Tetanus toxin: inhibitory action in chromaffin cells is initiated by specified types of gangliosides and promoted in low ionic strength solution

Adrenal medullary chromaffin cells preloaded with [3H]noradrenaline release the hormone by exocytosis upon stimulation with carbachol. This exocytosis is not influenced by tetanus toxin (Tetx) which can block the release of transmitters from nerve endings. Preincubation of chromaffin cells with gangliosides followed by toxin exposure in low ionic strength solution results in a decrease of stimulated catecholamine release. The inhibition of exocytosis is most evident when cells are pretreated with the ganglioside GD lb. Cells treated with GTlb are 10 times less sensitive, those treated with GDla and GMl even 30 times less sensitive to Tetx. Thus GDlb appears to be the principal carrier for Tetx in chromaffin cells whereas the other gangliosides only play a minor role.     

T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity

Gangliosides form a component of the glycosphingolipid-rich membrane microdomains recently shown to play an important role in receptor signal transduction. Specific gangliosides also serve as receptors for binding and internalization of bacterial toxins. In the course of characterizing the basis of the native tetanus toxin (TTx) reactivity of a human gamma delta T cell clone, we observed that transfer of the TCR was required to impart TTx reactivity on a TCR-negative recipient T cell. However, the reconstitution of toxin reactivity could be achieved regardless of the antigen specificity of the TCR chains. Further analysis showed that the T cell recognition of native TTx was dependent on the presence of its ganglioside receptor, GT1b, on the T cell surface. Incorporation of exogenous GT1b into plasma membrane conferred TTx reactivity on otherwise non-reactive T cells provided these cells expressed the TCR. Finally, reconstitution of TCR-negative Jurkat T cells with a CD8-CD3zeta chain chimera demonstrated that the cytoplasmic region of the CD3zeta chain was sufficient to couple ganglioside-mediated TTx binding to T cell activation. These data reveal a novel mode of TCR-dependent reactivity to a bacterial toxin that could mobilize a large subset of T cells, thus representing a form of innate immunity. Given the possibility that endogenous ligands may bind to cell surface gangliosides, regulation of their levels and topology on the cell surface may constitute an immunoregulatory mechanism.     

Retrograde axonal and transsynaptic transport of macromolecules: Physiological and pathophysiological importance

Anterograde and retrograde transport within axons and dendrites of nerve cells represent an integral part of the nerve cell function and biochemistry. A few exogenous macromolecules with most different molecular weights and physico-chemical properties (Nerve Growth Factor, tetanus toxin, cholera toxin, various lectins, antibodies against dopamine--hydroxylase) have been shown to be taken up and transported with the retrograde axonal transport in exceedingly high amounts if compared to most other macromolecules. Specific binding to membrane receptors seems to be the prerequisite for this highly efficient retrograde transport. Upon arrival at the cell body tetanus toxin is able to leave the neuron and to migrate transsynaptically to presynaptic nerve terminals of second-order neurons.For NGF, tetanus toxin and some neurotropic viruses retrograde axonal transport eventually followed by transsynaptic transport may be crucially involved in their mechanism of action. Indirect evidence suggests the existence of a variety of endogenous molecules carrying specific information from the target cell and the nerve terminal to the cell body and eventually transsynaptically into second- or third-order neurons.Dedicated to K. Bucher on the occasion of his 65th birthday.     

Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation

Polyclonal monospecific antibodies raised in sheep against rat testis calmodulin demonstrated cross-reactivity with bovine adrenal medullary chromaffin cell calmodulin. This antibody immunoprecipitated a [35S]methionine-labelled protein from chromaffin cell extracts prepared from [35S]methionine prelabelled cells that comigrated on a sodium dodecylsulfate gel electrophoresis system with calmodulin. In addition, an excess of non-radioactive exogenous calmodulin was shown to readily compete with this labelled endogenous protein for the antibodies' binding sites. Erythrocyte ghosts were used as vehicles for microinjecting either preimmune immunoglobulin G or anti-calmodulin immunoglobulin G into chromaffin cells following a polyethylene glycol-induced cell fusion procedure. The efficiency of ghost cell fusion was monitored and found to be 43.6 +/- 1% (n = 33). Cell morbidity subsequent to fusion and microinjection was negligible (87.8 +/- 0.6% of the total cell population were viable cells; n = 33) as determined by the Trypan Blue exclusion test. The delivery of intact antibodies raised against calmodulin directly into the cytoplasm of cultured chromaffin cells by erythrocyte ghost-mediated microinjection, inhibited catecholamine output in response to stimulation by either acetylcholine (10(-4) M) or a depolarizing concentration of potassium (56 mM). However, under these conditions, the chromaffin cell's ability to accumulate exogenous catecholamines through a high affinity uptake system, as well as the kinetic parameters that characterize this uptake mechanism remained unaltered. Furthermore, microinjection of preimmune immunoglobulin G did not modify either catecholamine uptake or stimulation-induced amine release from chromaffin cells. It therefore appears that calmodulin may play a role in the process of stimulus-secretion coupling in the chromaffin cell in culture while it is of little significance to the high affinity amine uptake mechanism.     

Bacterial toxins

Many bacterial toxins are proteins, encoded by the bacterial chromosomal genes, plasmids or phages. Lysogenic phages form part of the chromosome. The toxins are usually liberated from the organism by lysis, but some are shed with outer membrane proteins in outer membrane vesicles. An important non-protein toxin is lipopolysaccharide or endotoxin, which is a constituent of the cell wall of gram negative bacteria. Toxins may damage the eukaryotic cell membrane by combining with some structural component, or otherwise alter its function. Many toxins combine with specific receptors on the surface membrane, frequently glycoproteins or gangliosides, and penetrate the cell to reach their intracellular target. A common mechanism of entry is absorptive endocytosis. Many protein toxins have an A-B structure, B being a polypeptide which binds to the receptor and A being an enzyme. Many toxins are activated, either when produced by the bacterium or when bound to the membrane receptor, by proteases (nicking). An enzymatic process common to many toxins is adenosine diphosphate (ADP)-ribosylation of the adenylate cyclase regulatory proteins, leading to an increase in intracellular cyclic adenosine monophosphate (cAMP). This is the mechanism of action of cholera toxin. Diphtheria toxin catalyzes the transfer of ADP-ribose to elongation factor-2, inhibiting protein synthesis. Most toxins act on the target cells to which they bind, but tetanus toxin, and, to a lesser degree, botulinum toxin, ascend axons and affect more distant structures. Although many toxin effects caused by bacteria have been described, only a few toxins have been identified, characterized, and their mode of action determined at the molecular level. The best known of these are discussed.     

Ultrastructural study of plasma membrane GM1 in neuroectodermal cells using cholera-peroxidase

Summary Cholera toxin was coupled to peroxidase to yield a highly specific ultrastructural marker for G _m1 gangliosides. Study of embryonic brain cells in culture revealed intense binding of choleraperoxidase by plasma membranes of both neurons and glial cells. In contrast, long-term monolayer glioblastoma cultures, including one producing C-type virus, revealed virtually no labelling of their plasma membranes. Such cells were shown to be capable of incorporating exogenously applied G_{m 1} into their plasma membranes. Studies with fixed brain and synaptosomal fractions were in accord with results on embryonic brain cells in culture, and autoradiographic findings with¹²⁵I cholera supported observations made utilizing cholera—peroxidase. From our studies there is some indication that long-term propagation in vitro alters the plasma membrane G _m1.     

