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The Journal of Immunology, 2004, 173: 3653-3659.
Copyright © 2004 by The American Association of Immunologists

LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity

Domingo F. Barber1,2, Mathias Faure1,3 and Eric O. Long4

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. In this study, we describe an experimental system that circumvents some of the difficulties. Adhesion through {beta}2 integrin LFA-1 is a common requirement of CTLs and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 that was either expressed on a Drosophila insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells toward ICAM-1- and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells. Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive early activation signals directly through LFA-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lysis of virus-infected cells and of tumor cells by NK cells is controlled by multiple receptor-ligand interactions (1, 2). NK cells express many different activation receptors, which can contribute to lysis of target cells, including receptors that associate with ITAM motif-containing subunits (3, 4); receptor NKG2D, which signals through a distinct pathway by association with the small protein DAP10 (1, 5, 6); and a number of other receptors that have costimulatory function, such as CD2 and 2B4 (CD244) (7, 8). In addition, NK cell cytotoxicity is kept under control by inhibitory receptors, which recognize MHC class I molecules on target cells, and deliver an inhibitory signal through ITIM in their cytoplasmic tail (9, 10, 11). Signals from ITAM-containing receptors (e.g., CD16, CD94/NKG2C, NKp46, NKp30) are transmitted through recruitment of tyrosine kinases Syk or ZAP70, whereas NKG2D signals through a PI3K-binding motif. However, lysis of NK-sensitive target cells occurs with Syk/ZAP70 double-deficient NK cells, even with target cells that do not express ligands of NKG2D (12). These results highlight the possible redundancy among NK cell activation receptors and imply that alternative activation pathways, which are independent of ITAM and of DAP10 signals, exist in NK cells.

Strong adhesion to target cells mediated by the {beta}2 integrin LFA-1 (a heterodimer of CD11a/CD18, also called {alpha}L/{beta}2) is required for lysis by CTL and by NK cells (13, 14). In the absence of LFA-1 engagement, as in CD18-deficient leukocyte adhesion deficiency patients, and in mice with targeted mutations in CD11a or CD18, or after blocking LFA-1 with mAbs, target cell lysis by cytotoxic lymphocytes is impaired (15, 16, 17, 18, 19, 20, 21). Ligand engagement by LFA-1 induces a complex outside-in signaling cascade in T cells (22, 23, 24, 25, 26), but it is not clear whether such signals are required during cellular cytotoxicity.

It has been difficult to evaluate the minimal requirements for NK cell cytotoxicity because NK-sensitive target cells express ligands for multiple different NK cell receptors. For example, it is not known whether any one signaling pathway is required to activate cytotoxicity, and whether signaling by any one receptor is sufficient. High activation thresholds set by inhibitory signals could mask important activation signals required for target cell lysis. Furthermore, it has not been possible to determine whether LFA-1 is required for adhesion alone or whether it serves a dual role in adhesion and signaling for cytotoxicity. To circumvent the complexity inherent to the multiple receptor-ligand interactions between NK cells and mammalian target cells, we reconstructed a target cell from an evolutionary distant cell onto which ligands for human NK cell receptors could be expressed one at a time. We report in this work that expression of ICAM-1 (CD54), a ligand of LFA-1, on Drosophila insect cells is sufficient to induce lysis by human NK cells through LFA-1. In addition, ICAM-1 coupled to beads induced granule polarization in NK cells, implying that LFA-1 is sufficient to induce activation signals in NK cells.


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

For flow cytometry analysis and cell sorting, the following directly conjugated mouse mAbs were used: R-PE-conjugated (R-PE) CD11a (clone HI111), R-PE-CD11b (clone ICRF44), R-PE-CD18 (clone 6.7), R-PE-CD54 (clone HA58), CyChrome CD54 (clone HA58), and FITC anti-HLA-A,B,C (clone G46-2.6) were all from BD Pharmingen (San Diego, CA). R-PE-CD3 (clone UCHT1), R-PE-CD56 (clone N901), R-PE-CD158a (clone EB6), and R-PE-CD158b (clone GL183) were from Beckman Coulter (Miami, FL). FITC CD58 (clone TS2/9.1.4.3) was from Ancell (Bayport, MN). Isotype-matched PE-, FITC-, or CyChrome-conjugated mAbs were from BD Pharmingen. For blocking experiments, NK cells were incubated with 10 µg/ml purified mAbs 30 min before and during killing experiments. CD2 (clone TS2/18), CD11a (clone TS1/22), and CD18 (clone TS1/18) mAbs were from Pierce (Woburn, MD). The CD11b mAb (clone LM2/1) was from BioSource International (Camarillo, CA).

Cells

Culture and transfection of Drosophila SC2 cells have been described (27, 28, 29). Expression of transfected human cDNAs was induced for 24–48 h in 1 mM CuSO4, and was monitored by flow cytometry before every experiment. HLA-C*0304 on SC2 was loaded with 10 µM of the peptide GAVDPLLAL, and HLA-C*0401 with 2 µM of the peptide QYDDAVYKL or peptide QYDDAVYEL during the last 20 h of a 40-h induction period in 1 mM CuSO4 (29). The 721.221 cells were a gift of R. DeMars (University of Wisconsin, Madison, WI). Human NK cells were isolated from peripheral blood using the NK cell isolation kit (Miltenyi Biotec, Auburn, CA), expanded in IL-2, and cloned, as described (30). The A6-TCR CD8+ CTL clone RS56, specific for human T cell leukemia virus-1 (HTLV-1),5 Tax11–19, and the HMy2.C1R-A2 (gifts from W. Biddison, NINCDS, Bethesda, MD) were cultured, as described (31). For inhibition experiments, cells were preincubated for 40 min at 37°C with inhibitors and assayed in the continuing presence of inhibitors. Inhibitors used were cytochalasin D (20 µM), PP1 (30 µM), and wortmannin (400 nM), all from BIOMOL (Plymouth Meeting, PA).

Fc fusion proteins and beads

Human B7.1/Fc, ICAM-1/Fc, and ICAM-2/Fc fusion proteins were purchased from R&D Systems (Minneapolis, MN). Protein A-conjugated 5.6-µm-diameter beads were from Bangs Laboratories (Fishers, IN). A total of 10 x 106 beads was washed twice with H2O and coated with 5 µg of Fc fusion proteins (at 1 µg/ml) for 2 h at 4°C in 400 µl. Beads were then washed three times with H2O and resuspended at 20 x 106 beads/ml in HBSS. Coating was >98% efficient, as judged by flow cytometry.

Binding assays and cytotoxicity assays

Conjugate formation between NK cells and SC2 cells was determined, as described (32), with the following modifications: SC2 cells were resuspended in HBSS medium (BioSource International) + 5% FBS, and 1 x 105 effector cells and 4 x 105 target cells were mixed in a 200 µl final volume. For binding to beads experiments, 1 x 105 NK cells were resuspended with 6 x 105 beads for 20 min at 37°C in 200 µl. Effector cell-bead conjugates were fixed with paraformaldehyde and quantified by FACS analysis, according to the side scatter scale. For cytotoxicity assays (33), 2 x 104 target cells labeled with the green fluorescent cell linker PKH67-GLE (Sigma-Aldrich, St. Louis, MO) in a volume of 100 µl were added in duplicate to 12 x 75-mm round-bottom polystyrene tubes (BD Labware, Franklin Lakes, NJ). Effector cells, in a volume of 100 µl, were added to yield different E:T ratios. Effector and target cells were mixed by gently tapping, centrifuged at 25°C for 4 min at 300 rpm (25 x g), and incubated at 37°C in 5% C02 for 3 h. After incubation, the reaction was stopped by placing the samples for 2 min at 4°C. A total of 10 µl of propidium iodide (PI; BD Pharmingen) was added to each tube 10–15 min before data acquisition. Target cells were gated by side scatter and fluorescence (FL-1). PI uptake was determined within the gated cells. The percentage of cells labeled by PI in the absence of NK cells was subtracted from the number obtained in the presence of NK cells. Insect cells grow at temperatures below 30°C and undergo a heat shock response at 37°C. However, the duration (3 h) of the lysis assay at 37°C did not cause detectable increase of PI uptake in insect cells alone.

Immunostaining and confocal microscopy

A total of 1 x 106 NK cells was resuspended with 1 x 106 SC2-ICAM-1, SC2-CD48, or 4 x 106 uncoated or coated beads in HBSS/3% FBS in 12 x 75-mm round-bottom polystyrene tubes (BD Labware), and centrifuged at 4°C for 3 min at 300 rpm (25 x g). Samples were placed at 37°C for 20 min. Cells were allowed to settle on poly(lysine) (Roche Molecular Biochemicals, Indianapolis, IN)-coated cover glasses for 1 h, before fixation in PBS/4% paraformaldehyde, and permeabilized with PBS/0.5% Triton X-100. Nonspecific sites were saturated for 30 min with PBS/10% normal donkey serum/0.5% Triton X-100, and cells were stained with 5 µg/ml anti-perforin mAb (clone {delta} G9; Pierce), and revealed with a goat anti-mouse Alexa 568 secondary Ab (Molecular Probes, Eugene, OR) in PBS/3% normal donkey serum/0.5% Triton X-100. After three washes with PBS, the cells were mounted on slides using the Prolong antifade kit (Molecular Probes). Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exton, PA) using a x40 or x100 oil immersion objective NA 1.4, zoom 2. Differential interference contrast images were collected simultaneously with the fluorescence images. Images were processed using the Leica TCS-NT/SP software (version 1.6.587), Imaris 3.0.6 (Bitplane, Zurich, Switzerland), and Adobe Photoshop 6 (Adobe Systems, San Jose, CA). Perforin polarization was determined visually as the percentage of NK cell-bead conjugates with polarized perforin of the total number of individual conjugates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ICAM-1 on insect cells induces cytotoxicity by NK cells

The cDNA for human ICAM-1 was transfected into Drosophila SC2 cells using an inducible expression system (27, 28). A high expression level of ICAM-1 on SC2 cells was selected by cell sorting (Fig. 1). Lysis of SC2 cells after a 3-h incubation with IL-2-activated NK cells was determined by a flow cytometry assay (33). Very little lysis of untransfected SC2 cells was observed, even at high E:T cell ratios (Fig. 2). In contrast, expression of ICAM-1 on SC2 cells was sufficient to trigger a cytotoxic response by NK cells (Fig. 2). As NK cells express the two ICAM-1 receptors LFA-1 and Mac-1 (CD11b/CD18), blocking mAbs were used to determine the contribution of these two {beta}2 integrins to lysis of SC2-ICAM-1 cells. Lysis was inhibited completely by mAbs to CD11a and CD18, but not by mAbs to CD11b or to CD2 (Fig. 2b). Therefore, recognition of ICAM-1 on SC2 cells by LFA-1 alone accounts for the induction of NK cell cytotoxicity.



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  		FIGURE 1. Cell surface expression of human ICAM-1 and HLA-Cw4 on transfected Drosophila SC2 cells. The single transfectants expressing either ICAM-1 or HLA-Cw4, as indicated within each panel, are in the top panels. The double transfectant expressing both ICAM-1 and HLA-Cw4 is shown in the bottom panels. HLA-Cw4 was loaded with an exogenous peptide. Flow cytometry was performed with a PE-conjugated mAb for ICAM-1 (left panels) and FITC-conjugated mAb for HLA-Cw4 (right panels). Dotted lines represent controls with isotype-matched PE- or FITC-conjugated mAbs. The same background was obtained with the specific mAbs and untransfected SC2 cells (data not shown).

 


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  		FIGURE 2. ICAM-1 expression in SC2 cells is sufficient to induce lysis by NK cells through LFA-1. a, Specific lysis of SC2 cells (asterisks) and SC2-ICAM-1 cells (diamonds) by IL-2-activated human NK cells. b, Specific lysis of SC2 cells (asterisks), and of SC2-ICAM-1 cells (diamonds) in the presence of 10 µg/ml mAbs specific for CD2 (squares), CD11a (circles), CD11b (triangles), and CD18 (inverted triangles).

 
Signals forLFA-1-dependent cytotoxicity occur downstream of adhesion

Activated NK cells kill sensitive target cells by a Ca2+-dependent degranulation and release of perforin and granzymes. As expected, lysis of HLA class I-deficient human 721.221 cells was inhibited by EGTA/Mg2+ (Fig. 3a). Likewise, lysis of SC2-ICAM-1 cells was abrogated by EGTA/Mg2+ (Fig. 3b), consistent with a perforin-dependent pathway. Lysis of 721.221 cells was very sensitive to inhibition of actin polymerization by cytochalasin D, of Src family tyrosine kinases by PP1, and was partially sensitive to inhibition of PI3K by wortmannin (Fig. 3c). Lysis of SC2-ICAM-1 cells showed similar sensitivity to these three inhibitors (Fig. 3d). The similar properties of lysis of SC2-ICAM-1 and 721.221 cells suggest that the LFA-1-dependent activation of NK cells is characteristic of natural cytotoxicity. Activated NK cells form tight conjugates with SC2-ICAM-1 cells, as shown with a two-color adhesion assay (Fig. 3e). The time course and the extent of conjugate formation were very similar to those observed with a sensitive human target cell (32). The LFA-1-dependent adhesion to SC2-ICAM-1 cells was fairly resistant to inhibition of actin polymerization, Src family tyrosine kinases, and PI3K (Fig. 3f). Cytochalasin D delayed the formation of conjugates, but the inhibitory effect was largely overcome by 20 min (Fig. 3f, and data not shown). These results confirm earlier data on NK cell binding to solid-phase ICAM-1, which was also resistant to inhibition of actin polymerization, Src family kinase, and PI3K (28). These results show that signaling for cytotoxicity occurs downstream of adhesion.



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  		FIGURE 3. LFA-1-dependent signals for NK cell cytotoxicity are downstream of adhesion to SC2-ICAM-1 cells. a, Specific lysis of human 721.221 cells in the presence ({triangleup}) or absence ({diamondsuit}) of EGTA/Mg2+. b, Specific lysis of SC2 cells (asterisks) and SC2-ICAM-1 cells in the presence ({blacktriangleup}) or absence (diamonds) of EGTA/Mg2+. Adhesion of NK cells to SC2-ICAM-1 cells was not inhibited by EGTA/Mg2+ (data not shown). c and d, NK cells preincubated for 40 min with 20 µM cytochalasin D, 30 µM PP1, and 400 nM wortmannin were tested for lysis of 721.221 cells (c) and SC2-ICAM-1 cells (d). Lysis in the presence of inhibitors is shown relative to lysis without inhibitors. Error bars represent the SD in four independent experiments. e, Adhesion of NK cells to SC2 cells (asterisks) and SC2-ICAM-1 cells (diamonds) was determined by flow cytometry, and is represented as the fraction of NK cells that shifted into two-color conjugates. f, NK cells preincubated for 40 min with 20 µM cytochalasin D, 30 µM PP1, and 400 nM wortmannin were tested for conjugate formation with SC2-ICAM-1 cells. Data are represented as percentage of control. Error bars represent the SD in four independent experiments.

 
Formation of conjugates between NK cells and SC2-ICAM-1 cells in the presence of cytochalasin D, which did not result in extensive lysis (see Fig. 3, d and f), suggested that SC2-ICAM-1 cells are not simply sensitive to contact with cytotoxic cells or to cross-linking of ICAM-1. To confirm this point, lysis assays and conjugation assays were performed with a highly lytic CD8 T cell line, specific for the Tax11–19 peptide of HTLV-1 and restricted by HLA-A2 (31). SC2-ICAM-1 cells and SC2 cells expressing both ICAM-1 and CD58 (28) were not lysed by the CD8 T cell line (Fig. 4a), despite ICAM-1-dependent conjugate formation (Fig. 4b). Conjugate formation with SC2-ICAM-1-CD58 cells was greater than that with SC2-ICAM-1 cells (Fig. 4b). As expected, lysis of the HLA-A2 B cell line C1R-A2 was peptide dependent (Fig. 4a). Addition of specific peptide also induced strong conjugate formation (Fig. 4b), as expected from a TCR-dependent inside-out signal to LFA-1 (34). We conclude that lysis of SC2-ICAM-1 cells is not induced simply by contact of ICAM-1 with LFA-1, but requires an LFA-1-dependent signal in the NK cells.



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  		FIGURE 4. SC2-ICAM-1 cells are not lysed simply by contact with LFA-1. a, Lysis by an HLA-A2-restricted CD8+ CTL clone, specific for HTLV-1 Tax11–19, of SC2-ICAM-1 cells (diamonds), SC2-ICAM-1-CD58 cells (triangles), HMy2.C1R-A2 cells without peptide ({circ}), and HMy2.C1R-A2 cells loaded with Tax11–19 peptide (•). b, Conjugate formation, as determined in Fig. 3e, of the HLA-A2-restricted CD8+ CTL clone with SC2 cells (asterisks), SC2-ICAM-1 cells (diamonds), SC2-ICAM-1-CD58 cells (triangles), HMy2.C1R-A2 cells without peptide ({circ}), and HMy2.C1R-A2 cells loaded with Tax11–19 peptide (•).

 
LFA-1-dependent cytotoxicity is sensitive to inhibition by killer cell Ig-like receptors (KIR)

The KIR, CD158a and CD158b, inhibit human NK cells upon binding to HLA-C allotypes on human target cells (35, 36). It was therefore of interest to test whether NK cell cytotoxicity triggered by LFA-1 was also sensitive to inhibition by CD158a/b. SC2 cells were cotransfected with cDNAs encoding human {beta}2-microglobulin and the H chain of HLA-Cw3 or HLA-Cw4, either without or with ICAM-1 (29). As insect cells do not load MHC class I molecules with endogenous peptides (27), HLA-C expression at the surface of SC2 cells was stabilized by addition of specific peptides (29). CD158a+ (specific for HLA-Cw4) and CD158b+ (specific for HLA-Cw3) NK clones were used to test lysis of SC2 cells coexpressing ICAM-1 and cognate HLA-C. Peptide-loaded HLA-Cw3 inhibited lysis of SC2-ICAM-1 + HLA-Cw3 cells by a CD158b+ clone (Fig. 5a). Inhibition was specific because the same cells not loaded with peptide were lysed comparably to SC2-ICAM-1 cells (Fig. 5a). The recognition of HLA-Cw4 by a CD158a KIR is dependent on the side chain at position 8 in the bound peptide (37). This peptide selectivity in recognition by inhibitory CD158a KIR provided a perfect specificity control. SC2-ICAM-1 + HLA-Cw4 cells were loaded either with a peptide (QYDDAVYKL) that is compatible with recognition by CD158a KIR, or a peptide (QYDDAVYEL) that interferes with binding of CD158a KIR (37). Both peptides were equally effective in stabilizing cell surface HLA-Cw4 (37) (data not shown). Peptide-specific inhibition of lysis was observed with a CD158a+ NK cell clone incubated with SC2-ICAM-1 + HLA-Cw4 (Fig. 5b). Therefore, LFA-1-dependent NK cell cytotoxicity is sensitive to inhibition by ITIM-containing KIR.



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  		FIGURE 5. LFA-1-induced cytotoxicity of NK cells is inhibited by KIR. a, Specific lysis by a CD158b+ NK clone of SC2 cells (asterisks), SC2-ICAM-1 cells (diamonds), and SC2-ICAM-1/Cw3 cells without added peptide ({square}) and SC2-ICAM-1/Cw3 cells after loading HLA-Cw3 with a peptide ({blacksquare}). b, Specific lysis by a CD158a+ NK clone of SC2-ICAM-1 cells (diamonds), and SC2-ICAM-1/Cw4 cells after loading HLA-Cw4 with a peptide that prevents binding of KIR2DL1 ({square}) and after loading HLA-Cw4 with a peptide that promotes binding of KIR2DL1 ({blacksquare}). Similar results were obtained with other NK clones.

