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Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

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Ly-49A expression on Ly-49A+ and Ly-49A− LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into  Ly-49A+ and Ly-49A− populations by panning with anti–Ly-49A Ab.  Ly-49A− cells were additionally treated with rabbit anti–mouse Ab and  complement depletion. FACS® analysis was performed on day 9 LAK cells  using FITC–anti-Ly-49A (A1). Staining was performed in the presence of  unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 μg/106  cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS®  histograms (A–F). Dotted lines represent cells incubated with saline; solid  lines represent FITC–anti-Ly-49A staining.
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Figure 6: Ly-49A expression on Ly-49A+ and Ly-49A− LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into Ly-49A+ and Ly-49A− populations by panning with anti–Ly-49A Ab. Ly-49A− cells were additionally treated with rabbit anti–mouse Ab and complement depletion. FACS® analysis was performed on day 9 LAK cells using FITC–anti-Ly-49A (A1). Staining was performed in the presence of unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 μg/106 cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS® histograms (A–F). Dotted lines represent cells incubated with saline; solid lines represent FITC–anti-Ly-49A staining.

Mentions: The partial function of Ly-49A in mev/mev mice could reflect the residual activity of SHP-1 in these mice. To examine the effect of the complete absence of SHP-1, we next examined Ly-49A function in IL-2–activated NK cells isolated from me/me mice. Using spleen cells harvested from me/me mice (sacrificed just before their natural demise), +/me littermate heterozygote controls, and wild-type +/+ mice, we isolated Ly-49A+ and Ly-49A− cells. As shown in Fig. 6 (B, D, F), Ly-49A expression was equivalent on cells isolated from all mice. Ly-49A− cells (Fig. 6, C and E) contained <5% Ly-49A+ cells, whereas the +/+ Ly-49A− population (A) contained ∼10% Ly-49A+ cells. >95% of all cells were positive for NK1.1 and negative for CD3 by FACS® (data not shown).


Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Ly-49A expression on Ly-49A+ and Ly-49A− LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into  Ly-49A+ and Ly-49A− populations by panning with anti–Ly-49A Ab.  Ly-49A− cells were additionally treated with rabbit anti–mouse Ab and  complement depletion. FACS® analysis was performed on day 9 LAK cells  using FITC–anti-Ly-49A (A1). Staining was performed in the presence of  unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 μg/106  cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS®  histograms (A–F). Dotted lines represent cells incubated with saline; solid  lines represent FITC–anti-Ly-49A staining.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196152&req=5

Figure 6: Ly-49A expression on Ly-49A+ and Ly-49A− LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into Ly-49A+ and Ly-49A− populations by panning with anti–Ly-49A Ab. Ly-49A− cells were additionally treated with rabbit anti–mouse Ab and complement depletion. FACS® analysis was performed on day 9 LAK cells using FITC–anti-Ly-49A (A1). Staining was performed in the presence of unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 μg/106 cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS® histograms (A–F). Dotted lines represent cells incubated with saline; solid lines represent FITC–anti-Ly-49A staining.
Mentions: The partial function of Ly-49A in mev/mev mice could reflect the residual activity of SHP-1 in these mice. To examine the effect of the complete absence of SHP-1, we next examined Ly-49A function in IL-2–activated NK cells isolated from me/me mice. Using spleen cells harvested from me/me mice (sacrificed just before their natural demise), +/me littermate heterozygote controls, and wild-type +/+ mice, we isolated Ly-49A+ and Ly-49A− cells. As shown in Fig. 6 (B, D, F), Ly-49A expression was equivalent on cells isolated from all mice. Ly-49A− cells (Fig. 6, C and E) contained <5% Ly-49A+ cells, whereas the +/+ Ly-49A− population (A) contained ∼10% Ly-49A+ cells. >95% of all cells were positive for NK1.1 and negative for CD3 by FACS® (data not shown).

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Show MeSH
Related in: MedlinePlus