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*L-TYROSINE
The Journal of Immunology, 2003, 171: 2889-2895.
Copyright © 2003 by The American Association of Immunologists

Activating and Inhibitory Ly49 Receptors Modulate NK Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 10 and CXCL12 1

Marit Inngjerdingen2,*, Bent Rolstad{dagger} and James C. Ryan*

* Department of Arthritis and Immunology, Veterans Affairs Medical Center, Northern California Institute for Research and Education, and University of California, San Francisco, CA 94121; and {dagger} Department of Anatomy, University of Oslo, Oslo, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-Dd, led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-Dd led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune recognition of neoplastic or virally infected cells by NK lymphocytes is controlled by a compendium of NK receptors that activate or inhibit target cell killing (1). In rodents, these receptors include functionally divergent members of the Ly49 family (2, 3, 4). Mouse Ly49A is an inhibitory NK receptor that binds to some target MHC class I (MHC I) 3 molecules, such as H2-Dd (5, 6), recruiting inhibitory NK cell tyrosine phosphatases that prevent target killing (7, 8). Conversely, Ly49D can recruit cytoplasmic protein tyrosine kinases and can activate the killing of targets expressing H2-Dd (9, 10, 11, 12), and xenogeneic MHC ligands (13). These activating and inhibitory Ly49 receptors contribute to self-nonself discrimination, allowing NK cells to kill foreign or abnormal targets, while preventing the killing of normal cells expressing a full complement of self MHC I molecules (14). Activating and inhibitory Ly49 receptors that recognize the same MHC ligand are commonly expressed as pairs on NK cells, and Ly49D is found coexpressed with either Ly49A or the inhibitory Ly49G2 receptor (15).

NK cells can migrate into target sites within an affected animal, but the in vivo NK cell migration mechanisms and patterns are incompletely understood. However, NK cells can traffic in response to chemokines, small chemotactic cytokines that regulate the recruitment of leukocytes. Most chemokines are secreted during host inflammatory responses and are categorized as inflammatory chemokines, while some chemokines are involved in tissue homeostatic processes such as hemopoiesis, tissue homing, and embryogenesis (16). These latter chemokines are known as homeostatic chemokines. The proinflammatory chemokine CXC chemokine ligand (CXCL)10 (IFN-{gamma}-inducible protein-10), which binds to its receptor CXCR3, is involved in certain inflammatory conditions, such as CMV and hepatitis C infections (17, 18), and is reported to induce NK cell migration (19, 20). CXCL12 (stromal cell-derived factor-1{alpha}, which binds CXCR4, is a homeostatic chemokine that is chemoattractant for NK cells and may direct the homing of NK cells to the bone marrow (21, 22).

Limited experimental data suggest that specific subsets of NK cells preferentially migrate to effector sites (23, 24). NK subsets expressing Ly49D have been shown to elicit efficient graft rejection (12). In murine CMV, a subset of virus-specific NK cells enriched in the activating Ly49 receptors Ly49D and -H, preferentially migrates to the peritoneum or liver of infected mice (25, 26). A system allowing for the chemotaxis of specific NK subsets to sites of inflammation would be advantageous to an infected host, as only Ly49H+ NK cells are capable of killing murine CMV-infected targets (27). The expression of chemokine receptors in these models was not examined, and it is currently not known whether chemokine receptors are homogeneously expressed on all rodent NK cells or on NK-specific subsets. We speculated that the differential recruitment of NK cell-specific Ly49+ subsets to sites of ongoing viral infection might reflect modulation of chemotaxis by Ly49 receptors themselves.

