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Transcytosis of staphylococcal superantigen toxins.

Hamad AR, Marrack P, Kappler JW - J. Exp. Med. (1997)

Bottom Line: We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA.We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA.Our data suggest that these toxins can cross the epithelium in an immunologically intact form.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning.

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(a) SEB transcytosis  by Caco-2 cells is bidirectional  and time dependent. (Apical to  basal) Toxin and HRP (3 μg  each in 400 μl medium) were  added to the apical chamber.  Samples were collected from basal  chambers at indicated time points,  over a period of 24 h. Transcytosed toxin and HRP were then  measured as described in Materials and Methods. (Basal to apical)  The experiment was done as  above except the toxin and HRP  were added to the basal chamber and apical samples were collected. Each  point is the mean of three determinants. SEM was usually <15%. This  experiment is a representative of three similar experiments. (b) Transcytosis of SEB by Caco-2 cells is dose dependent but not saturable. Different  concentrations of SEB and HRP were added to the apical medium of the  same filter. Basal media were collected 90 min later and assayed for SEB  and HRP transcytosed. Each point was performed in triplicate. SEM was  usually <15%. This experiment is a representative of three separate experiments with similar results.
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Figure 2: (a) SEB transcytosis by Caco-2 cells is bidirectional and time dependent. (Apical to basal) Toxin and HRP (3 μg each in 400 μl medium) were added to the apical chamber. Samples were collected from basal chambers at indicated time points, over a period of 24 h. Transcytosed toxin and HRP were then measured as described in Materials and Methods. (Basal to apical) The experiment was done as above except the toxin and HRP were added to the basal chamber and apical samples were collected. Each point is the mean of three determinants. SEM was usually <15%. This experiment is a representative of three similar experiments. (b) Transcytosis of SEB by Caco-2 cells is dose dependent but not saturable. Different concentrations of SEB and HRP were added to the apical medium of the same filter. Basal media were collected 90 min later and assayed for SEB and HRP transcytosed. Each point was performed in triplicate. SEM was usually <15%. This experiment is a representative of three separate experiments with similar results.

Mentions: We prepared two chambered cultures separated by a monolayer of confluent Caco-2 cells as described in Materials and Methods (see Fig. 1). We initially studied SEB transcytosis to characterize this system, using HRP as an internal control to distinguish between specific transcytosis and nonspecific mechanisms such as paracellular diffusion with water (30). Two types of experiments were done. In both, SEB and HRP were added to either apical or basal media and their appearance in the opposite chamber assessed as described in the Materials and Methods. In the first experiment (Fig. 2 a), a fixed concentration of each protein (7.5 μg/ml) was used and the kinetics of transcytosis followed in either direction through the monolayer. In the second (Fig. 2 b), various concentrations of the protein were added to the apical media and the amount transcytosed to the basal medium assayed at 90 min. These results established several points. First, transcytosis of SEB was detected in both directions across the Caco-2 monolayer and the rate of SEB transcytosis was linear for the 24 h of the experiment. Second, SEB was transcytosed faster than HRP, especially in the apical to basolateral direction where the SEB transcytosis rate was usually three to six times faster than that of HRP. These results suggested that the transcytosis of SEB by Caco-2 was facilitated, perhaps by a specific surface receptor. However, we found no evidence in these experiments for saturation of the SEB transcytosis mechanism even using concentrations of SEB up to 300 μg/ml (Fig. 2 b).


Transcytosis of staphylococcal superantigen toxins.

Hamad AR, Marrack P, Kappler JW - J. Exp. Med. (1997)

(a) SEB transcytosis  by Caco-2 cells is bidirectional  and time dependent. (Apical to  basal) Toxin and HRP (3 μg  each in 400 μl medium) were  added to the apical chamber.  Samples were collected from basal  chambers at indicated time points,  over a period of 24 h. Transcytosed toxin and HRP were then  measured as described in Materials and Methods. (Basal to apical)  The experiment was done as  above except the toxin and HRP  were added to the basal chamber and apical samples were collected. Each  point is the mean of three determinants. SEM was usually <15%. This  experiment is a representative of three similar experiments. (b) Transcytosis of SEB by Caco-2 cells is dose dependent but not saturable. Different  concentrations of SEB and HRP were added to the apical medium of the  same filter. Basal media were collected 90 min later and assayed for SEB  and HRP transcytosed. Each point was performed in triplicate. SEM was  usually <15%. This experiment is a representative of three separate experiments with similar results.
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Related In: Results  -  Collection

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Figure 2: (a) SEB transcytosis by Caco-2 cells is bidirectional and time dependent. (Apical to basal) Toxin and HRP (3 μg each in 400 μl medium) were added to the apical chamber. Samples were collected from basal chambers at indicated time points, over a period of 24 h. Transcytosed toxin and HRP were then measured as described in Materials and Methods. (Basal to apical) The experiment was done as above except the toxin and HRP were added to the basal chamber and apical samples were collected. Each point is the mean of three determinants. SEM was usually <15%. This experiment is a representative of three similar experiments. (b) Transcytosis of SEB by Caco-2 cells is dose dependent but not saturable. Different concentrations of SEB and HRP were added to the apical medium of the same filter. Basal media were collected 90 min later and assayed for SEB and HRP transcytosed. Each point was performed in triplicate. SEM was usually <15%. This experiment is a representative of three separate experiments with similar results.
Mentions: We prepared two chambered cultures separated by a monolayer of confluent Caco-2 cells as described in Materials and Methods (see Fig. 1). We initially studied SEB transcytosis to characterize this system, using HRP as an internal control to distinguish between specific transcytosis and nonspecific mechanisms such as paracellular diffusion with water (30). Two types of experiments were done. In both, SEB and HRP were added to either apical or basal media and their appearance in the opposite chamber assessed as described in the Materials and Methods. In the first experiment (Fig. 2 a), a fixed concentration of each protein (7.5 μg/ml) was used and the kinetics of transcytosis followed in either direction through the monolayer. In the second (Fig. 2 b), various concentrations of the protein were added to the apical media and the amount transcytosed to the basal medium assayed at 90 min. These results established several points. First, transcytosis of SEB was detected in both directions across the Caco-2 monolayer and the rate of SEB transcytosis was linear for the 24 h of the experiment. Second, SEB was transcytosed faster than HRP, especially in the apical to basolateral direction where the SEB transcytosis rate was usually three to six times faster than that of HRP. These results suggested that the transcytosis of SEB by Caco-2 was facilitated, perhaps by a specific surface receptor. However, we found no evidence in these experiments for saturation of the SEB transcytosis mechanism even using concentrations of SEB up to 300 μg/ml (Fig. 2 b).

Bottom Line: We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA.We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA.Our data suggest that these toxins can cross the epithelium in an immunologically intact form.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning.

Show MeSH
Related in: MedlinePlus