 
ICAM induces granule polarization in NK cells

A contribution to the killing of SC2-ICAM-1 cells by receptors other than LFA-1 on NK cells, upon binding conserved determinants on insect cells, cannot be ruled out. Therefore, to test whether engagement of LFA-1 alone was sufficient to trigger signals, we investigated the interaction of NK cells with purified ICAM-1. Polarization of perforin-containing cytotoxic granules toward sensitive target cells (38, 39) was used as a measure for a late step in the activation of NK cell cytotoxicity. Perforin-containing granules polarized toward NK-sensitive SC2-ICAM-1 cells, but not toward NK-resistant SC2-CD48 cells, in a majority of NK cells (Fig. 6, a–c, and data not shown). This granule polarization assay was used with beads coated with soluble rICAM. Strong polarization of granules toward beads coated with ICAM-1 and ICAM-2, but not toward uncoated beads, was observed (Fig. 6, d–f). NK cells bound avidly to ICAM-1- and ICAM-2-coated beads, but did not bind to uncoated beads nor beads coated with B7.1 (Fig. 7a). A total of 25–30% of NK cells displayed polarization of granules in the absence of target cells and beads (Fig. 7b), consistent with another report (39). The frequency of granule polarization did not change after incubation with uncoated beads or beads coated with B7.1 (Fig. 7b). In contrast, ~70% of NK cells that were in contact with ICAM-1-coated beads displayed granule polarization toward the bead, which was similar to the polarization observed in NK cells incubated with sensitive 721.221 human target cells (Fig. 7b). Our results suggest that lysis of SC2-ICAM-1 cells occurs by targeted release of granules by NK cells, and demonstrate that LFA-1 binding to ICAM ligands is sufficient to signal for granule polarization in NK cells.



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  		FIGURE 6. Polarization of perforin-containing granules in NK cells toward ICAM. NK cells incubated with SC2-CD48 cells (a), with SC2-ICAM-1 cells (b and c), with uncoated beads (d), with beads coated with ICAM-1 (e), and beads coated with ICAM-2 (f) were fixed, permeabilized, and stained with an anti-perforin mAb and Alexa 568 secondary Ab. NK cells in conjugates (a–c) are marked by a star.

 


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  		FIGURE 7. LFA-1 is sufficient to induce perforin polarization in NK cells. a, Human NK cells were mixed with uncoated beads (–), and with beads coated with B7.1, ICAM-1, or ICAM-2, as indicated, for 20 min at 37°C. The percentage of NK cells bound to beads was determined by flow cytometry on the basis of side scatter. Error bars represent the SD in three independent experiments. b, NK cells with polarized perforin before (–) and after incubation with 721.221 cells (.221), with uncoated beads (–), and beads coated with ICAM-1 or B7.1, as indicated. Data are expressed as percentage of NK cells that display polarized perforin. The number of NK cells scored is given in parentheses above each bar.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of individual ligands of NK cell receptors on insect cells provides a very useful tool to dissect the requirements for target cell lysis by NK cells. Transfected insect cells have been used previously to define requirements for T cell activation, and revealed that ICAM-1 provides unique costimulatory function (40, 41). With this approach we show that ICAM-1 expression on insect cells is sufficient to induce LFA-1-dependent cytotoxicity by IL-2-activated NK cells. Furthermore, the use of beads coated with either ICAM-1 or ICAM-2 demonstrated that engagement of LFA-1 alone was sufficient to trigger polarization of cytotoxic granules toward ICAM. Earlier experiments had established an important role of LFA-1 in target cell killing by CTLs and NK cells, but could not distinguish between a role in adhesion alone and a dual role in adhesion and in signaling for cytotoxicity. The signals for LFA-1-dependent NK cell cytotoxicity are downstream of adhesion because inhibition of actin polymerization, of Src family kinase, and of PI3K inhibited lysis of target cells much more so than adhesion to target cells or binding to ICAM-1 on plates (28). The use of a Drosophila insect cell to reconstitute a target cell for NK cells was further validated by functional recognition of HLA-C by inhibitory KIR on NK cells.

It is possible that NK cell receptors recognize structures on insect cells once adhesion through LFA-1 occurs. If such high evolutionary conservation exists, it would more likely occur with nonprotein ligands. NK cells express an inhibitory member of the sialic acid-binding Siglec receptor family, Siglec-7 (or AIRM1) (42), and the hyaluronic acid-binding receptor CD44, which provides costimulation in NK cells (43, 44). Furthermore, the recent structure of activation receptor NKp44 suggests the possibility of a carbohydrate binding site (45). Using heat-inactivated influenza virus, which binds sialic acid via its hemagglutinin, as a probe for sialic acid, we found that SC2 cells do not express sialylated proteins at their surface (our unpublished observation), consistent with other data on Drosophila cells (46). In addition, treatment of SC2-ICAM-1 cells with neuraminidase did not reduce lysis by NK cells. Likewise, enzymatic removal of cell surface hyaluronic acid, which was monitored with a hyaluronic acid-binding protein, had no effect on lysis by NK cells (our unpublished observation). Therefore, sialic acid and hyaluronic acid are not required for lysis of SC2-ICAM-1 cells by NK cells.