Recent studies on T and B cells suggest that stimulation through the Ag receptors affects cell migration in response to chemokines, thus suggesting a cross-talk between these receptors and chemokine receptors. As rodent NK cells normally express a compendium of different activating and inhibitory Ly49 receptors, we chose to perform these studies on the rat NK cell line RNK16. RNK16 cells are phenotypically homologous to naive NK cells, and kill effectively NK-sensitive targets. We used RNK16 cells stably transfected with single mouse Ly49 receptors, enabling the study of one NK receptor in isolation. In the current study, we examined whether activating and inhibitory Ly49 receptors can differentially modulate NK cell migration in response to CXCL12 and CXCL10. We found that ligation of the inhibitory Ly49A receptor, which normally inhibits the killing of target cells expressing self MHC I, prevented NK cell migration in response to the CXCL10 and CXCL12 chemokines. In contrast, ligation of the activating Ly49D receptor augmented chemotaxis toward CXCL10, but inhibited chemotaxis toward CXCL12. These data suggest that NK cell target receptors may modulate chemotactic signals, enabling the preferential migration of NK cell subsets toward inflammatory chemokines, but not toward homeostatic chemokines, following activating Ly49 receptor ligation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Mouse chemokines CXCL12 (stromal cell-derived factor-1{alpha}) and CXCL10 (IFN-{gamma}-inducible protein-10) were purchased from PeproTech (Rocky Hill, NJ). Polyclonal goat Abs against CXCR3 (Y-16) and CXCR4 (G-19) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). mAbs to Ly49A (A1, mouse IgG2a) and Ly49D (12A8, rat IgG2a, cross-reactive with Ly49A) were produced from their respective hybridoma cell lines. mAb to H2-Dd-{alpha}3 (34-2-12S) was from BD PharMingen (San Diego, CA). Control Abs were goat IgG, mouse IgG (both from Sigma-Aldrich, St. Louis, MO), and rat IgG2a (generated in our laboratory).

Cell lines

RNK16, a rat leukemic NK cell line, and the rat myeloma cell line YB2/0 were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µM 2-ME (complete RPMI (cRPMI)). RNK16 stably transfected with Ly49A, Ly49D, or Ly49A-Y8F, and the YB2/0 transfectant YB2/0-Dd were kept in cRPMI, with the addition of 1 mg/ml G418 (Boehringer Mannheim, Indianapolis, IN) as previously described (8, 9). Transfected cells were cultured for at least 2 days in medium without G418 before experiments. There was no difference in the cell surface expression of CXCR4 or CXCR3 among the different RNK16 clones used in this study.

Construction of rat CXCR3

CXCR3 was cloned from a rat cDNA library and inserted into the EcoRI cloning site of the expression vector pEF-Bos-HA (kindly provided by K. Taskén, University of Oslo, Oslo, Norway) containing an N-terminal HA tag. Primers used for the cloning were 5'-ctcgaggaattcatgtaccttgaggtcagtgaa-3' (forward) and 5'-ctcgaggaattcttacaagcccaagtaggaggc-3' (reverse). Correct cloning of CXCR3 was verified by sequencing. Three micrograms of CXCR3 were transiently transfected into 293T cells using Lipofectamine (Invitrogen, Carlsbad, CA). Cells were tested for CXCR3 expression by flow cytometry 24 h post-transfection.

RT-PCR

mRNA expression of CXCR3 in RNK16 cells was analyzed by RT-PCR. Total RNK16 RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. One microgram of RNA was reversibly transcribed with oligo(dT)15 primer and 200 U Moloney murine leukemia virus reverse transcriptase (RT) supplied in a kit from Promega (Madison, WI). The resulting cDNA was subjected to PCR using the primers for CXCR3 described above.

Stimulation of cells

For Ab cross-linking experiments, RNK16.Ly49A, RNK16.Ly49A-Y8F, or RNK16.Ly49D cells (2 x 106 cells/ml) were incubated with 12A8 F(ab')2 or A1 mAb (1 µg/1 x 106 cells) for 30 min at 4°C. After a quick spin, cross-linking goat anti-rat or goat anti-mouse F(ab')2 Abs (1 µg/1 x 106 cells) were added at 37°C for the indicated times. Cells were washed, and resuspended in IMDM containing 0.5% BSA. For target cell stimulation of RNK16 cells, YB2/0 cells were prefixed with 4% formaldehyde in PBS for 15 min at room temperature, washed twice, and resuspended in RPMI 1640 supplemented with 2% FCS. Different RNK16 transfectants were mixed with YB2/0 cells at a 3:1 ratio, pelleted for 15 s at 1000 rpm, and incubated at 37°C for the indicated times, washed, and resuspended in IMDM with 0.5% BSA. The Syk kinase inhibitor Piceatannol (Calbiochem, La Jolla, CA) was incubated with RNK16 cells at 10 µM in 10% FCS in RPMI 1640 for 1 h at 37°C, and was kept with the cells while cross-linking with Abs.