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [(3)H]noradrenaline ([(3)H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca(2+)-evoked [(3)H]NA release but increased basal release (that occurring in the absence of Ca(2+)). Preincubation with antibodies against PLCgamma1 or PLCbeta3 (but not PLCdelta1, delta2, beta1 and beta2) also inhibited [(3)H]NA release evoked by Ca(2+) and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCgamma1 (but not PLCbeta3) antibodies inhibited the ability of Ca(2+) to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCgamma1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.     

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.     

Production and characterization of monoclonal antibodies to tetanus toxin

Monoclonal antibodies against tetanus toxin were produced to obtain highly specific antisera. Ten hybridoma cell lines producing monoclonal antibodies were derived from the fusion of rat myeloma cells and spleen cells from rats immunized with tetanus toxoid. Eight produced monoclonal antibodies specific for determinants on toxin and toxoid, whereas two were specific only for determinants on the toxoid. The antibodies produced by hybridomas were characterized by determination of the class of light and heavy chain components, epitope specificity, toxin neutralization, and subunit specificity. All of the antibodies contained kappa light chain, eight contained the gamma 1 heavy chain, and the remaining two contained the gamma 2a heavy chain. Five distinct epitopes were indicated by competition assay of paired monoclonal antibodies, and 4 of the 10 monoclonal antibodies neutralized the in vivo activity of tetanus toxin. The four neutralizing monoclonal antibodies and one other were specific for the C fragment of the heavy chain of the toxin molecule.     

Monoclonal antibodies as probes of tetanus toxin structure and function

Monoclonal antibodies specific for fragment B, fragment C, and light chain of tetanus toxin were prepared by fusion of P3X63Ag8 BALB/c myeloma cells with spleen cells from BALB/c mice immunized with tetanus toxoid or fragment B. Hybridoma colonies were assayed for antibody production by an enzyme-linked immunosorbent assay. Fourteen positive clones were identified, cloned by limiting dilution, and injected intraperitoneally into mice to obtain ascites fluids. Thirteen of the monoclonal antibodies were of the immunoglobulin G1 subclass and one was immunoglobulin G2. Two of the antibodies were directed against sites on fragment C, nine were directed against the light chain, and three were directed against the portion of fragment B which does not comprise the light chain of tetanus toxin. At least one antibody in each group exhibited significant toxin neutralization activity. However, only one of these neutralizing antibodies strongly inhibited the binding of 125I-tetanus toxin to ganglioside-coated plates. These data indicate that interference with receptor recognition is not the only means of neutralizing tetanus toxin. Monoclonal antitoxins as potential therapeutic and prophylactic reagents are discussed.     

Evidence for calcium-dependent vesicular transmitter release insensitive to tetanus toxin and botulinum toxin type F

Whether exocytosis evoked by a given releasing stimulus from different neuronal families or by different stimuli from one neuronal population occurs through identical mechanisms is unknown. We studied the release of [3H]noradrenaline, [3H]acetylcholine and [3H]dopamine induced by different stimuli from superfused rat brain synaptosomes pretreated with tetanus toxin or botulinum toxin F, known to block exocytosis by cleaving VAMP/synaptobrevin. The external Ca2(+)-dependent [3H]transmitter overflows evoked by KCl were similarly inhibited by tetanus toxin or botulinum toxin F; the toxins cleaved similar amounts of synaptosomal synaptobrevin, as determined by western blot analysis, suggesting prevalent involvement of synaptobrevin-II. GABA uptake-mediated release of the three [3H]transmitters was that differentially sensitive to the toxins: only the release of [3H]noradrenaline, which is dependent on external Ca2+, but not of [3H]acetylcholine and [3H]dopamine was blocked. Neither toxin affected the [3H]transmitter overflows evoked by the Ca2(+) ionophore ionomycin. Cadmium blocked the K(+)-evoked release of all [3H]transmitters and the GABA-evoked release of [3H]noradrenaline; the GABA-evoked releases of [3H]acetylcholine and [3H]dopamine and those elicited by ionomycin were insensitive to cadmium. The results suggest that tetanus toxin and botulinum toxin F selectively affect exocytosis linked to activation of voltage-sensitive Ca2(+) channels; the Ca2(+)-dependent, exocytotic-like release induced by stimuli not leading to activation of voltage-sensitive Ca2+ channels seems insensitive to these clostridial toxins.     

