s), which resulted in melanoma cell lysis and growth inhibition. Cytotoxin-mediated melanoma cell cytolysis was determined by counting the number of viable mitomycin-treated target cells remaining after a 40-hour incubation period in the absence or presence of various concentrations of cell-released factors, and cytotoxin-mediated cytostasis was performed with the use of the same procedures without mitomycin pretreatment of targets. The factors released from both activated macrophages and J774 cells were more effective against the poorly metastatic B16-F1 cells than against the highly metastatic B16-F10 or B16-F14b cells. In addition, the activity of the factors from both activated effector cells was inhibited by fetal bovine serum. The J774 cells and the activated peritoneal macrophages demonstrated similar activities against B16 melanoma variants, indicating that the J774 cell line may be suitable as a model for the study of macrophage cytotoxicity. The results suggested that the potential of the highly metastatic melanoma cells to implant, survive, and grow at secondary sites may be due, in part, to their ability to circumvent host antitumor mechanisms.

Inhibition of nonspecific tumoricidal activity by activated macrophages with antiserum against a soluble cytotoxic factor.

Mouse peritoneal macrophages activated for tumor cytotoxicity by any of several in vivo or in vitro treatments released a soluble cytotoxin into culture fluids only after exposure to small amounts of bacterial lipopolysaccharides. This cytotoxic factor was physicochemically similar to the cytotoxic factor (tumor necrosis factor) in sera of BCG-infected mice injected with lipopolysaccharide. A rabbit antiserum against partially purified serum cytotoxic factor also inhibited the activity of macrophage-derived cytotoxic factor. Of special interest was the observation that rabbit anti-cytotoxic factor inhibited the cytotoxic activity of macrophages both in the presence and in the absence of exogenously added lipopolysaccharide. Inhibition was not complete but was consistent in all experiments. Thus, cytotoxic factor was implicated as a possible effector molecule in the nonspecific tumoricidal activity of activated macrophages.

Cytotoxic Activity of Lymphocytes

Abstract Macrophage cell-mediated cytolysis (M-CMC) of tumor cells may be measured by culturing target cells on macrophage monolayers and determining target cells on macrophage monolayers and determining target cell survival by either 86Rb or 3H-thymidine incorporation. Using these techniques, one can demonstrate cytotoxicity of non-immune human and rat macrophages to a variety of tumor cell lines. Toxicity becomes maximal 8 to 16 hr after attaching macrophages to plastic surfaces and disappears after 36 to 48 hr. Addition of Escherichia coli endotoxin at 48 hr restores macrophage toxicity. Culture of macrophages on plastic surfaces also results in the release, non-specifically, of a soluble cytotoxin whose kinetics of release, disappearance, and reappearance after endotoxin all parallel the M-CMC. The amount of toxin released into cultures containing target cells is markedly reduced, suggesting that it is the mediator of the M-CMC and that it is utilized or adsorbed during target destruction. Sephadex G-100 chromatography and treatment with heterologous anti-lymphotoxin can distinguish this toxin from lymphotoxin.

Alloimmune peritoneal macrophages as specific effector cells: Characterization of specific macrophage cytotoxin

Specific macrophage cytotoxin (SMC) is a soluble cytotoxin detected in the culture medium after a 2-hr interaction of alloimmune peritoneal macrophages and specific target cells at 37°C. The cytotoxicity of SMC affects only specific allogeneic cells and is not mediated through heat labile serum components. SMC is inactivated by heating at 56°C for 30 min and it is trypsin sensitive. The specificity and characteristics of SMC show it to be unique from other reported mouse in vitro cytotoxins.

Binding of human lymphotoxin to target-cell membranes and its relation to cell-mediated cytodestruction.

One of the lymphocyte effector molecules produced in vitro by antigen- and mitogen-stimulated lymphocytes is lymphotoxin, which has been proposed to be the cytodestructive mediator in cell-mediated immune reactions. Our in vitro findings suggest that lymphotoxin binds to sites on the plasma membrane of sensitive target cells, and this binding represents the earliest detectable effect in cell destruction induced by lymphotoxin. The kinetics of binding are rapid, and the affinity of lymphotoxin for binding sites is strong. Furthermore, cytodestruction can be localized, for lymphotoxin-coated cells cannot release lymphotoxin to affect adjacent (51)Cr-labeled, noncoated cells in mixed cultures. This evidence suggests how a nonspecific soluble cytotoxin could cause highly specific localized tissue destruction.

Detection of lymphotoxin produced in mixed lymphocyte cultures (MLC): Variation in target cell sensitivity

In an attempt to resolve some of the uncertainty as to whether soluble cytotoxin—lymphotoxin (LT)—is produced in mixed leukocyte cultures (MLC), we established one-way MLC between the lymphocytes of normal individuals and two lines of lymphoblastoid cell lines (LCL). Cell-free culture fluid harvested after 5 days was tested for LT employing not only the stimulating LCL cells as targets, but also two lines of cells known to be sensitive to LT. The LCL cells—RPMI 8866 and NHDL-2—were found to be completely resistant to LT under the conditions of our assay. By contrast, when the sensitive cells were used in the cytotoxicity assay, LT was readily detected. These results emphasize the importance of intrinsic target cell sensitivity in the study of lymphocyte-mediated cytotoxic phenomena and raise questions as to the mechanism whereby cells may be resistant to LT.

Purification of Human Lymphotoxin

Abstract This paper describes the first preparation of highly purified lymphotoxin, a soluble cytotoxin produced by lymphocytes. Lymphotoxin was produced in vitro by phytohemagglutinin-stimulation of human lymphocytes and was then purified greater than 2000-fold by a sequence of column chromatography (Sephadex G-150 followed by DEAE-cellulose) and preparatory acrylamide gel electrophoresis. Ten per cent of the cytotoxic activity was recovered in the final product. The results of the purification steps were evaluated by a new quantitative assay for cytotoxic activity.

Target cell lysis mediated by soluble cytotoxin released from stimulated lymphocytes.

MANY investigations have shown that lymphocytes stimulated with a specific antigen, or non-specifically by plant mitogens such as phytohaemagglutinin (PHA), release soluble mediators of various reactions1–6. One of these mediators has been identified as a cytotoxic factor5, 6, largely on the basis of its ability to inhibit 14C-amino-acid incorporation into L cells. The actual effect of this cytotoxic factor remains uncertain because other evidence of its cytotoxicity is meagre and because the observed decline in protein synthesis does not distinguish between cell lysis, growth inhibition and/or cell detachment. Furthermore, its biological significance has been questioned7, because other investigators have stated that intimate contact between lymphocyte and target cell is essential for lysis of the latter.