Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of lysis of P388D1 (H-2Dd) targets correlates with  the level of expression of mouse Ly-49A on RNK-16 cells. The various  levels of Ly-49A expression are shown in FACS® histograms (A–D). Cells  were incubated with anti–Ly-49A (solid line) with FITC–goat anti–mouse  Ab (FITC–GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E–H) or YAC-1  targets (I–L). Effectors were wild-type RNK-16 cells (closed symbols) or  RNK-16 transfected with Ly-49A (open symbols). Effector cells used were  RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low  expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L).  Assays were carried out in the absence of antibody (squares), or in the  presence of anti-Ly-49A F(ab′)2 (diamonds) and control F(ab′)2 (antiNK1.1) (circles). The dotted line in F–H is the killing curve for wild-type  RNK-16 without mAb (from E for comparison).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196152&req=5

Figure 1: Inhibition of lysis of P388D1 (H-2Dd) targets correlates with the level of expression of mouse Ly-49A on RNK-16 cells. The various levels of Ly-49A expression are shown in FACS® histograms (A–D). Cells were incubated with anti–Ly-49A (solid line) with FITC–goat anti–mouse Ab (FITC–GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E–H) or YAC-1 targets (I–L). Effectors were wild-type RNK-16 cells (closed symbols) or RNK-16 transfected with Ly-49A (open symbols). Effector cells used were RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L). Assays were carried out in the absence of antibody (squares), or in the presence of anti-Ly-49A F(ab′)2 (diamonds) and control F(ab′)2 (antiNK1.1) (circles). The dotted line in F–H is the killing curve for wild-type RNK-16 without mAb (from E for comparison).

Mentions: To examine the intracellular signaling pathways that mediate the inhibitory function of mouse Ly-49A, we transfected mouse Ly-49A into RNK-16, a rat cell line with phenotypic and functional characteristics of rat NK cells (31). Nine RNK-mLy-49A clones expressing Ly-49A at different levels were obtained, three of which are represented in Fig. 1. Clones 2, 8, and 9 are representative of clones with low, medium, or high expression of Ly-49A, respectively (Fig. 1, B, C, D). Wild-type RNK-16 effector cells lysed P388D1 (H-2Dd) tumor cells (Fig. 1 E), but RNK-16 cells transfected with Ly-49A (RNK-mLy-49A cells) demonstrated reduced lysis of P388D1 (F, G, H). Inhibition of lysis of P388D1 cells was proportional to the level of Ly-49A expression on the RNK-16 transfectants. RNK-mLy-49A.2 had low expression and demonstrated only minimal inhibition of lysis (Fig. 1 F), whereas RNK-mLy49A.8 and RNK-mLy-49A.9, with progressively higher levels of expression, demonstrated marked inhibition of lysis (G and H). Inhibition of lysis in Ly-49A transfectants was reversible by the addition of anti–Ly-49A F(ab′)2 fragments, while control F(ab′)2 fragments (anti-NK1.1) had no effect. Addition of intact antibody had the same effect as F(ab′)2 fragments (data not shown). YAC-1 targets have previously been shown to be susceptible to lysis by mouse Ly-49A+ NK cells (6). YAC-1 target cells were lysed equally well by wild-type RNK-16 cells and by RNK-16 cells expressing Ly-49A at various levels, and lysis of YAC-1 targets was not altered by the presence of antibodies to Ly-49A (Fig. 1, I–L). These studies demonstrate that expression of Ly-49A renders RNK-16 cells ineffective in the killing of H-2Dd targets. The data indicate that the clonal RNK-mLy-49A transfectants behave similarly to freshly isolated murine Ly49A+ NK cells, and that they are a valid model in which to study Ly-49A function.


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)

Inhibition of lysis of P388D1 (H-2Dd) targets correlates with  the level of expression of mouse Ly-49A on RNK-16 cells. The various  levels of Ly-49A expression are shown in FACS® histograms (A–D). Cells  were incubated with anti–Ly-49A (solid line) with FITC–goat anti–mouse  Ab (FITC–GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E–H) or YAC-1  targets (I–L). Effectors were wild-type RNK-16 cells (closed symbols) or  RNK-16 transfected with Ly-49A (open symbols). Effector cells used were  RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low  expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L).  Assays were carried out in the absence of antibody (squares), or in the  presence of anti-Ly-49A F(ab′)2 (diamonds) and control F(ab′)2 (antiNK1.1) (circles). The dotted line in F–H is the killing curve for wild-type  RNK-16 without mAb (from E for comparison).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196152&req=5

Figure 1: Inhibition of lysis of P388D1 (H-2Dd) targets correlates with the level of expression of mouse Ly-49A on RNK-16 cells. The various levels of Ly-49A expression are shown in FACS® histograms (A–D). Cells were incubated with anti–Ly-49A (solid line) with FITC–goat anti–mouse Ab (FITC–GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E–H) or YAC-1 targets (I–L). Effectors were wild-type RNK-16 cells (closed symbols) or RNK-16 transfected with Ly-49A (open symbols). Effector cells used were RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L). Assays were carried out in the absence of antibody (squares), or in the presence of anti-Ly-49A F(ab′)2 (diamonds) and control F(ab′)2 (antiNK1.1) (circles). The dotted line in F–H is the killing curve for wild-type RNK-16 without mAb (from E for comparison).
Mentions: To examine the intracellular signaling pathways that mediate the inhibitory function of mouse Ly-49A, we transfected mouse Ly-49A into RNK-16, a rat cell line with phenotypic and functional characteristics of rat NK cells (31). Nine RNK-mLy-49A clones expressing Ly-49A at different levels were obtained, three of which are represented in Fig. 1. Clones 2, 8, and 9 are representative of clones with low, medium, or high expression of Ly-49A, respectively (Fig. 1, B, C, D). Wild-type RNK-16 effector cells lysed P388D1 (H-2Dd) tumor cells (Fig. 1 E), but RNK-16 cells transfected with Ly-49A (RNK-mLy-49A cells) demonstrated reduced lysis of P388D1 (F, G, H). Inhibition of lysis of P388D1 cells was proportional to the level of Ly-49A expression on the RNK-16 transfectants. RNK-mLy-49A.2 had low expression and demonstrated only minimal inhibition of lysis (Fig. 1 F), whereas RNK-mLy49A.8 and RNK-mLy-49A.9, with progressively higher levels of expression, demonstrated marked inhibition of lysis (G and H). Inhibition of lysis in Ly-49A transfectants was reversible by the addition of anti–Ly-49A F(ab′)2 fragments, while control F(ab′)2 fragments (anti-NK1.1) had no effect. Addition of intact antibody had the same effect as F(ab′)2 fragments (data not shown). YAC-1 targets have previously been shown to be susceptible to lysis by mouse Ly-49A+ NK cells (6). YAC-1 target cells were lysed equally well by wild-type RNK-16 cells and by RNK-16 cells expressing Ly-49A at various levels, and lysis of YAC-1 targets was not altered by the presence of antibodies to Ly-49A (Fig. 1, I–L). These studies demonstrate that expression of Ly-49A renders RNK-16 cells ineffective in the killing of H-2Dd targets. The data indicate that the clonal RNK-mLy-49A transfectants behave similarly to freshly isolated murine Ly49A+ NK cells, and that they are a valid model in which to study Ly-49A function.

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