Chemotaxis assays

Chemotaxis of RNK16 cells was assayed using a 48-well Transwell chemotaxis chamber, with 8 µm pore-sized polyvinylpyrrolidone-free filters (NeuroProbe, Gaithersburg, MD). The bottom chambers were filled with 28 µl of IMDM containing 0.5% BSA with or without CXCL10 or CXCL12 (100 ng/ml). RNK16 cells (1 x 105) in 50 µl were loaded in the upper chambers. After a 2-h incubation at 37°C and 5% CO2, transmigrated cells in the bottom chambers were harvested and counted in a Bürker chamber. All experiments were performed in triplicate.

Flow cytometry

Cell surface staining for receptor expression was performed with primary Abs at 1 µg/1 x 106 cells, and secondary FITC-conjugated Abs at 1 µg/1 x 106 cells. To determine changes in chemokine receptor surface levels after Ly49 ligation, RNK16 cells were prestained with 50 nM of the intracellular dye CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. The cells were then incubated at 37°C with prefixed YB2/0-Dd cells at the indicated times, and surface expression of CXCR3 or CXCR4 on the CFSE-stained RNK16 cell population was determined using primary Abs toward CXCR3 and CXCR4 and a PE-conjugated secondary Ab. All analyses were performed using a FACScan (BD Biosciences, San Jose, CA).

Statistical analysis

Data were analyzed using the unpaired Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chemotaxis is modulated by Ly49 receptor cross-linking

The effects of Ly49 ligation on chemotaxis were studied using the rat NK leukemic cell line RNK16. The expression of chemokine receptors by rat NK cells has not been previously investigated. Therefore, we initially determined whether RNK16 cells express functional CXCR3 and CXCR4 receptors. We found RNK16 cells to express high levels of CXCR4 (45–50% increase in mean fluorescence intensity, Fig. 1A) and, low but significant levels of CXCR3 (5–8% increase of mean fluorescence intensity, Fig. 1B). This expression pattern is similar to that found on resting human NK cells (28). More importantly, RNK16 cells migrated toward both CXCL10 and CXCL12 (Fig. 1, A and B) in a dose-dependent manner, with the most efficient migration at 100 ng/ml of either chemokine, after which the response leveled out. Curiously, the response to CXCL12 was relatively low, considering the high surface expression of CXCR4. In contrast, the response to CXCL10 was more pronounced than for CXCL12, even though the surface staining of CXCR3 was low. The low surface staining of RNK16 with anti-CXCR3 calls into question the specificity of our anti-CXCR3 polyserum, and low RNK16 staining might represent nonspecific binding of anti-CXCR3 to rat cells, irrespective of rat CXCR3 expression. To demonstrate that our commercially available anti-mouse CXCR3 Ab efficiently cross-reacts with rat CXCR3, as stated in the Santa Cruz Biotechnology product literature, we selectively amplified the rat CXCR3 cDNA and transiently transfected this construct into 293T cells. As shown in Fig. 1C, the anti-mouse CXCR3 Ab Y16 brightly stained 293T cells transiently transfected with the rat CXCR3 construct, confirming the cross-reactivity of the Santa Cruz Biotechnology Y16 Ab with rat CXCR3. Finally, we show the presence of mRNA for CXCR3 in RNK16 cells, in support of its expression (Fig. 1D). These results show that, although the surface expression of CXCR3 is low, RNK16 cells respond well to its ligand CXCL10. Although we cannot exclude the possibility that CXCL10 can bind yet another receptor, as has been suggested by Soejima and Rollins (29), these experiments demonstrate that the rat NK cell line RNK16 responds to CXCL10 and CXCL12 in a dose-dependent manner similar to that seen in human NK cells (28).



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  		FIGURE 1. RNK16 cells respond to the chemokines CXCL10 and CXCL12. A, Left panel, Expression of CXCR4 on RNK16 cells was analyzed by flow cytometry using a polyclonal goat anti-CXCR4 Ab (black line), control goat IgG in gray. Right panel, Chemotaxis of RNK16 cells in response to CXCL12 was measured in Boyden chambers as described in Materials and Methods. B, Left panel, CXCR3 surface expression on RNK16 cells, measured by flow cytometry using a goat polyclonal Ab toward CXCR3. Right panel, Chemotaxis of RNK16 cells in response to increasing concentrations of CXCL10 is demonstrated. Chemotaxis results are presented as the mean ± SE of three experiments. C, The specificity of the anti-CXCR3 Ab was tested on a rat CXCR3 construct, transiently transfected into 293T cells. D, RT-PCR analysis of RNK16 mRNA for CXCR3. Lane 2, mRNA reversibly transcribed with RT; lane 3, reverse transcription without RT.

