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The Journal of Immunology, 2007, 178: 1261-1267.
Copyright © 2007 by The American Association of Immunologists, Inc.

Activating Ly-49 Receptors Regulate LFA-1-Mediated Adhesion by NK Cells1

Mohammed S. Osman, Deborah N. Burshtyn2 and Kevin P. Kane2

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors, and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions, yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore, we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1, and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors, leading to target cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are large granular lymphocytes that are important components of innate resistance to tumors and viruses (1, 2). NK cells mediate protective functions through cytolysis of virally infected cells or tumor cells, and by release of cytokines and chemokines (3, 4, 5). NK cells are controlled through a balance of signals generated by inhibitory and activating receptors, with response resulting from reduction of inhibitory signals or enhancement of activating signals (6).

NK cells express a variety of activating receptors that trigger cell-mediated cytotoxicity and cytokine release (6). Activating NK cell receptors of mouse that trigger cell-mediated cytotoxicity and/or cytokine release include Fc{gamma}RIII, natural cytotoxicity receptors, NKG2D, and activating Ly-49 receptors that directly recognize MHC class I molecules (6, 7, 8, 9, 10, 11, 12). Activating Ly-49 molecules are disulphide-linked homodimeric lectin-like receptors that contain an arginine residue in the transmembrane segment, which facilitates association with the signaling adapter protein DAP12 (13). DAP12 contains an ITAM, which recruits Syk family tyrosine kinases to trigger the cytolytic cascade and cytokine release (14).

NK cells also express LFA-1 ({alpha}Lbeta2), a beta2-integrin that binds ICAM-1-ICAM-5 and is important for adhesion to target cells (15, 16, 17). In various cell types, signals emanating from activating receptors regulate the affinity and/or avidity of LFA-1 ultimately resulting in increased binding of ICAM-1 (18, 19, 20). LFA-1 binding of ICAM-1 stabilizes the intercellular adhesion between cytotoxic cells and their targets promoting the delivery of cytotoxic granule contents toward susceptible targets (15, 21). Inside-out signaling producing firm LFA-1 mediated adhesion, for example, between CTLs and their targets, is triggered by signaling resulting from TCR engagement (19). In NK cells, LFA-1 provides adhesion and contributes an early activating signal that facilitates polarization of cytolytic granules toward target cells (15, 17, 21). Although the signaling by activating Ly-49 engagement has some similarities with that by the TCR, such as involvement of an ITAM and Syk family kinases, there are no reports to date demonstrating inside-out signaling by an NK cell activating receptor.

In this study, we show that Ly-49 activating receptors promote adhesion of NK cells through a DAP12-dependent inside-out regulation of LFA-1 binding to ICAM-1. Furthermore, we find that cross-linking with Ly-49 receptor-specific Abs can mimic cognate ligand interaction and trigger LFA-1 mediated NK cell binding to purified ICAM-1. Together, these results establish a role for Ly-49-activating receptors in regulating adhesion to target cells through LFA-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and cell lines

The following Abs were produced from the hybridomas as described (22) including A1 (IgG2a), anti-Ly-49A and anti-Ly-49P (23); B27M1 (IgG2a), anti-HLA-B7 (24); M1/42, anti-mouse H-2 (rat IgG2a) (25); 2.4G2 (rat IgG2b) anti-mouse FcR{gamma} (26); M17/5.2 (rat IgG2b) anti-mouse LFA-1 (27); and anti-mouse F4/80 Ag (rat IgG2b) (28). Purified G28, (IgG2a) anti-rat CD8{alpha}; WT.1, (IgG2a) anti-rat CD11a; and 4E5, (rat IgG2b), anti-Ly-49D FITC were purchased from BD Pharmingen. FITC-coupled rat anti-mouse IgG and mouse anti-rat IgG were purchased from Jackson ImmunoResearch Laboratories. Purified goat anti-mouse secondary Ab, goat anti-rat secondary Ab, and unmodified rat IgG were purchased from Sigma-Aldrich. Rabbit anti-DAP12 serum (14) was provided by Dr. D. McVicar (National Cancer Institute, Frederick, MD). The pharmacological inhibitors PP2 and piceatannol were purchased from Calbiochem, and reconstituted in DMSO then stored at 4°C. RNK-16, a spontaneous rat NK leukemia, was maintained as described (22, 29). The P815 mouse mastocytoma was maintained in RPMI 1640 supplemented with 5% FCS and 5 x 10–5 M 2-ME.

Flow cytometry

For the detection of cell surface Ly-49P, CD8{alpha}, or LFA-1, RNK or RNK transfectants (~0.3 x 106 cells/sample) were incubated with normal rat serum for 10 min at room temperature, then the indicated mAb was added (2 µg/ml) for 30–45 min on ice. Following the incubation, the cells were washed three times using 1x PBS, then FITC-coupled secondary rat anti-mouse IgG Abs were added, incubated for 20–30 min on ice, washed three times with 1x PBS, fixed with 4% p-formaldehyde in PBS, then analyzed using a FACScan flow cytometer. For the detection of Ly-49D on ex vivo NK cells, 4E5-FITC (2 µg/ml) was added to DX5-enriched NK cells preincubated with 2.4G2 (10 µg/ml, 10 min at room temperature) for 30 min on ice, washed three times with 1x PBS, then fixed and analyzed as previously described.

Mutagenesis of Ly-49P

Ly-49P (22) was mutated using the QuickChange Mutagenesis kit (Stratagene) to encode Leu for Arg57 in Ly-49P. The altered cDNA was verified by DNA sequencing and inserted into XhoI/XbaI sites of the BSR{alpha}EN vector, which was provided by Dr. A. Shaw (Washington University, St. Louis, MO). RNK-16 were transfected with wild-type or mutant Ly-49P cDNAs as described (22), to generate RNK-P and RNK-P(R57L).

Immunoblotting

A total of 1.5–2.0 x 107 cells was lysed in 1% Triton X-100 (w/v), 0.15 M NaCl, 20 mM Tris (pH 8) with protease inhibitors. Lysates were immunoprecipitated with A1 or B27M1 and protein G-agarose, separated by 12% SDS-PAGE under reducing conditions, and transferred to Immobilon-P. Membranes were blotted with rabbit antiserum and detected by chemiluminescence (Pierce).

Cytotoxicity assays

Target cells were labeled at 37°C with 100 µCi of Na51CrO4 (51Cr) for 1–2 h. Following washing, 1 x 104 51Cr-labeled targets were incubated for 4 h at 37°C in V-bottom microtiter plates with RNK-16, RNK-P, or Ly49P(R57L) at various E:T ratios in triplicate as described (22).

Animals

Female C57BL/6 mice at 6–8 wk of age were purchased from The Jackson Laboratory. Experiments were approved by the Animal Welfare and Policy Committee of the University of Alberta (Edmonton, Alberta, Canada).

Conjugate assays

The cell conjugate assays were performed as described (30), using red (PKH26) and green (PKH67) membrane linker dyes (Sigma-Aldrich). For assays in the presence of blocking mAbs, the Abs were prebound to soluble protein-A/G (1:1 ratio) for 15 min at room temperature, then added to RNK-16, RNK-P, or RNK-P(R57L) effector cells and incubated at 4°C for 10 min. Following incubation, labeled target cells were added to the effector cells preincubated with normal rat serum, then the number of tight conjugates was determined (30). Results represent the mean ± SD. For adhesion assays with ex vivo NK cells, mouse ICAM-1 was affinity-purified from mouse A20 cells using a YN-1/1.7.4 Sepharose column as described (31). NK cells were prepared using the Easy Sep DX5+ selection kit (StemCell Technologies) from RBC-depleted single cell suspensions of C57BL/6 spleens. Cells were stained for Ly-49D expression using 4E5-FITC Abs. DX5+ cells were treated with anti-Ly-49D FITC or anti-H2 mAbs (1 µg/106 cells), then mixed with cell-size beads immobilized with BSA or ICAM-1 (2 µg/107 beads) in RPMI 1640 or PBS containing 0.5 mM MgCl2 at an effector to bead ratio of 2:1, and centrifuged at 30 x g for 3 min. Cell to bead mixtures treated with mAbs were cross-linked using goat anti-rat secondary Ab (0.5 µg/ml) for 2.5 min, then fixed with 1% p-formaldehyde. Ly-49D+ cell conjugates were determined as the percentage of DX5+ to Ly49D+ cells shifted to a side light scatter corresponding to that of the beads (see Fig. 4A).


Figure 4
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  		FIGURE 4. Cross-linking of Ly-49P leads to DAP12 and Src/Syk kinase-dependent up-regulation of LFA-1 binding to isolated ICAM-1. A, RNK-16, RNK-P, or RNK-P(R57L) were prelabeled with 51Cr, and the Fc receptors were blocked with 10 µg/ml normal rat serum. Anti-Ly49P (A1) or anti-CD8{alpha} mAbs (5 µg/ml) were mixed with the cells, which were then added to the wells immobilized with mouse ICAM-1 (1 µg/ml), or 2% FCS containing goat anti-mouse secondary Ab (g{alpha}m IgG, 0.5 µg/ml), as indicated. B, RNK or RNK-P cells were incubated for 30 min at 37°C in the absence or presence of PP2 or piceatannol at the indicated concentrations, and then used as in A. Results represent mean ± SD of each triplicate sample. Data are representative of three (A) or two (B) independent experiments.

 
Plate ICAM-1 adhesion assay

Purified ICAM-1 was immobilized on flat-bottom microtiter plates at 1 µg/ml overnight at 4°C in PBS supplemented with 900 µM CaCl2 and 500 µM MgCl2. RNK-16, RNK-P, or RNK-P(R57L) cells were labeled with 51Cr, incubated with normal rat serum for 10 min at room temperature, then with 5 µg/ml A1 or isotype control Abs (as indicated) for 10 min at room temperature, and the cells (1.1 x 105) were added to immobilized ICAM-1 or 2% FCS, with or without 0.5 µg/ml goat anti-mouse secondary Ab for 30 min at 37°C. At the end of incubation, an aliquot was harvested to determine spontaneous release, then unbound cells were removed by pipetting shear force, and the number of bound cells determined by beta counting. In all adhesion experiments, cell binding was calculated as the percentage of specific cell binding by the formula 100 x ((bound counts)/(total counts – spontaneous release)), with results representing the mean ± SD of each triplicate. For experiments using pharmacological inhibitors, 51Cr-labeled RNK-P cells were pretreated with the indicated concentration of the inhibitor or DMSO alone for 30 min at 37°C. Following incubation with either inhibitor, the cells were treated as described with normal rat serum and A1, then added to immobilized ICAM-1-immobilized wells containing goat anti-mouse secondary Ab for 30 min at 37°C. Subsequently, the cells were harvested as previously described, and the number of bound cells was determined by beta counting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ly-49P-dependent lysis requires LFA-1

To study the potential role of activating Ly-49 receptors in triggering NK cell adhesion to target cells, we expressed the NOD activating Ly-49P on rat RNK-16 leukemia cells by transfection and ensured that the transfectants expressed similar levels of CD11a and CD8{alpha} as parental cells (Fig. 1A, top). To monitor the expression of Ly-49P on the surface of RNK-16 cells, we used the A1 mAb that we have previously shown to recognize both Ly-49A and Ly-49P (22). RNK cells transfected with Ly-49P (RNK-P) expressed uniform levels of Ly-49P as expected, whereas no mouse Ly-49P expression was detected on the untransfected rat cells (Fig. 1A, bottom). We used P815 mastocytoma cells as target cells for the Ly-49P transfectants because they express endogenous H-2Dd and ICAM-1, ligands for Ly-49P and LFA-1, respectively (Fig. 1A, right). The P815 target cells were efficiently lysed by RNK-P effector cells, but not parental RNK-16 (Fig. 1B). These results indicate that lysis of target cells is dependent on Ly-49P recognition of H-2Dd expressed by P815. Lysis of P815 was blocked by Abs to Ly-49P, as well as LFA-1 (Fig. 1C) and ICAM-1 (Fig. 1D), but not by isotype control Abs (Fig. 1, C and D), indicating that by interacting with their respective ligands, Ly-49P and LFA-1 both contribute to P815 lysis.


Figure 1
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  		FIGURE 1. Ly-49P and LFA-1 control cytolysis by RNK-P cells. A, Expression of receptors and ligands on RNK-16, RNK-P, and P815 cells. RNK-16 or RNK-P cells were stained with anti-rat CD11a (dark gray histogram), anti-rat CD8{alpha} (light gray histogram) (top) or anti-Ly-49P (filled histogram) (bottom). P815 cells were stained for H-2Dd (light gray histogram) or ICAM-1 (filled histogram) (right). Corresponding isotype controls are unshaded. B, RNK-16- or RNK-P-mediated cytotoxicity was measured by 51Cr release from P815-labeled target cells. C and D, Ly-49 and LFA-1 both contribute to RNK-P lysis of P815 cells. Cytotoxicity was measured in the presence or absence of the indicated Ab protein-A/G complexes at an E:T ratio of 12.5:1. Error bars indicate SD for each of triplicate samples.

 
Ly-49P promotes adhesion to susceptible target cells in a LFA-1-dependent manner

Studies using NK cells have demonstrated that tight adhesion is an important step preceding cytotoxicity (15). Therefore, we tested whether Ly-49P promotes formation of conjugates between effector cells and susceptible targets, using two-color flow cytometry (30). RNK-P, but not parental RNK-16, rapidly formed tight conjugates with P815, with maximum heteroconjugates detected by 10 minutes (Fig. 2A). Cell adhesion was dependent on engagement of Ly-49P with H-2Dd, as blocking this interaction significantly reduced the number of tight conjugates (Fig. 2B). The tight cell adhesion was also dependent on interactions between LFA-1 and ICAM-1 as Abs to these molecules also reduced the number of NK cell conjugates (Fig. 2, B and C). Thus, Ly-49P-mediated recognition regulates cell binding in a LFA-1-ICAM-1-dependent manner and promotes cytolysis of susceptible target cells.


Figure 2
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  		FIGURE 2. Ly-49P regulates adhesion of RNK cells to P815 cells in a LFA-1/ICAM-1-dependent manner. A, RNK or RNK-P cells were labeled with a red membrane linker dye, PKH26, then incubated with P815 cells labeled with the green dye, PKH67, for the indicated times at an E:T of 1:2. The percentage of NK cells in conjugates was determined using two-color flow cytometry. B and C, Ly-49P facilitates tight adhesion to target cells that is LFA-1/ICAM-1-dependent. Anti-LFA-1 (1.25 µg/ml), anti-Ly-49P (A1, 2.5 µg/ml), control anti-CD8{alpha} (2.5 µg/ml), or isotype control (B27M1, 1.25 µg/ml) Abs were preincubated with RNK-P cells. Alternatively, anti-ICAM-1 or anti-F4/80 (5 µg/ml) were incubated with P815 cells preincubated with 2.4G2 (10 µg/ml). Conjugate assays were conducted at the 10 min time point. For samples analyzed at the 0 min time point, cells were immediately fixed then analyzed for heteroconjugates. Data are representative of at least three independent experiments. Results represent mean ± SD of triplicate samples.

 
Ly-49P-mediated adhesion is DAP12 dependent

The interaction between inhibitory Ly-49 and MHC class I is sufficient to mediate binding of cells to ligands on plates or on other cells (32, 33). To distinguish the contribution of direct binding of H-2Dd by Ly-49P toward tight cell to cell adhesion, from Ly-49P-mediated signaling events that alter LFA-1 binding, we constructed a Ly-49P mutant with an amino acid substitution at position 57, where the positively charged Arg was replaced with Leu and stably expressed this receptor on RNK-16 cells by transfection. This mutation, R57L, abrogates the ability of the receptor to associate with DAP12, and in turn its ability to signal following ligand recognition, but still allow Ly-49P(R57L) surface expression (13). RNK-P(R57L) cells expressed similar levels of Ly-49P using the A1 mAb, as well as CD11a, and CD8{alpha} as RNK-P cells (Fig. 3A and data not shown). However, the DAP12 signaling adapter co-immunoprecipitates with Ly49P but not Ly-49P(R57L) from the RNK transfectants (Fig. 3B). Furthermore, RNK-P(R57L), unlike RNK-P, was unable to lyse P815 (Fig. 3C), despite retaining the ability to lyse YB2/0 directly and EL-4 cells by Ab-dependent cellular cytotoxicity via CD16 (data not shown). Importantly, the tight adhesion typically observed between RNK-P and P815 was instead negligible between RNK-P(R57L) and P815 and similar to background levels obtained with untransfected RNK-16 (Fig. 3D). These results indicate that the ligand binding capacity of Ly-49P does not significantly increase adhesion to H-2Dd bearing target cells on its own, and instead suggests that Ly-49P promotes tight binding to susceptible target cells through DAP12-dependent signals.


Figure 3
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  		FIGURE 3. DAP12 association with Ly-49P is required for RNK-P cell adhesion to P815 target cells. A, RNK-P (black line histogram) or RNK-P(R57L) (gray line histogram) cells were stained with A1 or isotype-matched control (filled histogram) followed by secondary rat anti-mouse Abs. B, DAP12 association with Ly-49P but not Ly-49P(R57L). Precleared cell lysates were immunoprecipitated using A1 or isotype control (B27M1), and blotted with an anti-DAP12 serum. Cytolysis of 51Cr-labeled P815 targets (C) and adhesion to P815 targets (D) are induced by Ly-49P but not Ly-49P(R57L). Error bars indicate SD for each of triplicate samples. The percentage of NK cell conjugates was determined at an E:T of 1:2 using two-color flow cytometry for the indicated incubation periods. Results are representative of three independent experiments.

 
Cross-linking of Ly-49P leads to DAP12-dependent inside-out signals regulating LFA-1 binding to mouse ICAM-1

To directly test whether Ly-49P could trigger changes in LFA-1 binding to ICAM-1 we examined binding of RNK-P to plate-bound mouse ICAM-1, upon stimulation of Ly-49P. To mimic signals generated by Ly-49P recognition of H-2Dd, we cross-linked Ly-49P, Ly-49P(R57L), or CD8{alpha} expressed on RNK-16 or its transfectants with primary Abs specific for Ly-49P or rat CD8{alpha}, with goat anti-mouse secondary Ab, then determined cell binding to mouse ICAM-1. Little binding was evident to wells immobilized with serum, or following CD8{alpha} cross-linking (Fig. 4A). RNK-P, but not untransfected RNK-16 or RNK-P(R57L), bound to immobilized ICAM-1 when stimulated by anti-Ly-49P (A1) and secondary Abs (Fig. 4A). Furthermore, cross-linking with secondary Abs was required (data not shown), suggesting that activating Ly-49 clustering may promote events leading to induced ICAM-1 binding. RNK transfectants stably expressing the activating Ly-49D receptor also bound immobilized ICAM-1 following cross-linking with anti-Ly-49D and secondary Abs (data not shown). Therefore, engagement of activating Ly-49 receptors on RNK-16 cells triggers DAP12-dependent LFA-1 binding to ICAM-1. Furthermore, RNK-P triggered binding to ICAM-1 required both Syk and Src family kinase (SFK)3 activities as the specific inhibitors piceatannol and PP2, respectively, significantly inhibited binding of RNK-P cells to ICAM-1 in a dose-dependent manner (Fig. 4B).

Ex vivo NK cells regulate LFA-1 binding to ICAM-1 via Ly-49D

To determine whether triggering of LFA-1 binding to ICAM-1 is a property of an activating Ly-49 receptor expressed by normal mouse NK cells, we developed a flow cytometry based ICAM-1 binding assay using resting ex vivo NK cells. NK cells enriched from C57BL/6 spleen by DX5 mAb selection, of which 25–30% express the activating Ly-49D receptor (34), were stimulated with anti-Ly-49D or an anti-H-2 control and secondary Ab in the presence of ICAM-1 or BSA beads. The number of DX5+/Ly-49D+ NK cells of each experimental group bound to the beads immobilized with ICAM-1 or BSA was determined, as diagrammed (Fig. 5A). The ICAM-1 was efficiently displayed on the cell size beads as detected by flow cytometry with the YN1/1.7.4 Ab (Fig. 5B), and beads were easily distinguished from NK cells due to very different forward and side scatter properties (Fig. 5B). Conjugates between beads and DX5+/Ly49D+ NK cells were readily detected by a shift to enhanced side scatter of gated DX5+/Ly-49D+ cells, as a consequence of bead binding (Fig. 5C). Whereas only 4% of Ly-49D+ NK cells stimulated with the control anti-H2 Ab bound ICAM-1 beads, ~23% of Ly-49D+ NK cells bound ICAM-1 beads upon stimulation by Ly-49D engagement (Fig. 5, C and D). These results show that the specific engagement of an activating Ly-49 NK cell receptor expressed on ex vivo NK cells up-regulates NK cell adhesion to ICAM-1. Furthermore, the adhesion to ICAM-1 by the ex vivo NK cells was mediated by LFA-1 as Abs to this receptor reduced NK cell binding to ICAM-1 beads to background levels (Fig. 5D). These results demonstrate that LFA-1 adhesion to ICAM-1 can be up-regulated on normal NK cells, and by a naturally expressed activating Ly-49 receptor.


Figure 5
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  		FIGURE 5. Cross-linking of Ly-49D induces ex vivo NK cells to bind ICAM-1 via LFA-1. A, Procedure to measure Ly-49D-induced LFA-1 binding to isolated ICAM-1 with primary cells. Erythrocyte-depleted splenocytes from C57BL/6 mice were enriched for DX5+ cells, then treated as indicated. B, ICAM-1-immobilized beads express a uniform level of ICAM-1, and have a distinct side light scatter (SSC) profile from cells. Cell-sized Sepharose beads were immobilized with either BSA (top left) or mouse ICAM-1 (bottom left), blocked with BSA, then stained for mouse ICAM-1 using specific anti-mouse ICAM-1 (filled histogram) or isotype control (open histogram) mAbs, followed by staining with secondary FITC goat anti-rat secondary mAbs. Forward and side light scatter (FSC and SSC) profiles of beads (top right) or cells (bottom right). C and D, Ly-49D cross-linking results in a LFA-1-dependent increase in cellular adhesion to ICAM-1 beads. DX5-enriched cells were treated as outlined in A, then the percentage of adhesion of DX5+/Ly-49D+ cells was determined by first gating on the PE-FITC-positive cells as shown in C, then calculating the percentage of cell/bead heteroconjugates based on forward and side light scatter histogram profiles in the presence or absence of anti-LFA-1 (M17/5.2) or isotype control (F4/80) mAbs (5 µg/ml). Data in D represent the mean percentage ± SD of cell binding for triplicate samples. Results are representative of three (C) or two (D) independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that engagement of activating Ly-49 molecules stimulates NK cell binding to ICAM-1. Triggering LFA-1 adhesion by DAP12 coupled receptors is likely to play an important role in how NK cells recognize target cells. Our demonstration of activating Ly-49s triggering LFA-1 binding adds a new dimension to our understanding of how these receptors stimulate cytolysis. NK cell LFA-1 binding to target cell ICAM-1 is known to be an important and likely early event in NK cell recognition of a target cell (21). Activating Ly-49 engagement may precede or more likely follow initial low affinity LFA-1 interactions with ICAM-1, but once engaged, activating Ly-49 then significantly up-regulate LFA-1 binding to ICAM-1. In turn, the substantially strengthened interaction with target cells will greatly enhance opportunities for increased interaction of activating Ly-49 and other low affinity activating NK cell receptors to engage their ligands, amplifying NK cell activation signals. Furthermore, LFA-1 engagement by ICAM-1 is known to induce cytolytic granule polarization toward target cells (21). Up-regulation of LFA-1 binding to ICAM-1 through activating Ly-49 engagement may also promote this event, which combined with amplified activation signals, may lead to rapid target cell cytotoxicity. Thus, activating Ly-49 receptors may act as sensors to amplify signals for adhesion and activation.

It is well established that multiple distinct receptors expressed on NK cells cooperate to induce cytolytic function and release of cytokines (7, 21, 35, 36). In fact, resting human ex vivo NK cells appear to require engagement of at least two independent receptors to become activated (37). These same investigators also demonstrated that when LFA-3 or CD48, ligands for CD2 and 2B4, respectively, are coexpressed on target cells with ICAM-1, a ligand for LFA-1, this led to enhanced adhesion of resting NK cells, significantly beyond that observed to target cells expressing only ICAM-1 (38). Furthermore, in the case of LFA-1 expressed on human NK cells, it also can physically associate with DNAM-1, another adhesion and signaling receptor and regulate its phosphorylation state and ability to induce NK cell cytotoxicity (39). Although NK cell receptors clearly cooperate and may even associate, it has not previously been demonstrated that one defined NK activating cell receptor can regulate the adhesive function of another NK cell receptor for its ligand.

Human NK cells can bind ICAM-1 directly with no direct stimulus, albeit to a much lesser extent for resting human NK cells, therefore it has been proposed that NK cells may be unique (38), and unlike T cells, in not requiring stimulation through an independent activating receptor before LFA-1 can bind tightly to ICAM-1. We did not observe significant binding of resting mouse NK cells or the rat RNK-16 cell to ICAM-1 in the absence of stimulation via an activating receptor. We do not know why there are apparent differences in regulation and/or activation state of human and mouse LFA-1 on NK cells, however, our results show that LFA-1 adhesion can be up-regulated by mouse Ly-49 activating NK cell receptors, similar to LFA-1 regulation by T cells. It remains to be determined whether LFA-1 adhesion is regulated in a similar fashion on resting human NK cells. However, given that LFA-1-mediated adhesion in human NK cells can be negatively regulated by inhibitory receptors, it seems likely that LFA-1 adhesion is modulated in human NK cells as well (30, 40).

