The protective mechanism of action of amines in diphtheria toxin treated Vero cells.

The mechanisms by which a number of alkylamines and ethylenediamine derivatives protect Vero cells from diphtheria toxin were studied. The protective alkylamines and ethylenediamine compounds blocked the cellular degradation of diphtheria toxin, but did not prevent bulk toxin uptake. Specific antibody neutralized the inhibitory effects of toxin on protein synthesis in amine-treated cells when added at 37 degrees C, but was ineffective when added at 4 degrees C. Analogous results were obtained when inositol hexaphosphate was used in the place of specific antibody. Both experiments suggested that, in the presence of the amine compounds studied, diphtheria toxin is sequestered intracellularly and is recycled to the surface for antibody or inositol hexaphosphate neutralization. Finally, it was shown that the protective amines markedly increased the intralysosomal pH of Vero cells and that when the pH of the culture medium was lowered to 4.5, the amine-mediated protective effect was bypassed. In general, the results suggest that the amines exert their protective effect at the level of some acidic intracellular vesicle population, possibly the lysosomes, and thus may indicate a crucial role for the lysosomes in the generation of cytotoxicity.

Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine.

Chloroquine was found to prevent the cytotoxic action of diphtheria toxin on cultured monkey kidney cells. Analysis of the cellular processing of 125I-labeled diphtheria toxin showed that chloroquine does not affect the rate or extent of toxin uptake but substantially blocks degradation. These studies provide strong evidence that diphtheria toxin enters monkey kidney cells primarily by adsorptive endocytosis and suggest that lysosomal processing is involved in intracellular activation of the proenzyme form of the toxin.

Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells.

The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect.

Protection of mammalian cells from diphtheria toxin by exogenous nucleotides.

Exogenous nucleotides were found to protect mammalian cells from the lethal effects of diphtheria toxin. Protective potency of a given nucleotide was base specific and phosphate chain length dependent. Full expression of protective potency required an intact nucleotide, but the effect did not appear to be mediated by nucleotide-induced phosphorylation. Nucleotides antagonized the binding of diphtheria toxin to its cell surface receptor in a manner that correlated with the degree of protection. It was concluded that cellular protection from diphtheria toxin by nucleotides results from inhibition of toxin-receptor binding and that nucleotides therefore may serve as valuable research tools for future studies.

Association of diphtheria toxin with Vero cells. Demonstration of a receptor.

The interaction of radiolabeled diphtheria toxin with highly sensitive mammalian cell lines was studied. Toxin bound to (or was taken up by) Vero cells at 4 and 37 degrees C in a highly specific manner. At both temperatures, excess unlabeled toxin competed for up to 90% of the cell-associated label. The association at 37 degrees C was biphasic, increasing to a peak at 1 to 2 h and falling thereafter. At 4 degrees C, association increased with time to a steady state. Both fragment B and CRM-197 competed for the association of labeled toxin with cells. The magnitude of association correlated with the cytotoxic sensitivity of several cell lines. Both pH and exogenous nucleotides affected the association in a manner consistent with effects on cytotoxicity. The label associated with cells at 4 degrees C was largely intact toxin, while that at 37 degrees C was degraded. At 4 degrees C, the association was saturable (K = 9 X 10(8) liters/mol), was reversible, and indicated about 1 to 2 X 10(5) binding sites/cell.

Differential chemical protection of mammalian cells from the exotoxins of Corynebacterium diphtheriae and Pseudomonas aeruginosa.

Many drugs or chemicals had markedly different effects on the cytotoxicity induced by Pseudomonas aeruginosa exotoxin A (PE) or Corynebacterium diphtheriae exotoxin (DE). The glycolytic inhibitor NaF protected cells from DE but potentiated the cytotoxicity of PE. Another energy inhibitor, salicylic acid, also protected cells from DE but had no effect with PE. Colchicine and colcemid did not affect the cytotoxicity of either toxin. Cytochalasin B exhibited a modest protection from DE but no effect with PE. Ouabain, a specific inhibitor of the Na+, K+-dependent adenosine 5'-triphosphatase (ATPase), did not affect the cytotoxicity of either toxin. Ruthenium red, a specific inhibitor of the Ca2+, Mg2+,-dependent ATPase, conferred marked protection from DE-induced cytotoxicity but did not affect PE-induced cytotoxicity. A number of local anesthetics were tested, and they too presented differential results with PE and DE. Most chemicals that affected toxin-induced cytotoxicity had little or no influence on the in vitro adenosine 5'-diphosphate-ribosylation catalyzed by either toxin. This work presents further evidence that PE and DE have different mechanisms of intoxication and suggests that these differences lie in the attachment or internalization stages of intoxication.

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.

Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.