 
RNK16 cells stably transfected with either the inhibitory Ly49A or the activating Ly49D receptors express these receptors at high levels (8, 9). The rat Ab 12A8 binds to both Ly49D (Fig. 2A) and to Ly49A (Fig. 2E), while the mAb A1 binds specifically to Ly49A (Fig. 2C). When Ly49D was cross-linked with 12A8 F(ab')2 Abs, an increase in the chemotactic response to CXCL10 was observed after stimulation for 1 h (Fig. 2B). In contrast, CXCL12-induced chemotaxis was reduced almost to background levels under the same conditions (Fig. 2B). Cross-linking of Ly49A with the mAb A1 led to a decrease in chemotaxis toward both CXCL10 and CXCL12 after 10 min (Fig. 2D). To demonstrate that these are Ly49A-specific effects, rather than effects mediated through FcRs, Ly49A was cross-linked with 12A8 F(ab')2. The same changes in chemotaxis were obtained as with mAb A1 cross-linking (Fig. 2F). These data suggest that the same ligand can induce differential effects on chemotaxis through either Ly49A or Ly49D. In control experiments, cross-linking of either Ly49A or Ly49D had no effect on the spontaneous migration of RNK16 effectors.



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  		FIGURE 2. Cross-linking of Ly49 receptors modulates chemotaxis toward CXCL10 and CXCL12. A, Expression of Ly49D on RNK16.Ly49D cells, measured by flow cytometry using anti-rat 12A8 mAb. B, Cross-linking of RNK16.Ly49D cells with 12A8 F(ab')2 Abs for 0, 10, or 60 min, followed by a chemotaxis assay toward either 100 ng/ml CXCL10 or CXCL12. C, RNK16.Ly49A cells stained with the Ly49A-specific Ab A1. D, RNK16.Ly49A cross-linked with mAb A1 for 0, 10, or 60 min, were subjected to chemotaxis toward 100 ng/ml CXCL10 or CXCL12. E, RNK16.Ly49A cells were stained with the 12A8 Ab. F, Ly49A were cross-linked with 12A8 F(ab')2 Abs for 0, 10, or 60 min, and chemotaxis of RNK16.Ly49A cells against 100 ng/ml CXCL10 or CXCL12 was measured. Results are presented as the mean ± SE of three experiments.

 
Ly49 receptor ligation with H2-Dd modulates chemotaxis

The activating Ly49D and inhibitory Ly49A receptors both recognize the MHC I molecule H2-Dd. They elicit different responses upon ligation, however, either promoting (Ly49D) or inhibiting (Ly49A) target cell lysis (4, 9, 12). We examined the effect on chemotaxis of RNK16 transfectants after their interaction with H2-Dd on target cells. YB2/0 cells stably transfected with H2-Dd (Fig. 3A) were used as targets for either RNK16.Ly49A or RNK16.Ly49D. Although YB2/0 cells have very low or no CXCR3 or CXCR4 surface expression (data not shown), these target cells still spontaneously migrate toward both chemokines. Therefore, YB2/0 cells were fixed with 4% formaldehyde before use. The fixed YB2/0 cells were not able to migrate through the filter in the chemotaxis chamber toward either CXCL10, CXCL12, or control medium (data not shown). Either RNK16.Ly49A or RNK16.Ly49D were incubated at 37°C with fixed YB2/0-Dd for the indicated times, then added to the chemotaxis chamber. Cell bound H2-Dd had similar effects on chemokine-induced migration as 12A8 F(ab')2 Abs. Ligation of Ly49D with cell-bound H2-Dd reduced CXCL12 chemotaxis and enhanced CXCL10 chemotaxis (Fig. 3B), while H2-Dd ligation of RNK16.Ly49A cells decreased chemotaxis toward both CXCL10 and CXCL12 (Fig. 3C). In control experiments, preincubation of untransfected YB2/0 cells with either RNK16.Ly49D or RNK16.Ly49A cells had no effect on chemotaxis toward CXCL10 or CXCL12 (Fig. 3D, and data not shown). Similarly, the incubation of untransfected RNK16 cells with YB2/0-Dd targets had no effect on chemotaxis (data not shown). Thus, modulation of NK cell chemotaxis through Ly49A and Ly49D by cell bound H2-Dd was similar to the modulation induced by anti-Ly49 cross-linking. These results collectively show that divergent Ly49 receptors differentially regulate NK cell responses to chemokines.