Selective uptake of 3H tyramine by chromaffin cells in vitro and simultaneous release of noradrenaline

Summary Cultures of adrenal explants derived from rat and mouse embryos were exposed to³H tyramine for intervals of 5 to 25 minutes. Light and electron microscopic autoradiography revealed a selective up-take of³H tyramine in chromaffin cells, and not in adrenocortical cells or fibroblasts. During this same period release of noradrenaline by chromaffin cells was demonstrated. Grains were observed over dense core vesicles at all times, and labelled vesicles were always distributed throughout the cytoplasm with no concentrations close to the plasma membrane. The mechanism of tyramine-induced secretion is discussed in the light of these findings.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannicacid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane. Our results provide the first ultrastructural demonstration of exocytosis in bovine chromaffin cells with the aid of transmission electron microscopy. It is also clear that exocytosis is the mechanism of release in both intact and permeabilized cells.     

Effects of cations and osmotic protectants on cytolytic activity of Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans leukotoxin permeabilized the plasma membrane of HL-60 promyelocytic leukemia cells, resulting in colloid osmotic lysis. These events were associated with efflux of 51chromium (from prelabeled cells), influx of propidium iodide, and ultrastructural evidence of cellular damage. Target cell lysis was inhibited by procedures which may interfere with the initial interaction of the toxin with the plasma membrane. For example, washing cultures (to dilute and remove toxin) or the addition of monoclonal antibodies (to neutralize toxin) or trypsin (to inactivate toxin) limited lysis when undertaken within the first 5 min of the reaction. The extent of injury was also diminished when radiolabeled HL-60 cells were exposed to toxin in the presence of unlabeled, toxin-sensitive cells (e.g., HL-60 cells or human neutrophils) or certain toxin-resistant target cells (e.g., human K562 erythroleukemia cells). This suggests that the association of the toxin with the cell membrane may not be sufficient to cause lysis without activation of additional effector mechanisms. The addition of specific trivalent (e.g., La3+) or divalent (e.g., Ca2+ and Zn2+) cations to toxin-treated cells appeared to enhance their capacity to repair or minimize the extent of toxin-mediated membrane damage. Depending on size, certain saccharides served as osmotic protectants: maltose almost completely inhibited radiolabel release, while smaller molecules provided correspondingly less protection. The results imply that the leukotoxin has membranolytic activity, producing pores in target cells with a functional diameter approximately the size of maltose (0.96 nm).     

Presence of a high affinity uptake system for catecholamines in cultured bovine adrenal chromaffin cells

The uptake of catecholamines was investigated in bovine adrenal chromaffin cells cultured for 2 and 7 days. These cells, after their attachment onto the collagen-coated plates, began to develop processes which progressively increased in length with time in culture. Process outgrowth of a few cells was apparent on day 2 in culture. However, by day 7 most of the chromaffin cells possessed processes with a mean length of52.68 ± 1.27μm(n= 202). There was found to exist in both 2-day and 7-day-old cultures an uptake mechanism for (?)noradrenaline which in many aspects simulated the neuronal uptake₁ transport system in that it was saturable, followed Michaelis-Menten kinetics, had a high affinity for (?)noradrenaline with apparentK_m values ranging from 0.35–0.48 μm (n = 4) and 0.46 ? 0.67 μm (n = 4) on day 2 and day 7 respectively. This uptake process was blocked by low concentrations of desipramine (10^?7 M), a specific inhibitor of uptake₁, and like the neuronal uptake mechanism exhibited absolute Na⁺ dependency in which Li⁺ could not adequately replace Na⁺. However, uptake by the chromaffin cells did not show any stereochemical specificity towards the (?) form of noradrenaline nor did it exhibit structural specificity for (?)noradrenaline over (?)adrenaline.Therefore, it appeared that a high affinity accumulating mechanism for noradrenaline was present in cultured chromaffin cells isolated from adult bovine adrenal medulla. The properties of this uptake system did not vary significantly from day 2 to day 7 and thus did not parallel the morphological changes seen in culture.     

