have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal m... more have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal macrophages (M#{248}) to produce both tumor necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to 3T1 2 target cells. The present study identifies the roles of both enzymatic activity and contaminating lipopolysacharides (LPS) ( 1 ng) on these activities. The addition of 100 ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNF production but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effective than HRP in both TNF production and MMC but was not affected by PB. The inability of PB to modify DHRP-induced TNF suggests that LPS was not required. The induction of TNF and MMC in the absence of LPS was also corroborated by similar studies using M#{248} from endotoxin-resistant C3H/HeJ mice. Glycosylated proteins such as HRP, DHRP, and mannosylated bovine serum albumin (M-BSA) are known to bind to mannose receptors (mannosyl-fucosyl receptor [MFRJ) on the surface of MO. In the present studies, M-BSA behaved similarly to DHRP in that it induced both TNF secretion and MMC. These results suggest that binding to the MFR may be sufficient to induce TNF secretion and MMC. In addition, the data suggest that neither enzymatic activity nor LPS was required for DHRP-induced TNF.
This report describes the utilization of lllindium.oxin e chelate ([lllIn]Ox) for studies of macr... more This report describes the utilization of lllindium.oxin e chelate ([lllIn]Ox) for studies of macrophage-mediated cytotoxicity. [I i lin ]Ox efficiently labeled both nonadherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intraeellularly incorporated [ I I iin ]Ox was very slow (0.25--0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [l I I In ]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [tllIn]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [l ilin ]Ox released in response to cytolytic macrophages correlated well with those observed for the s I Cr and [3H ]TdR radiolabels. Therefore, [ 111 In ]Ox can be utilized for relatively short-term (less than 20 h) assays with ]ymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.
have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal m... more have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal macrophages (M#{248}) to produce both tumor necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to 3T1 2 target cells. The present study identifies the roles of both enzymatic activity and contaminating lipopolysacharides (LPS) ( 1 ng) on these activities. The addition of 100 ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNF production but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effective than HRP in both TNF production and MMC but was not affected by PB. The inability of PB to modify DHRP-induced TNF suggests that LPS was not required. The induction of TNF and MMC in the absence of LPS was also corroborated by similar studies using M#{248} from endotoxin-resistant C3H/HeJ mice. Glycosylated proteins such as HRP, DHRP, and mannosylated bovine serum albumin (M-BSA) are known to bind to mannose receptors (mannosyl-fucosyl receptor [MFRJ) on the surface of MO. In the present studies, M-BSA behaved similarly to DHRP in that it induced both TNF secretion and MMC. These results suggest that binding to the MFR may be sufficient to induce TNF secretion and MMC. In addition, the data suggest that neither enzymatic activity nor LPS was required for DHRP-induced TNF.
This report describes the utilization of lllindium.oxin e chelate ([lllIn]Ox) for studies of macr... more This report describes the utilization of lllindium.oxin e chelate ([lllIn]Ox) for studies of macrophage-mediated cytotoxicity. [I i lin ]Ox efficiently labeled both nonadherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intraeellularly incorporated [ I I iin ]Ox was very slow (0.25--0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [l I I In ]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [tllIn]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [l ilin ]Ox released in response to cytolytic macrophages correlated well with those observed for the s I Cr and [3H ]TdR radiolabels. Therefore, [ 111 In ]Ox can be utilized for relatively short-term (less than 20 h) assays with ]ymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.
Uploads
Papers by Aaron Castro