10:727-746. 16, 19). These cytokines include interleukin 1 (IL-1), tumor necrosis element alpha (TNF-), and gamma interferon (IFN-). These proinflammatory mediators have potent immunoenhancing effects and are known to be pathogenic at high levels (12). The massive production of inflammatory mediators from both T cells and monocytes appears early, within hours after the SE bind to these cells, while T-cell proliferation happens later on. Recently, caspase inhibitors were used to prevent allergic airway swelling (7) and to decrease the death rate from acute experimental pancreatitis (18). Caspase inhibitors also clogged murine liver injury (13) and attenuated bleomycin-induced pneumopathy (14). Additional studies indicated that caspase activation is required for mitogen- or anti-CD3-stimulated T-cell proliferation (1, 8, 17). Caspases belong to a family of autocatalytic cysteine proteases which regulate many cellular processes, including apotosis, cell differentiation, and swelling (5). They share sequence homologies and are grouped into three subfamilies based on their function and substrate specificity (5, 6, 18, 20). Group 1 caspases, which include caspase-1, -4, -5, and -14, mediate cytokine processing and swelling. Caspase-1, originally known as the IL-1-transforming enzyme, is the first-discovered caspase and is essential in the proteolytic processing of cytokines IL-1 and IL-18. The additional major groups of caspases, organizations 2 and 3, function mostly in apotosis and in additional cellular proteolytic cascades resulting in DNA fragmentation and degradation of multiple cellular substrates (5). To elucidate further the cellular mechanisms contributing to harmful shock, caspase activation in SE-stimulated human being peripheral blood mononuclear cells (PBMC) was examined through the use of specific and pan-caspase inhibitors. Purified TSST-1 and SEB were from Toxin Technology (Sarasota, Fla.). The endotoxin content of these preparations was 1 ng of endotoxin per mg of protein, as determined by the amoebocyte lysate gelation test (BioWhittaker, Walkersville, Md.). Human being recombinant TNF- (hrTNF), antibodies against hrTNF, peroxidase-conjugated anti-rabbit immunoglobulin G, and peroxidase-conjugated anti-goat immunoglobulin G were from Roche (Indianapolis, Ind.). hrIL-1 was kindly provided by J. Oppenheim (National Malignancy Institute, Frederick, Md.). hrIFN- and hrIL-6 were from Collaborative Study (Boston, Mass.). Antibodies against IFN- and MCP-1 were from BD PharMingen (San Diego, Calif.). hrMCP-1, hrMIP-1, and hrMIP-1, and antibodies against IL-1, IL-6, MIP-1, and MIP-1 were purchased from R&D Systems (Minneapolis, Minn.). The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk), Z-Asp-(2,6-dichlorobenzoyl)oxymethane (Z-D-CH2-DCB), Z-Asp-Glu-Val-Asp-fmk (Z-DEVD-fmk), and Z-Ile-Glu-Thr-Asp-fmk (Z-IETD-fmk) were from Calbiochem (San Diego, Calif.) and dissolved in dimethyl sulfoxide. All other common reagents were from Sigma (St. Louis, Mo.). Human being PBMC were isolated by Ficoll-Hypaque denseness gradient centrifugation of heparinized blood from normal human being donors. PBMC were cultured in 24-well plates at 37C in RPMI 1640 supplemented with 10% fetal bovine serum at a concentration of 106 cells/ml. Cells were stimulated with either TSST-1 (200 ng/ml) or SEB (200 ng/ml) for 16 h. The exotoxins and the various concentrations of caspase inhibitors were added simultaneously. Supernatants were harvested and analyzed for IL-1, TNF-, IL-6, IFN-, MCP-1, MIP-1, and MIP-1. Cytokines and chemokines were measured by an enzyme-linked immunosorbent assay with cytokine- or chemokine-specific antibodies, as previously explained (10, 11). Human being recombinant cytokines and chemokines (20 to 1 1,000 pg/ml) were used as calibration requirements for each plate. The detection limit of each assay was 20 pg/ml. Cytotoxicity was measured by the launch of lactate dehydrogenase (LDH) from your cytosol into the tradition supernatant. LDH was measured by using a cytotoxicity kit (Roche) in accordance with the manufacturer’s instructions. T-cell proliferation was assayed with PBMC (105 cells/well), which were plated in triplicate with TSST-1 or SEB (200 ng/ml), with or without caspase inhibitors, for 48 h at 37C in 96-well microtiter plates. Cells were pulsed with 1 Ci of [3H]thymidine (New England Nuclear, Boston, Mass.) per well during the last 5 h of tradition (10). Cells were harvested onto glass dietary fiber.235:68-76. to