Uptake of diphtheria toxin and its fragment A moiety by mammalian cells in culture.

Evidence suggesting that diphtheria toxin reaches the cytoplasm of susceptible mammalian cells by two independent mechanisms is presented. A schematic model describing the two processes of toxin entry into the cell is developed. One process of toxin uptake considered to by physiologically significant is passage of the protein toxin through the plasma membrane. This most likely happens by binding of fragment B to receptors on the membrane and by subsequent toxin-membrane interaction so that ultimately fragment A, the enzymatically active moiety, is transported tothe cell interior. This process, which ultimately leads to cessation of protein synthesis and cell death, involves a comparatively small number of toxin molecules. A second mechanism of toxin uptake is by classical pinocytosis. The majority of toxin taken into the cell is accomplished by this process. The fate of toxin taken into HEp-2 cells via pinocytosis is proteolysis by lysosomal enzymes. Thus, such vesicle-bound toxin is ordinarily not expressed biologically. Evidence suggesting that ammonium chloride provides total protection to diphtheria toxin-susceptible cells by preventing entry of toxin by the specific receptor-associated process is also provided; data showing that the ammonium salt immobilizes bound toxin on the plasma membrane of HEp-2 cells are presented. Finally, it is suggested that actively endocytic cells such as guinea pig macrophages interact with toxin in a significantly different manner than do nonphagocytic cells.     

Cloning and expression of functional fragment C of tetanus toxin.

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.     

Cholera toxin and its B subunit inhibit interferon effects on virus production and erythroid differentiation of Friend leukemia cells

Treatment of Friend leukemia cells with cholera toxin or with its purified B subunit inhibits the interferon (IFN)-induced reduction of Friend virus release and of virus yields after exogenous infection with vesicular stomatitis virus. Likewise, the “enhancement” of erythroid differentiation of DMSO-treated Friend cells induced by low doses of crude or semipurified IFN is completely abolished. These results, confirmed with pure IFN preparations, are in keeping with the hypothesis that an early interaction with the cell membrane is a necessary step for the establishment of these different IFN effects. In addition, they indicate that the increased cyclic AMP levels, induced by the whole toxin, do not play a significant role in these phenomena; rather they may be due to the lectin-like activity of the B subunit. Evaluation of reciprocal dose-response curves of IFN and cholera toxin shows that treatment with toxin concentrations as low as 10¹³M induces a major inhibition of IFN effects. A millionfold higher toxin concentration does not substantially increase the toxin actions. The toxin-induced redistribution of membrane gangliosides is discussed as a possible mechanism of the observed inhibition of IFN activities.     

Development of functional neurons from postnatal stem cells in vitro

In order for stem cells to fulfill their clinical promise, we must understand their developmental transitions and it must be possible to control the differentiation of stem cells into specific cell fates. To understand the mechanism of the sequential restriction and multipotency of stem cells, we have established culture conditions that allow the differentiation of multipotential neural stem cells from postnatal stem cells. We used immunocytochemistry, fluorescence microscopy, and calcium imaging to demonstrate that progeny of adult rat neural stem cells develop into functional neurons that release excitatory neurotransmitters. We also found that the nontoxic heavy chain fragment of tetanus toxin, a toxin that targets neurons with high specificity, retained the specificity toward neural stem cell-derived neurons. These studies show that neural stem cells derived from adult tissues retain the potential to differentiate into functional neurons with morphological and functional properties of mature central nervous system neurons.     

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected.