Monocyte-mediated drug-dependent cellular cytotoxicity: effects on different WEHI 164 Target cell lines

Summary The contribution of monocyte cytotoxic protein factor (CF) to monocyte-mediated drug-dependent cellular cytotoxicity (DDCC) has been investigated. Cell lines which have been derived from murine WEHI 164 cells (termed WEHI 164 parental) by selecting for high (WEHI 164 clone 3) and low (R-WEHI 164) sensitivity to CF-mediated cytotoxicity were used as target cells in DDCC. By comparing the CF doses which produced 50% dead cells (LD 50) we found that WEHI 164 clone 3 was approximately 30 times more sensitive than WEHI 164 parental which in turn was 70 times more sensitive than R-WEHI 164. Actinomycin D (Act D) treatment of WEHI 164 parental and R-WEHI 164 greatly increase susceptibility to CF-mediated cytotoxicity. The susceptibility of WEHI 164 clone 3 was apparently somewhat increased at low dilutions of CF, whereas no significant increase was observed at high dilutions. The susceptibility to DDCC of the three target cell lines (WEHI 164 parental, WEHI 164 clone 3, and R-WEHI 164) correlated with the sensitivity pattern obtained in CF-mediated cytotoxicity of Act D-treated target cells. Monocyte- and CF-mediated cytotoxicity against Act D-treated WEHI 164 clone 3 and R-WEHI 164 was inhibited by neutralizing CF antiserum. These data indicate that CF is an effector molecule in monocyte-mediated DDCC.     

Human monocyte cytotoxic factor mediates cytolysis of WEHI 164 cells

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.     

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.     

Effect of pyridoxine on tumor necrosis factor activitiesin vitro

Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occuring vitamin B₆ compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line (WEHI 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone WEHI 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agentin vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients.     

Effect of antisera against recombinant tumor necrosis factor and the monocyte-derived cytotoxin(s) on monocyte-mediated killing of various tumor cells

Antisera raised against recombinant tumor necrosis factor (TNF) and against the monocyte-derived cytotoxic/cytostatic protein factor (CF), which is related to recombinant TNF, have been compared with respect to their ability to inhibit monocyte-mediated killing of various types of cells which differ in their sensitivity to recombinant TNF. During 6 hr of coculturing monocytes and target cells, the recombinant TNF antiserum inhibited killing of the extremely TNF-sensitive WEHI 164 clone 13 cells and actinomycin D-treated WEHI 164 cells from which the clone 13 cells were derived (parental WEHI 164 cells (P-WEHI 164 cells]. The CF antiserum also inhibited monocyte-mediated killing of these cells during 6 hr of coculturing with monocytes, but on a per volume basis it was less potent than the recombinant TNF antiserum, consistent with the fact that the CF antiserum also was much less potent in inhibiting the cytotoxic activity of recombinant TNF. However, during 72 hr of coculturing with monocytes and target cells, the CF antiserum inhibited monocyte-mediated killing of P-WEHI 164 cells more efficiently than the recombinant TNF antiserum. Moreover, during 72 hr of coculturing with monocytes, only the CF antiserum was able to significantly inhibit monocyte-mediated killing of the relatively recombinant TNF-resistant K562 cells. This suggests that a factor immunologically different from recombinant TNF, perhaps a form of natural TNF differing somewhat immunologically from recombinant TNF, was involved in the killing of K562 cells, and possibly in the killing of P-WEHI 164 cells, during 72 hr of coculturing with monocytes. Although this factor was present extracellularly, it appears that it may act as a monocyte-associated factor in monocyte-mediated killing of K562 cells, since exposure to recombinant interferon-gamma (rIFN-gamma) in the absence of Escherichia coli endotoxin (lipopolysaccharide, LPS) activated the monocytes to mediate killing of K562 cells more efficiently than exposure to LPS alone, despite the fact that only little cytotoxic/cytostatic activity was released from the monocytes without the addition of LPS. The ability of rIFN-gamma and LPS to activate monocytes to produce and release CF has also been studied.     

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA-induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.     

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.     