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  		FIGURE 3. Ligation of Ly49 receptors with H2-Dd modulate CXCL10 and CXCL12 chemotaxis. A, Analysis of surface expression of H2-Dd on either YB2/0 (left panel) or YB2/0-Dd (right panel) cells. B, RNK16.Ly49D cells were stimulated with prefixed YB2/0-Dd cells at 37°C for 0, 10, or 60 min at a 3:1 ratio, and then subjected to a chemotaxis assay toward 100 ng/ml CXCL10 or CXCL12. C, RNK16.Ly49A cells were incubated at a 3:1 ratio with prefixed YB2/0-Dd cells at 37°C for 0, 10, or 60 min at a 3:1 ratio, and chemotaxis toward 100 ng/ml CXCL10 or CXCL12 was measured. D, RNK16.Ly49A or Ly49D cells were incubated with untransfected YB2/0 cells at a 3:1 ratio for 0, 10, or 60 min and assayed for chemotaxis against 100 ng/ml CXCL10. Results are presented as the mean ± SE of three experiments.

 
Surface expression of CXCR3 and CXCR4 is unaltered

We next tried to determine the mechanisms behind Ly49-induced modulation of chemotaxis. First, we examined whether Ly49 receptor ligation could regulate surface expression of either CXCR3 or CXCR4. Again, we ligated either RNK16.Ly49A or RNK16.Ly49D cells with cell bound H2-Dd. RNK16 transfectants were prestained with the intracellular dye CFSE to distinguish RNK16 cells from YB2/0-Dd cells. Surface expression of CXCR3 or CXCR4 before and after ligation was analyzed by flow cytometry. No significant changes in CXCR3 or CXCR4 surface expression were observed after ligation of either Ly49A (Fig. 4A) or Ly49D (Fig. 4B). These data indicate that Ly49 receptors regulate chemokine receptor-induced responses rather than their surface expression.



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  		FIGURE 4. Unaltered cell surface expression of CXCR3 and CXCR4 after Ly49 ligation. RNK16.Ly49A (A) or RNK16.Ly49D (B) were prestained with 50 nM CFSE, then mixed with prefixed YB2/0-Dd cells for either 0 (solid lines) or 60 min (dotted lines) at 37°C at a 3:1 ratio. Surface expression of either CXCR3 or CXCR4 (black lines) on RNK16 cells was measured using secondary PE-conjugated Abs. Control staining is shown in gray.

 
Ly49A regulates chemotaxis through its immune receptor tyrosine-based inhibitory motif (ITIM)

The above data suggests that the Ly49A and Ly49D receptors modulate CXCL10 and CXCL12 chemotaxis through mechanisms independent of Ly49/H2-Dd adhesion and independent of chemokine receptor down-regulation. We speculated that Ly49-dependent signaling events might modulate chemokine receptor signaling and function. Ly49A delivers signals primarily through the tyrosine phosphatase Src homology phosphatase (SHP)-1 (3), which is recruited to the ITIM VxYxxV. Mutating the ITIM tyrosine to phenylalanine (Ly49A-Y8F) has previously been shown to completely abrogate the binding of SHP-1 and the inhibitory effects of Ly49A on NK cytotoxicity (8). Therefore, we examined whether the Ly49A ITIM motif was required for its inhibitory effects on chemotaxis. Using RNK16 cells stably transfected with Ly49A-Y8F (Fig. 5A), we demonstrated that cross-linking with F(ab')2 12A8 had no effect on either CXCL10- or CXCL12-induced chemotaxis (Fig. 5B, compared with Fig. 2F). The same results were obtained when Ly49A-Y8F was ligated with H2-Dd bound to YB2/0 cells (Fig. 5C, compared with Fig. 3C). This also shows that inhibition of chemotaxis is not due to simple adhesion and entrapment of RNK16-Ly49A cells by YB2/0-Dd cells in the upper wells of the Boyden chamber. Rather, these results suggest that tyrosine phosphatases such as SHP-1 may be directly involved in the modulation of chemotactic signals by Ly49A, as phosphorylation of the ITIM tyrosine is required for Ly49A function and SHP-1 binding.



