Vol. 58, No. 7
INFECTION AND IMMUNITY, JUlY 1990, p. 2392-2396
0019-9567/90/072392-05$02.00/0 Copyright C) 1990, American Society for Microbiology
Binding Competition of Toxic Shock Syndrome Toxin 1 and Other Staphylococcal Exoproteins for Receptors on Human Peripheral Blood Mononuclear Cells RAYMOND H. SEE,"2 GERALD KRYSTAL,2'3 AND ANTHONY W. CHOWl.4* Departments of Medicine,' Microbiology,4 and Pathology,2 Division of Infectious Diseases, University of British
Columbia, and Terry Fox Laboratory, British Columbia Cancer Research Center,3 Vancouver, British Columbia, Canada VSZ IM9 Received 13 December 1989/Accepted 13 April 1990
Binding of toxic shock toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) to human peripheral blood mononuclear cells (PBMC) was investigated by using 125I-labeled ligands. Scatchard analyses revealed similar numbers of receptors (approximately 5,000 to 8,000) and similar dissociation constants (Kd, approximately 20 to 25 nM) per PBMC. SEA but not enterotoxin B, Cl, C2, C3, D, or E significantly inhibited binding of 12-5-TSST-1 to PBMC. Cross-competition of TSST-1 and SEA in binding assays suggests that they may bind to overlapping or separate epitopes on the same receptor.
Toxic shock syndrome (TSS) is a multisystem disease associated with Staphylococcus aureus infection. A 22kilodalton exoprotein, TSS toxin 1 (TSST-1), is thought to play a central role in this disease. TSST-1 has a wide variety of biological effects, including T-lymphocyte mitogenicity (20), induction of interleukin-1 (10, 19) and tumor necrosis factor release from human monocytes (11), suppression of immunoglobulin M response against sheep erythrocytes (21), stimulation of lymphokine release by T lymphocytes (11, 21), and enhancement of host susceptibility to lethal endotoxic shock (27). These properties are shared by a group of related exoproteins, staphylococcal enterotoxin A (SEA), SEB, SEC, SED, and SEE (1). The molecular means by which TSST-1 and staphylococcal enterotoxins exert these various effects are still unclear. The first action may be binding to a specific cell surface receptor. Binding sites for TSST-1 have been demonstrated for several cell types, including human conjunctival epithelial cells (13), human T lymphocytes (22, 28), and endothelial cells from umbilical cord veins and arteries (14). Recently, TSST-1, SEA, and SEB have been shown to bind to specific receptors on human peripheral blood mononuclear cells (PBMC). These receptors have been identified as the class II major histocompatibility complex antigens as demonstrated on B and T cell lines as well as on class II transfectants (6, 8, 16, 28). In the present study, we show that among enterotoxins A, B, Cl, C2, C3, D, and E, only SEA was able to significantly inhibit TSST-1 binding to the TSST-1 receptor on human PBMC. In addition, although both TSST-1 and SEA bind to the same receptor, the binding epitopes appear to be separate or overlapping. TSST-1 was purified from culture supernatants of S. aureus MN8 by ion-exchange chromatography, chromatofocusing, and gel filtration, as previously described (9, 24). The final concentration of TSST-1 in the purified preparation was determined by a noncompetitive enzyme-linked immunosorbent assay (23). Purity of TSST-1 was >95%, as determined by silver staining of sodium dodecyl sulfate-polyacrylamide gels (15) and by immunoblotting (2) with pooled human serum. Highly purified (>95%) staphylococcal enterotoxins *
Corresponding author.
A, B, Cl, C2, C3, D, and E were purchased from Toxin Technology, Inc. (Madison, Wis.). Fresh human PBMC were obtained by centrifugation of plateletpheresis buffy coats from healthy adult donors over Histopaque 1.077 (Sigma Chemical Co., St. Louis, Mo.). Cells at the interface were washed three times in Hanks balanced salt solution and suspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Viability of cells was >96%, as judged by trypan blue exclusion. TSST-1 and SEA were iodinated by a modified chloramine T procedure, as previously described (18). Briefly, 0.1 M sodium phosphate buffer (pH 7.4) containing 100 ,g of polyethylene glycol 6000 per ml and 10% dimethyl sulfoxide was added to 100 pkg of TSST-1 or SEA in a total volume of 135 ,1. A quantity (10 ,lI) of freshly prepared chloramine T solution (1 mg/ml) was added, and this was followed by 20 ,ul (2 mCi) of 1251 (carrier-free Nal; 100 mCi/ml; ICN Radiochemicals, Irvine, Calif.). After 20 min at room temperature, the reaction was stopped by the addition of 10 ,ul of sodium metabisulfite (3 mg/ml) and the mixture was put on ice for 5 min. lodinated toxins were separated from free iodine by gel filtration through 10-ml Sephadex G-25 (Pharmacia Fine Chemicals, Dorval, Quebec, Canada) columns equilibrated with phosphate-buffered saline containing 0.25% (wt/vol) gelatin. Pooled fractions corresponding to the protein peak were adjusted to 0.1% with bovine serum albumin and stored in 200-,ul aliquots at -70°C. More than 95% of the radioactivity in the pooled fractions was precipitable with 10% trichloroacetic acid. Typically, the specific activity of radioiodinated TSST-1 or SEA ranged from 30 to 55 puCi/,ug of protein, with
INFECTION AND IMMUNITY, JUlY 1990, p. 2392-2396
0019-9567/90/072392-05$02.00/0 Copyright C) 1990, American Society for Microbiology
Binding Competition of Toxic Shock Syndrome Toxin 1 and Other Staphylococcal Exoproteins for Receptors on Human Peripheral Blood Mononuclear Cells RAYMOND H. SEE,"2 GERALD KRYSTAL,2'3 AND ANTHONY W. CHOWl.4* Departments of Medicine,' Microbiology,4 and Pathology,2 Division of Infectious Diseases, University of British
Columbia, and Terry Fox Laboratory, British Columbia Cancer Research Center,3 Vancouver, British Columbia, Canada VSZ IM9 Received 13 December 1989/Accepted 13 April 1990
Binding of toxic shock toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) to human peripheral blood mononuclear cells (PBMC) was investigated by using 125I-labeled ligands. Scatchard analyses revealed similar numbers of receptors (approximately 5,000 to 8,000) and similar dissociation constants (Kd, approximately 20 to 25 nM) per PBMC. SEA but not enterotoxin B, Cl, C2, C3, D, or E significantly inhibited binding of 12-5-TSST-1 to PBMC. Cross-competition of TSST-1 and SEA in binding assays suggests that they may bind to overlapping or separate epitopes on the same receptor.
