Inhibition of class II major histocompatibility complex antigen processing by Escherichia coli heat-labile enterotoxin requires an enzymatically active A subunit - PubMed (original) (raw)
Inhibition of class II major histocompatibility complex antigen processing by Escherichia coli heat-labile enterotoxin requires an enzymatically active A subunit
M P Matousek et al. Infect Immun. 1998 Jul.
Abstract
Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.
Figures
FIG. 1
Overnight treatment of macrophages with LT inhibits intracellular processing of HB101.Crl-HEL but does not inhibit the presentation of preexisting surface peptide–MHC-II complexes. Macrophages were incubated with viable HB101.Crl-HEL for 2 h either after (A) or before (B) overnight treatment with LT (1 μg/ml). (A) Macrophages were treated with or without LT overnight, washed, incubated with HB101.Crl-HEL for 2 h at 37°C, fixed with 1% paraformaldehyde, and washed extensively. (B) Macrophages were incubated with HB101.Crl-HEL for 2 h at 37°C, washed, treated with or without LT overnight, fixed, and washed extensively. Antigen presentation was determined by incubation with HEL-specific 3A9 T hybridoma cells (105/well) for 20 to 24 h at 37°C, followed by a bioassay for interleukin 2 production (16). Interleukin 2-dependent CTLL-2 cells were incubated for 24 h at 37°C with supernatants collected from antigen presentation assays. The cells were then pulsed for 18 to 24 h with Alamar blue. Both reduced and oxidized forms of Alamar blue have high absorbance near 570 nm, whereas only the oxidized form has high absorbance near 600 nm. Production of the reduced form (a measure of cell growth and metabolic activity) can be measured by subtracting the optical density at 600 nm (OD600) from OD570 (2) or subtracting OD595 from OD550. All data points are presented as mean (OD550 − OD595) ± standard deviation for triplicate points.
FIG. 2
LT and CT do not inhibit macrophage catabolism of HB101.Crl-HEL. Macrophages (2 × 106 cells/well in 24-well plates) were incubated overnight with or without LT or CT (1 μg/ml). 125I-labeled HB101.Crl-HEL was centrifuged onto the macrophages at 2,500 × g for 10 min at 4°C. The plates were then incubated at either 4°C (negative control) or 37°C for 20 min, washed to remove extracellular bacteria, and then incubated for 2 h at either 4 or 37°C to allow for processing and catabolism of intracellular bacteria. High-molecular-weight proteins were precipitated from both the media and cell lysates (cells solubilized in 1% Triton X-100 in phosphate-buffered saline) with 10% trichloroacetic acid at 4°C. Bacterial catabolism was reflected by trichloroacetic acid-soluble radioactivity in the medium, shown here as a mean percentage of the total counts per minute in the well plus or minus the standard deviation of duplicate samples.
FIG. 3
Ribosyltransferase activity of the A subunit is necessary for inhibition of antigen processing, whereas antigen presentation is enhanced by toxin preparations that lack A subunit enzymatic activity. Macrophages were treated overnight with or without the toxin preparations (1 μg/ml), washed, incubated with viable HB101.Crl-HEL for 2 h at 37°C, and fixed. Antigen presentation was determined by incubation with 3A9 T hybridoma cells for 20 to 24 h at 37°C.
FIG. 4
Elevation of intracellular cAMP levels in macrophages after treatment with toxin preparations. Macrophages were treated overnight with the indicated toxin preparations (1 μg/ml), the cells were lysed in 66% ethanol, and cAMP levels were determined by using a TiterZyme dual-range cAMP enzyme immunoassay kit from PerSeptive Diagnostics (Cambridge, Mass.).
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