We showed a requirement for DAP12 association in activating Ly-49 regulation of LFA-1 ligand binding activity, indicating that ITAM-dependent signals are initiating Ly-49 triggered "inside-out" signaling for LFA-1 regulation. Regulation of LFA-1 adhesion is a complex process and Ly-49 may alter LFA-1 avidity through changes in LFA-1 membrane mobility or the affinity of LFA-1 for ICAM-1 (41). Signaling for LFA-1 regulation in lymphocytes is only partially understood, but a few key components known to be involved in TCR-induced LFA-1 regulation are also expressed by mouse and rat NK cells, e.g., linker for activation of T cells (LAT) (42, 43), leukocyte protein SLP-76 (44), and ADAP (45). TCR signaling uses LAT to recruit and activate SLP-76 thereby linking to degranulation-promoting adapter protein ADAP, with ADAP specifically required for LFA-1 clustering (reviewed in Ref. 46). The Ly-49-DAP12 receptor complex in NK cells uses LAT, but in the absence of LAT, the adapter LAB, which is not expressed in T cells, is recruited to trigger cytotoxicity (47). Given that LAT and LAB have some overlapping but many distinct activities, NK cells may have redundancy or greater flexibility in LFA-1 regulation, compared with T cells, which may exclusively use LAT. Our data indicate that Ly-49-induced regulation of LFA-1 binding to ICAM-1 requires SFK and Syk tyrosine kinase activities as the binding of RNK-P cells to ICAM-1 was effectively inhibited with doses of PP2 and piceatannol within their respective IC50 values (2 and 10 µM, respectively) in RNK-16 cells. We observe a modest increase in binding to ICAM-1 using drug doses lower than the IC50 values for which we presently have no obvious explanation. The Syk and SFKs have been shown to play important roles in the regulation of Ly-49D-dependent responses (14, 48, 49). SFKs have been shown to play a pivotal role in DAP12 phosphorylation (49) and may promote formation and stability of ADAP/SKAP-55/SLP-76 complexes (45, 50, 51). Similarly, SFKs may modulate LFA-1 binding to ICAM-1 following Ly-49/DAP12 induction by stimulating the activation of Syk kinases directly, and RAP-1 indirectly (49, 52, 53). Interestingly, NKG2D also associates with DAP12 and DAP10 in mouse NK cells (54, 55), and it was very recently shown that the NKG2D-dependent elimination of certain tumor cells requires LFA-1 recognition of ICAM-1 (56). It remains to be determined whether NK receptors other than Ly-49s, which also associate with ITAM-containing signaling adapters, or receptors such as NKG2D that can associate with distinct signaling adapters, also regulate LFA-1 adhesive properties.

In summary, we reveal a novel aspect of the regulation of NK cell effector functions by demonstrating that activating Ly-49 receptors up-regulate NK cell binding to ICAM-1 through a DAP12-dependent mechanism. The Ly-49-driven mobilization of LFA-1 binding capacity likely represents an important proximal event leading to the eventual death of a target cell.


   Acknowledgments
 
We thank Dr. D. McVicar for providing the anti-DAP12 serum, and Andy Kokaji (Medical Microbiology and Immunology Department, University of Alberta, Edmonton, Alberta, Canada) for providing purified mouse ICAM-1.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Canadian Institutes of Health Research grants (to D.N.B. and K.P.K.). D.N.B. is an Alberta Heritage Foundation for Medical Research senior scholar and K.P.K. is an Alberta Heritage Foundation for Medical Research scientist. M.S.O. was supported by an Alberta Heritage Foundation for Medical Research studentship. Back

2 Address correspondence and reprint requests to Dr. Deborah N. Burshtyn, Department of Medical Microbiology and Immunology, 6-59 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada; E-mail address: burshtyn{at}ualberta.ca or Dr. Kevin P. Kane, Department of Medical Microbiology and Immunology, 660 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada; E-mail address: kevin.kane{at}ualberta.ca Back

3 Abbreviations used in this paper: SFK, Src family kinase; LAT, linker for activation of T cell. Back

Received for publication August 22, 2006. Accepted for publication October 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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