Toxic shock syndrome (TSS) is a multisystem disease associated with Staphylococcus aureus infection. A 22kilodalton exoprotein, TSS toxin 1 (TSST-1), is thought to play a central role in this disease. TSST-1 has a wide variety of biological effects, including T-lymphocyte mitogenicity (20), induction of interleukin-1 (10, 19) and tumor necrosis factor release from human monocytes (11), suppression of immunoglobulin M response against sheep erythrocytes (21), stimulation of lymphokine release by T lymphocytes (11, 21), and enhancement of host susceptibility to lethal endotoxic shock (27). These properties are shared by a group of related exoproteins, staphylococcal enterotoxin A (SEA), SEB, SEC, SED, and SEE (1). The molecular means by which TSST-1 and staphylococcal enterotoxins exert these various effects are still unclear. The first action may be binding to a specific cell surface receptor. Binding sites for TSST-1 have been demonstrated for several cell types, including human conjunctival epithelial cells (13), human T lymphocytes (22, 28), and endothelial cells from umbilical cord veins and arteries (14). Recently, TSST-1, SEA, and SEB have been shown to bind to specific receptors on human peripheral blood mononuclear cells (PBMC). These receptors have been identified as the class II major histocompatibility complex antigens as demonstrated on B and T cell lines as well as on class II transfectants (6, 8, 16, 28). In the present study, we show that among enterotoxins A, B, Cl, C2, C3, D, and E, only SEA was able to significantly inhibit TSST-1 binding to the TSST-1 receptor on human PBMC. In addition, although both TSST-1 and SEA bind to the same receptor, the binding epitopes appear to be separate or overlapping. TSST-1 was purified from culture supernatants of S. aureus MN8 by ion-exchange chromatography, chromatofocusing, and gel filtration, as previously described (9, 24). The final concentration of TSST-1 in the purified preparation was determined by a noncompetitive enzyme-linked immunosorbent assay (23). Purity of TSST-1 was >95%, as determined by silver staining of sodium dodecyl sulfate-polyacrylamide gels (15) and by immunoblotting (2) with pooled human serum. Highly purified (>95%) staphylococcal enterotoxins *
Corresponding author.
A, B, Cl, C2, C3, D, and E were purchased from Toxin Technology, Inc. (Madison, Wis.). Fresh human PBMC were obtained by centrifugation of plateletpheresis buffy coats from healthy adult donors over Histopaque 1.077 (Sigma Chemical Co., St. Louis, Mo.). Cells at the interface were washed three times in Hanks balanced salt solution and suspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Viability of cells was >96%, as judged by trypan blue exclusion. TSST-1 and SEA were iodinated by a modified chloramine T procedure, as previously described (18). Briefly, 0.1 M sodium phosphate buffer (pH 7.4) containing 100 ,g of polyethylene glycol 6000 per ml and 10% dimethyl sulfoxide was added to 100 pkg of TSST-1 or SEA in a total volume of 135 ,1. A quantity (10 ,lI) of freshly prepared chloramine T solution (1 mg/ml) was added, and this was followed by 20 ,ul (2 mCi) of 1251 (carrier-free Nal; 100 mCi/ml; ICN Radiochemicals, Irvine, Calif.). After 20 min at room temperature, the reaction was stopped by the addition of 10 ,ul of sodium metabisulfite (3 mg/ml) and the mixture was put on ice for 5 min. lodinated toxins were separated from free iodine by gel filtration through 10-ml Sephadex G-25 (Pharmacia Fine Chemicals, Dorval, Quebec, Canada) columns equilibrated with phosphate-buffered saline containing 0.25% (wt/vol) gelatin. Pooled fractions corresponding to the protein peak were adjusted to 0.1% with bovine serum albumin and stored in 200-,ul aliquots at -70°C. More than 95% of the radioactivity in the pooled fractions was precipitable with 10% trichloroacetic acid. Typically, the specific activity of radioiodinated TSST-1 or SEA ranged from 30 to 55 puCi/,ug of protein, with