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  		FIGURE 5. A mutation in the Ly49A ITIM abrogates the Ly49A-mediated decrease in chemotaxis. A, Surface expression of Ly49A-Y8F (ITIM mutant) measured with the 12A8 Ab. B, Ly49A-Y8F was cross-linked with 12A8 F(ab')2 Abs for 0, 10, or 60 min, and subjected to chemotaxis assays toward 100 ng/ml CXCL10 or CXCL12. C, RNK16.Ly49A-Y8F cells were incubated with prefixed YB2/0-Dd cells at 3:1 ratios for 0, 10, and 60 min, and chemotaxis was measured using 100 ng/ml CXCL10 or CXCL12. Results are presented as the mean ± SE of three experiments.

 
Syk regulates Ly49D modulation of chemotaxis

Ly49D activation leads to rapid recruitment of the protein tyrosine kinase Syk through the adapter DAP12 (10). Treating NK cells with the Syk inhibitor Piceatannol leads to abrogation of NK cytotoxicity (30), demonstrating the requirement of Syk signaling for this function. Therefore, we examined whether abrogation of Syk signaling might interrupt Ly49D-dependent regulation of chemotaxis. Chemotaxis through either CXCR3 or CXCR4 was not affected by the pretreatment of RNK16 cells with up to 50 µM Piceatannol for 1 h (Fig. 6A). Piceatannol treatment did not alter the surface expression levels of either CXCR3 or CXCR4 (data not shown). We next examined whether Syk was required for the Ly49D-induced modulation of chemotaxis through either CXCR3 or CXCR4. RNK16.Ly49D cells were pretreated with 10 µM Piceatannol for 1 h before cross-linking with 12A8 F(ab')2. Although the carrier DMSO had no effect on Ly49D-mediated enhancement of CXCL10-induced chemotaxis, pretreatment with Piceatannol efficiently abrogated the increase in CXCL10 chemotaxis (Fig. 6B) as well as the decrease in CXCL12-induced chemotaxis (Fig. 6B). Surprisingly, Piceatannol abrogated the Ly49A-dependent decrease in chemotaxis toward CXCL10, but not to CXCL12 (Fig. 6C), suggesting a unique role of Syk in modulation of chemotaxis through CXCR3.



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  		FIGURE 6. Syk is required for Ly49D-mediated effects on chemotaxis. A, RNK16 cells were preincubated for 1 h with increasing amounts of Piceatannol, and chemotaxis in response to 100 ng/ml CXCL10 or CXCL12 was measured. B, RNK16.Ly49D cells were pretreated with either DMSO or 10 µM Piceatannol for 1 h, before cross-linking with 12A8 F(ab')2 Abs for 1 h. Chemotaxis was measured toward either 100 ng/ml CXCL10 or CXCL12. C, RNK16.Ly49A cells pretreated with either DMSO or 10 µM Piceatannol were cross-linked for 1 h with 12A8 F(ab)2 Abs and subjected to chemotaxis assay against 100 ng/ml CXCL10 or CXCL12. Results are presented as the mean ± SE of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that ligation of the activating NK cell receptor Ly49D enhances migration toward the inflammatory chemokine CXCL10 and diminishes migration toward the homeostatic chemokine CXCL12. In addition, cross-linking of the inhibitory Ly49A receptor diminishes migration toward both CXCL10 and CXCL12. The modulation of chemotaxis by Ly49 receptors is not dependent upon chemokine receptor down-regulation on the surface of NK cells, but rather depends upon cross-talk between chemokine receptor and Ly49 receptor signaling events. Ly49A-dependent inhibition of chemotaxis was abrogated by mutation of the ITIM on Ly49A, which interrupts functional recruitment of the inhibitory SHP-1 tyrosine phosphatase. Ly49D-dependent effects on NK cell migration were abrogated by Piceatannol, a pharmacologic inhibitor of the Syk tyrosine kinase which couples to Ly49D through the DAP12 adapter protein. In addition, Piceatannol abrogated Ly49A-dependent inhibition of chemotaxis through CXCR3, but not through CXCR4, suggesting a unique role for Syk in modulation of signaling through CXCR3. These experiments were conducted in a transfection system using the rat NK cell tumor RNK16. However, it is reasonable to assume that the system investigated here also applies to normal NK cells, as mouse NK cells express both Ly-49 members and CXCR3/CXCR4.