harmful shock, including the cascade of inflammatory cytokines and chemokines (3, 4, 9, 16, 19). These cytokines include interleukin 1 (IL-1), tumor necrosis element alpha (TNF-), and gamma interferon (IFN-). These proinflammatory mediators have potent immunoenhancing effects and are known to be pathogenic at high levels (12). The massive production of inflammatory mediators from both T cells and monocytes appears early, within hours after the SE bind to these cells, while T-cell proliferation happens later. Recently, caspase inhibitors were used to prevent allergic airway swelling (7) and to decrease the death rate from acute experimental pancreatitis (18). Caspase inhibitors also clogged murine liver injury (13) and attenuated bleomycin-induced pneumopathy (14). Additional studies indicated Ecteinascidin-Analog-1 that caspase activation is required for mitogen- Ecteinascidin-Analog-1 or anti-CD3-stimulated T-cell proliferation (1, 8, 17). Caspases belong to a family of autocatalytic cysteine proteases which regulate many cellular processes, including apotosis, cell differentiation, and swelling (5). They share sequence homologies and are grouped into three subfamilies based on their function and substrate specificity (5, 6, 18, 20). Group 1 caspases, which include caspase-1, -4, -5, and -14, mediate cytokine processing and swelling. Caspase-1, originally known as the IL-1-transforming enzyme, is the first-discovered caspase and is essential in the proteolytic processing of cytokines IL-1 and IL-18. The additional major groups of caspases, organizations 2 and 3, function mostly in apotosis and in additional cellular proteolytic cascades resulting in DNA fragmentation and degradation of multiple cellular substrates (5). To elucidate further the cellular mechanisms contributing to harmful shock, caspase activation in SE-stimulated human being peripheral blood mononuclear cells (PBMC) was examined through the use of specific and pan-caspase inhibitors. Purified TSST-1 and SEB were obtained from Toxin Technology (Sarasota, Fla.). The endotoxin content of these preparations was 1 ng of endotoxin per mg of protein, as determined by the amoebocyte lysate gelation test (BioWhittaker, Walkersville, Md.). Human recombinant TNF- (hrTNF), antibodies against hrTNF, peroxidase-conjugated anti-rabbit immunoglobulin G, and peroxidase-conjugated anti-goat immunoglobulin G were obtained from Roche (Indianapolis, Ind.). hrIL-1 was kindly provided by J. Oppenheim (National Malignancy Institute, Frederick, Md.). hrIFN- and hrIL-6 were obtained from Collaborative Research (Boston, Mass.). Antibodies against IFN- and MCP-1 were obtained from BD PharMingen (San Diego, Calif.). hrMCP-1, hrMIP-1, and hrMIP-1, and antibodies against IL-1, IL-6, MIP-1, and MIP-1 were purchased from R&D Systems (Minneapolis, Minn.). The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk), Z-Asp-(2,6-dichlorobenzoyl)oxymethane (Z-D-CH2-DCB), Z-Asp-Glu-Val-Asp-fmk (Z-DEVD-fmk), and Z-Ile-Glu-Thr-Asp-fmk (Z-IETD-fmk) were obtained from Calbiochem (San Diego, Calif.) and dissolved in dimethyl sulfoxide. All other common reagents were obtained from Sigma (St. Louis, Mo.). Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of heparinized blood from normal human donors. PBMC were cultured in 24-well plates at 37C in RPMI 1640 supplemented with 10% fetal bovine serum at a concentration of 106 cells/ml. Cells were stimulated with either TSST-1 (200 ng/ml) or SEB (200 ng/ml) for 16 h. The exotoxins and the various concentrations of caspase inhibitors were added simultaneously. Supernatants were harvested and analyzed for IL-1, TNF-, IL-6, IFN-, MCP-1, MIP-1, and MIP-1. Cytokines and chemokines were measured by an enzyme-linked immunosorbent assay with cytokine- or chemokine-specific antibodies, as previously described (10, 11). Human recombinant cytokines and chemokines (20 to 1 1,000 pg/ml) were used as calibration standards for each plate. The detection limit of each assay was 20 pg/ml. Cytotoxicity was measured by the release of lactate dehydrogenase (LDH) from the cytosol into the culture supernatant. LDH was measured by using a cytotoxicity kit (Roche) in accordance with the manufacturer’s instructions. T-cell proliferation was assayed with PBMC (105 Ecteinascidin-Analog-1 cells/well), which were plated in triplicate with TSST-1 or SEB (200 ng/ml), with or without caspase inhibitors, for 48 h at 37C in 96-well microtiter plates. Cells were pulsed with 1 Ci of [3H]thymidine (New England Nuclear, Boston, Mass.).Oppenheim. molecules on antigen-presenting cells (9). These superantigens polyclonally activate T cells (4), resulting in proliferation and, eventually, apotosis. Multiple components of the host inflammatory response brought on by SE have been shown to contribute to toxic shock, including the cascade of inflammatory cytokines and chemokines (3, 4, 9, 16, 19). These cytokines include interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-), and gamma interferon (IFN-). These proinflammatory mediators have potent immunoenhancing effects and are known to be pathogenic at high levels (12). The massive production of inflammatory mediators from both T cells and monocytes appears early, within hours after the SE bind to these cells, while T-cell proliferation occurs later. Recently, caspase inhibitors were used to prevent allergic airway inflammation (7) and to decrease the death rate from acute experimental pancreatitis (18). Caspase inhibitors also blocked Ecteinascidin-Analog-1 murine liver injury (13) and attenuated bleomycin-induced pneumopathy (14). Other studies indicated that caspase activation is required for mitogen- or anti-CD3-stimulated T-cell proliferation (1, 8, 17). Caspases belong to a family of autocatalytic cysteine proteases which regulate many cellular processes, including apotosis, cell differentiation, and inflammation (5). They share sequence homologies and are grouped into three subfamilies based on their function and substrate specificity (5, 6, 18, 20). Group 1 caspases, which include caspase-1, -4, -5, and -14, mediate cytokine processing and inflammation. Caspase-1, originally known as the IL-1-converting enzyme, is the first-discovered caspase and is essential in the proteolytic processing of cytokines IL-1 and IL-18. The other major groups of caspases, groups 2 and 3, function mostly in apotosis and in other cellular proteolytic cascades resulting in DNA fragmentation and degradation of multiple cellular ESR1 substrates (5). To elucidate further the cellular mechanisms contributing to toxic shock, caspase activation in SE-stimulated human peripheral blood mononuclear cells (PBMC) was examined through the use of specific and pan-caspase inhibitors. Purified TSST-1 and SEB were obtained from Toxin Technology (Sarasota, Fla.). The endotoxin content of these preparations was 1 ng of endotoxin per mg of protein, as determined by the amoebocyte lysate gelation test (BioWhittaker, Walkersville, Md.). Human recombinant TNF- (hrTNF), antibodies against hrTNF, peroxidase-conjugated anti-rabbit immunoglobulin G, and peroxidase-conjugated anti-goat immunoglobulin G were obtained from Roche (Indianapolis, Ind.). hrIL-1 was kindly provided by J. Oppenheim (National Malignancy Institute, Frederick, Md.). hrIFN- and hrIL-6 were obtained from Collaborative Research (Boston, Mass.). Antibodies against IFN- and MCP-1 were obtained from BD PharMingen (San Diego, Calif.). hrMCP-1, hrMIP-1, and hrMIP-1, and antibodies against IL-1, IL-6, MIP-1, and MIP-1 were purchased from R&D Systems (Minneapolis, Minn.). The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk), Z-Asp-(2,6-dichlorobenzoyl)oxymethane (Z-D-CH2-DCB), Z-Asp-Glu-Val-Asp-fmk (Z-DEVD-fmk), and Z-Ile-Glu-Thr-Asp-fmk (Z-IETD-fmk) were obtained from Calbiochem (San Diego, Calif.) and dissolved in dimethyl sulfoxide. All other common reagents were obtained from Sigma (St. Louis, Mo.). Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of heparinized Ecteinascidin-Analog-1 blood from normal human donors. PBMC were cultured in 24-well plates at 37C in RPMI 1640 supplemented with 10% fetal bovine serum at a concentration of 106 cells/ml. Cells were stimulated with either TSST-1 (200 ng/ml) or SEB (200 ng/ml) for 16 h. The exotoxins and the various concentrations of caspase inhibitors were added simultaneously. Supernatants were harvested and analyzed for IL-1, TNF-, IL-6, IFN-, MCP-1, MIP-1, and MIP-1. Cytokines and chemokines were measured by an enzyme-linked immunosorbent assay with cytokine- or chemokine-specific antibodies, as previously described (10, 11). Human recombinant cytokines and chemokines (20 to 1 1,000 pg/ml) were used as calibration standards for each plate. The detection limit of each assay was 20 pg/ml. Cytotoxicity was measured by the release of lactate dehydrogenase (LDH) from the cytosol into the culture supernatant. LDH was measured by using a cytotoxicity kit (Roche) in accordance.
