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Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Brumbaugh KM, Binstadt BA, Billadeau DD, Schoon RA, Dick CJ, Ten RM, Leibson PJ - J. Exp. Med. (1997)

Bottom Line: Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function.Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk.These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

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Piceatannol pretreatment inhibits NK cell– mediated cytotoxicity. NK cells  were treated for 15 min at 37°C  with the indicated concentration of piceatannol, washed, and  then incubated for 4 h with either 51Cr-labeled K562 cells or  51Cr-labeled P815 cells coated  with 0.15 μg/ml of the anti-FcR  mAb 3G8. Data are expressed as percentage of inhibition of lytic U/106  cells calculated from NK cells incubated in vehicle alone.
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Figure 5: Piceatannol pretreatment inhibits NK cell– mediated cytotoxicity. NK cells were treated for 15 min at 37°C with the indicated concentration of piceatannol, washed, and then incubated for 4 h with either 51Cr-labeled K562 cells or 51Cr-labeled P815 cells coated with 0.15 μg/ml of the anti-FcR mAb 3G8. Data are expressed as percentage of inhibition of lytic U/106 cells calculated from NK cells incubated in vehicle alone.

Mentions: Although the above data demonstrate biochemically that Syk is modified in NK cells after target cell stimulation, a functional requirement of Syk for cytotoxicity remained to be demonstrated. To assess whether Syk family tyrosine kinase activity is required for natural cytotoxicity, we used the PTK inhibitor piceatannol (38). Syk has previously been shown to be 10-fold more sensitive to piceatannol treatment than Lyn, an Src family member, in in vitro kinase assays (38). Pretreatment of NK cell clones with piceatannol inhibited natural cytotoxicity and FcR-initiated killing in a concentration-dependent fashion with half-maximal inhibition at a concentration of ∼6 μg/ml (Fig. 5). This 50% inhibitory concentration is consistent with the concentration that exhibits specific inhibition of Syk in vitro kinase activity. These results suggest that the tyrosine kinase activity of the Syk family is functionally required for natural cytotoxicity.


Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Brumbaugh KM, Binstadt BA, Billadeau DD, Schoon RA, Dick CJ, Ten RM, Leibson PJ - J. Exp. Med. (1997)

Piceatannol pretreatment inhibits NK cell– mediated cytotoxicity. NK cells  were treated for 15 min at 37°C  with the indicated concentration of piceatannol, washed, and  then incubated for 4 h with either 51Cr-labeled K562 cells or  51Cr-labeled P815 cells coated  with 0.15 μg/ml of the anti-FcR  mAb 3G8. Data are expressed as percentage of inhibition of lytic U/106  cells calculated from NK cells incubated in vehicle alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199178&req=5

Figure 5: Piceatannol pretreatment inhibits NK cell– mediated cytotoxicity. NK cells were treated for 15 min at 37°C with the indicated concentration of piceatannol, washed, and then incubated for 4 h with either 51Cr-labeled K562 cells or 51Cr-labeled P815 cells coated with 0.15 μg/ml of the anti-FcR mAb 3G8. Data are expressed as percentage of inhibition of lytic U/106 cells calculated from NK cells incubated in vehicle alone.
Mentions: Although the above data demonstrate biochemically that Syk is modified in NK cells after target cell stimulation, a functional requirement of Syk for cytotoxicity remained to be demonstrated. To assess whether Syk family tyrosine kinase activity is required for natural cytotoxicity, we used the PTK inhibitor piceatannol (38). Syk has previously been shown to be 10-fold more sensitive to piceatannol treatment than Lyn, an Src family member, in in vitro kinase assays (38). Pretreatment of NK cell clones with piceatannol inhibited natural cytotoxicity and FcR-initiated killing in a concentration-dependent fashion with half-maximal inhibition at a concentration of ∼6 μg/ml (Fig. 5). This 50% inhibitory concentration is consistent with the concentration that exhibits specific inhibition of Syk in vitro kinase activity. These results suggest that the tyrosine kinase activity of the Syk family is functionally required for natural cytotoxicity.

Bottom Line: Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function.Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk.These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

Show MeSH
Related in: MedlinePlus