Previous studies from other groups have shown that the functional effects of seven-transmembrane spanning, G protein-coupled chemokine receptors can be modulated by tyrosine kinase-coupled receptors in leukocytes. In particular, the divergent effects of Ly49D on chemotaxis through CXCR3 and CXCR4 are reminiscent of experiments showing a modulation of chemokine receptor signals by stimulation through the Ag receptor complexes on B and T lymphocytes (31, 32, 33, 34). Like Ly49D, both the B cell receptor (BCR) and the TCR signal through adapter proteins that recruit stimulatory tyrosine kinases. Stimulation of either the TCR or the BCR reduces lymphocyte migration toward CXCL12 (31, 32, 33, 34). Conversely, chemotaxis toward the homeostatic chemokine CC chemokine ligand (CCL)19 is enhanced upon BCR ligation (34). The mechanisms by which Ag receptors alter chemokine responsiveness are not yet entirely clear. TCR- and BCR-induced inhibition of CXCL12 signaling has been proposed to result from protein kinase C-mediated down-regulation of CXCR4 surface expression (32, 33). In contrast, IL-16-induced stimulation through CD4 decreases T cell responses to the chemokines CXCL12 and CCL4 (CCR5 ligand), but does not affect the surface levels of either CXCR4 or CCR5 (35, 36). In our studies, we have not been able to detect any changes in the surface levels of CXCR4 or CXCR3 after Ly49D ligation. It is possible that the ligation of Ly49D in NK cells modulates early events in chemokine receptor signaling or modulates the downstream cellular and cytoskeletal machinery involved in cellular migration. We have tested whether the rate of actin polymerization in response to either CXCL10 or CXCL12 changes if the cells have been preligated through either Ly49D or Ly49A, but we were not able to detect any significant changes (data not shown). CXCR3 and CXCR4 activate similar downstream chemotactic machinery, but Ly49D has opposing effects on migration triggered through these two chemokine receptors. Thus, the divergent effects of Ly49D on CXCR3- and CXCR4-induced chemotaxis more likely reflect differential regulation of early, rather than late, chemokine receptor signal transduction events by Ly49D-associated tyrosine kinases, such as Syk.

Chemokines bind to structurally similar receptors. Different chemokine receptors can couple the same G proteins, and can activate similar auxiliary signaling pathways such as phosphatidylinositol-3 kinase, Akt/protein kinase B, and Janus kinase/STAT (37, 38, 39). Nevertheless, it is assumed that divergent chemokine receptors might differentially modulate or activate different signaling pathways and that receptor-specific signals might underlie discrete functions. The differential regulation of chemotaxis by tyrosine kinase-coupled receptors implies that individual chemokine receptors must possess unique structural features. Although CXCR3 and CXCR4 are structurally similar, currently undefined differences in these receptors likely underlie their divergent modulation by Ly49D-coupled signaling intermediates. Possible mechanisms whereby G protein-associated chemokine signals might integrate with signals generated through Ly49 receptors are not entirely clear and are currently under investigation. Two recent reports showed an increased expression of the regulator of G protein signaling (RGS)1 after BCR ligation (40, 41), with an associated diminution in B lymphocyte chemotaxis toward the homeostatic chemokine CXCL12 (41). Interestingly, it has also been suggested that a decrease in RGS3 mRNA might account for increased chemotaxis toward CCL19 after BCR stimulation (41). The expression and function of specific RGS proteins in NK cells are currently not known. It is intriguing to speculate that Ly49D-induced modulation of RGS proteins could underlie the mechanisms whereby it specifically augments chemotaxis through CXCR3 and decreases chemotaxis through CXCR4.