Cationized ferritin as a platelet-stimulating surface probe. Binding to platelets and effects on platelet function

Polycationic derivatives of ferritin containing primary amino groups (CFah) or tertiary amino groups (CFdmp) were potent platelet agonists inducing shape change, aggregation and secretion, but also agglutination in the presence of EDTA. Pretreatment of platelets with neuraminidase, PGE1, indomethacin, or creatine kinase/creatine phosphate inhibited CF-induced activation. In contrast, neuraminidase and PGE1 increased the agglutination by CF, indicating an inverse relationship between activation and CF-induced agglutination. At pH 7.4, the cationic charges of CFdmp exceeded those of CFah by a factor of 1.5 and the platelets bound approximately 1.5 times more CFah than CFdmp, suggesting the same number of anionic surface sites for both CF preparations. The capacity of the platelets to bind CF was diminished by 55% at 0 degree C or by 62% after aldehyde fixation and by 13% with PGE1. This suggests that the binding capacity depends on the mobility of the binding sites in the plane of the membrane but is only slightly increased by platelet activation. Binding to fixed or cold platelets approached equilibrium within a few seconds whereas saturation required several minutes at 37 degrees C. Neuraminidase preferentially reduced the slow binding and much less the rapid binding. Since activation by CF developed during seconds, suppressible by a brief treatment with neuraminidase 25 mU/ml, a small portion of neuraminidase-sensitive sites appears to be necessary for CF-induced platelet activation. Full activation and agglutination occurred at CF concentrations far below saturating concentrations. The results show that neither CF-induced activation nor agglutination depend on a simple neutralization of the negative surface charge.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.     

Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.     

Cytotoxic effector function of B lymphoblasts

EBV-transformed B lymphoblastoid cell lines and clones were cytotoxic in vitro against Actinomycin D-pretreated WEHI 164 sarcoma cells, a system in which mononuclear phagocytes were previously identified as effectors. Similarly, unlike resting B cells, normal B lymphoblasts stimulated for 72 hr with Staphylococcus aureus Cowan I and purified according to the expression of the B cell surface marker B1, demonstrated appreciable cytolytic activity. B lymphoblastoid cells and derived supernatants were cytotoxic for Actinomycin D-pretreated WEHI 164 cells, and killing was inhibited by an anti-lymphotoxin but not an anti-tumor necrosis factor antiserum. Thus, B lymphoblasts have cytotoxic potential, mediated by lymphotoxin or a lymphotoxin-like soluble product.     

Induction of tumor cell resistance to macrophage-mediated lysis by phorbol esters: a postbinding event

Activated macrophages can recognize, bind to, and lyse tumor cells in an antibody-independent manner. We have found that tumor cells pretreated with phorbol esters are markedly less susceptible to macrophage-mediated cytolysis, although the initial binding step is unaffected. Tumor cells preincubated with tumor-promoting phorbol esters (10(-8)-10(-6) M) were rendered resistant to macrophage kill whereas non-tumor-promoting derivatives were inactive in protecting tumor cells against cytolysis. Inhibition of [3H]phorbol-12,13-dibutyrate binding by other phorbol esters correlated with their potency as tumor promoters and their ability to render tumor cells resistant to macrophage killing. The role of protein kinase C as the receptor to phorbol esters was implicated by inhibition of PDBu binding by phenothiazine derivatives. This suggests a possible mechanism for the resistance of phorbol ester-treated tumor cells to macrophage-mediated cytolysis.     

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.     

Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity of CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normala cells, which may be useful for the diagnosis of carriers of this defective gene.     

Changes in adsorption and permeability of mitoxantrone on plasma membrane of BCRP/MXR resistant cells

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.     

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.     

Schistosoma mansoni and S. japonicum worm numbers in 129/J mice of two types and dominance of susceptibility in F1 hybrids

In a study on the genetics of resistance to schistosomiasis in WEHI 129/J mice, susceptibility to either Schistosoma mansoni or Schistosoma japonicum was shown to be unequivocally dominant in F1 hybrid crosses between genetically resistant WEHI 129/J and susceptible BALB/c mice. The operation of only 1 or 2 genes in the expression of resistance to S. mansoni was suggested by backcross analysis. Thus, approximately 25% of (BALB/c x WEHI 129/J) F1 x WEHI 129/J mice were resistant to S. mansoni infection, whereas resistance was manifest in approximately 50% of WEHI 129/J mice. The data are consistent with resistance being controlled by 1 recessive gene having 50% penetrance. We also report that 129/J mice obtained directly from the Jackson Laboratories (Bar Harbor, Maine) (designated JAX 129/J), differ from locally bred WEHI 129/J in being entirely susceptible to S. mansoni infection. However, both WEHI 129/J and JAX 129/J are relatively resistant to S. japonicum infection.     

The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.

Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.