In contrast to the effects of Ly49D ligation on CXCL10-induced chemotaxis, ligation of the inhibitory Ly49A receptor leads to diminished chemotaxis toward CXCL10. Ly49A ligation also inhibits CXCL12-mediated chemotaxis, suggesting that inhibitory receptors such as Ly49A deliver strong negative signals that may interfere with common chemokine signaling pathways or with chemotactic cellular machinery. Ly49A delivers inhibitory signals predominantly through the cytoplasmic tyrosine phosphatase SHP-1, which is recruited to a phosphorylated ITIM motif within the cytoplasmic tail of Ly49A (8). SHP-1 mediates its functions by countering the actions of stimulatory tyrosine kinases at the effector-target interface. Mutational disruption of the ITIM prevents Ly49A-mediated inhibition of NK cell cytotoxicity (8). Similarly, we here show that mutation of the ITIM in Ly49A abolishes its inhibitory effect on both CXCL10 and CXCL12 chemotaxis. These data support a role for tyrosine phosphatases such as SHP-1 as negative regulators of chemokine signaling. Moreover, they suggest that the recently reported increase in CXCL12-induced migration of thymocytes in moth-eaten (SHP-1 mutant) mice may be due to direct inhibitory effects of SHP-1 on chemokine receptor function (42). These data indicate that SHP-1 might dephosphorylate molecules important for chemokine signaling or that it interferes with cellular processes required for leukocyte migration. This is, to our knowledge, the first demonstration of a regulatory role for an ITIM-containing immune receptor on chemokine function, although the exact mechanisms by which tyrosine phosphatases modulate chemokine signaling are not yet clear.

The physiologic relevance of Ly49 modulation of chemotaxis is not yet known, but our data suggest that tyrosine kinase activation through activating Ly49 receptors can specifically attenuate or augment chemotaxis toward certain chemokines. Mature peripheral NK cells express pairs of activating and inhibitory Ly49 receptors that may recognize the same MHC ligand, and subsets of NK cells express Ly49D in combination with either Ly49A or Ly49G2 (15). The inhibitory receptors are thought to functionally dominate over activating receptors. Thus, we expect that ligation of murine NK cells expressing both Ly49A and Ly49D will lead to decreased chemotaxis toward CXCL12 and CXCL10, as observed with ligation through Ly49A alone. This may at first present a paradox, as the effects of the inflammatory chemokine CXCL10 on cells coexpressing these receptors could cancel each other out. However, a recent paper by Ortaldo and Young (43) describes that IL-12 and IL-18 are able to deliver a costimulating signal enabling Ly49D signaling to override the inhibitory Ly49 receptors. Therefore, in the appropriate cytokine milieu in vivo, NK cell chemotaxis may be influenced more by activating than inhibitory receptors. Also, the finding that distinct NK cell subsets might specialize in combating certain infections (25) presents the possibility that interactions of NK cell receptors with their ligands might give NK cells new migratory properties. Modulation of cellular migration in this way might be advantageous during an immune response. Activated lymphocytes could preferentially chemotax toward sites of inflammation rather than toward sites of tissue homeostasis once their activating receptors have been triggered by cells expressing altered MHC ligands such as tumor cells or infected cells. A similar economy during immune responses might be derived from the inhibitory effects of Ly49A on chemotaxis. Ly49A-induced attenuation of migration toward both inflammatory (CXCL10) and homeostatic (CXCL12) chemokines could make nonreactive NK lymphocytes remain at homeostatic sites, promoting migration neither toward nor away from sites of inflammation.


   Acknowledgments
 
We thank Mary Nakamura, Eréne Niemi, and Mike Daws for practical advice and Jason Cyster for helpful discussion.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1 AI 44126 and by the U.S. Veterans Administration (to J.C.R.). M.I. is a research fellow of the Norwegian Cancer Society. Back

2 Address correspondence and reprint requests to Dr. Marit Inngjerdingen, Department of Arthritis and Immunology 111R, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. E-mail address: marit.inngjerdingen{at}basalmed.uio.no Back

3 Abbreviations used in this paper: MHC I, MHC class I; CXCL, CXC chemokine ligand; RT, reverse transcriptase; ITIM, immune receptor tyrosine-based inhibitory motif; SHP, Src homology phosphatase; BCR, B cell receptor; CCL, CC chemokine ligand; RGS, regulator of G protein signaling. Back

Received for publication November 27, 2002. Accepted for publication July 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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