To date, no receptor has been shown to be either necessary or sufficient in signaling for NK cell cytotoxicity. The importance of certain receptors, such as NKG2D and the natural cytotoxicity receptor NKp46, in lysis of tumor cells and virus-infected cells has been well documented with specific combinations of target cells and NK cells, indicating a possible redundancy of activation receptors and activation pathways in NK cells (1, 4). Our data suggest that LFA-1 may be an important signaling receptor for cytotoxicity by NK cells. ITAM- and NKG2D-independent natural cytotoxicity was observed in mouse NK cells with a double Syk and ZAP70 deficiency and with target cells lacking NKG2D ligands (12). This cytotoxicity was sensitive to inhibition of Src family kinases and PI3K, as was the LFA-1-triggered target cell killing reported in this work. Such results are consistent with a contribution of LFA-1 signals to ITAM/NKG2D-independent natural cytotoxicity.

The interaction of NK cells with mammalian target cells is very complex, involving many receptor-ligand interactions, some of which exert a negative effect on cytotoxicity. Recent data on mouse NK cells have revealed that natural cytotoxicity is controlled not only by inhibitory receptors specific for MHC class I, but also by inhibitory NKR-P1B/D, which bind to Clr-b/Ocil (47, 48). As Clr-b/Ocil molecules are widely expressed (48), activation thresholds for lysis of many target cells by NK cells may be quite high. The simplified target cell system used in this study has eliminated most of this complexity and has the potential to reveal important receptor-ligand interactions, which are not easily detected with mammalian target cells. Even though induction of NK cell cytotoxicity by LFA-1 signals alone may rarely occur in vivo, our approach has revealed a signaling function for LFA-1, which could provide essential activation signals together with other cytotoxicity receptors in many NK-target cell combinations.

Several recent studies on LFA-1 in NK cells and T cells indicate that binding to ICAM is sufficient to trigger signaling pathways (26, 49, 50, 51, 52). Taking advantage of the insect cell system described in this work, we have shown that incubation of NK cells with SC2-ICAM-1 cells resulted in a Src family kinase-dependent activation of the guanine exchange factor Vav1, upstream of actin polymerization (50). In contrast, the enhancement of Vav1 phosphorylation induced by SC2 cells coexpressing ICAM-1 and CD48, the ligand of receptor 2B4, was downstream of actin polymerization (50). Thus, our data suggest a model for regulation of NK cell cytotoxicity whereby LFA-1 provides an early signal for actin polymerization, through activation of the GTPase Rac1 by Vav1, which is necessary for clustering and signaling by other receptors such as 2B4 (53). LFA-1 engagement is sufficient to initiate signaling because ICAM-1 on beads induced a Pyk2-dependent activation of Rac1 in NK cells (51). Furthermore, Ab-mediated cross-linking of LFA-1 on human NK cells induced phosphorylation of Vav and of the MAPK ERK1/2 (52). In T cells, binding of the active form of LFA-1 to ICAM-1 promotes actin polymerization (49). Binding of human T lymphoblasts to ICAM-1 on plates and concomitant activation of LFA-1 resulted in transient stimulation of Rac1 and in PI3K-dependent activation of Pyk2 (26). Furthermore, binding of soluble, dimeric ICAM-2 to LFA-1 on Jurkat cells leads to a cytohesin-dependent activation of ERK1/2 (25). This signaling pathway induced by LFA-1, although not sufficient to activate cytotoxicity in T cells, could be important in triggering NK cell cytotoxicity (52). The LFA-1 signaling pathway in NK cells may be similar to a novel pathway attributed to {beta}3 integrin in transfected Chinese hamster ovary cells: binding of {beta}3 integrin to fibrinogen resulted in a Syk-dependent phosphorylation of Vav1, and activation of ERK2 independently of actin polymerization (54). Early LFA-1 signals in NK cells, upstream of signaling by other receptors that are dependent on actin polymerization, may be a common requirement of otherwise redundant activation pathways for natural cytotoxicity.

Signaling events triggered by ligation of LFA-1 in NK cells, such as costimulation of CD16 signals for TNF-{alpha} secretion (55), and induction of NK cell migration (56) and NK cell adhesion (28, 57), have been reported, but not in the context of target cell lysis. Association of LFA-1 with other cell surface molecules, such as CD87 and DNAM-1, has been observed (21, 58). It is possible that LFA-1 signaling for cytotoxicity is dependent on such associations.

Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells resulted in inhibition by KIR of the LFA-1-dependent cytotoxicity. We have recently shown in a transfected cell system that KIR engagement by HLA-C on target cells leads to dephosphorylation of Vav1 by the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1, which is recruited by phosphorylated ITIMs in KIR. Vav1 was the only tyrosine-phosphorylated protein associated with a trapping mutant of Src homology region 2 domain-containing phosphatase-1 during inhibition of NK cells by HLA-C on target cells; and Vav1 trapping was independent of actin polymerization (59). These results, together with the actin polymerization-independent activation of Vav1 by SC2-ICAM-1 cells (50), suggest that inhibitory KIR block NK cell activation at a very early step, upstream of signals from other receptors.

Our results point to ICAM as important ligands for stimulation of natural cytotoxicity. The redistribution of ICAM-2 by the protein ezrin into uropods of target cells resulted in increased sensitivity to lysis by NK cells, in support of an important role for LFA-1 ligands in natural cytotoxicity (60). Expression of ICAM-1 is enhanced by inflammatory cytokines, including IFN-{gamma}, and by infections of endothelial and epithelial cells with bacteria and viruses (61, 62). ICAM down-regulation could be a mechanism used by pathogens to evade lysis by NK cells. Indeed, down-regulation of ICAM-1 and B7.1 by protein K5 of Kaposi sarcoma-associated herpesvirus reduces sensitivity to NK cells (63). Regulation of natural cytotoxicity, at least in part, by expression of ICAM on target cells could serve useful functions. As many different cell types require protection from lysis by NK cells, low expression levels of ICAM could diminish sensitivity to natural cytotoxicity as an alternative protection mechanism to the expression of ligands for NK cell inhibitory receptors. Conversely, up-regulation of ICAM during inflammation or infection may serve to modulate NK cell responses at the proper time and place.

The contribution of early signals for cytotoxicity in NK cells by LFA-1 is very different from integrin-mediated activation of T cells. T cells do not adhere to ICAM-bearing cells unless an inside-out signal has been received, which up-regulates the avidity of adhesion through LFA-1 (34, 64). In contrast to T cells, NK cells can use LFA-1 directly to bind to target cells (28) and to transmit signals for cytotoxicity. In this respect, triggering of NK cell cytotoxicity is more akin to the innate response of neutrophils, in which engagement of LFA-1 can induce degranulation (65).


   Acknowledgments
 
We thank M. Sandusky for nucleotide sequencing, W. Biddison and R. DeMars for gifts of reagents, and S. Rajagopalan for advice and comments on the manuscript.


   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 D.F.B. and M.F. contributed equally to this work and share joint first authorship. Back

2 Current address: Centro Nacional de Biotecnologia, Madrid, Spain 28049. Back

3 Current address: U548 Institut National de la Santé et de la Recherche Médicale Commissariat à l’Energie Atomique-Grenoble, 17 rue des Martyrs, 38054 Grenoble, France. Back

4 Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases-National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address: eLong{at}nih.gov Back

5 Abbreviations used in this paper: HTLV, human T cell leukemia virus; KIR, killer cell Ig-like receptor; PI, propidium iodide. Back

Received for publication April 20, 2004. Accepted for publication July 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cerwenka, A., L. L. Lanier. 2001. Natural killer cells, viruses, and cancer. Nat. Rev. Immunol. 1:41.[Medline]
  2. Moretta, L., A. Moretta. 2004. Unravelling natural killer cell function: triggering and inhibitory human NK receptors. EMBO J. 23:255.[Medline]
  3. Lanier, L. L.. 2001. On guard-activating NK cell receptors. Nat. Immunol. 2:23.[Medline]
  4. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19:197.[Medline]
  5. Billadeau, D. D., J. L. Upshaw, R. A. Schoon, C. J. Dick, P. J. Leibson. 2003. NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat. Immunol. 4:557.[Medline]
  6. Zompi, S., J. A. Hamerman, K. Ogasawara, E. Schweighoffer, V. L. Tybulewicz, J. P. Di Santo, L. L. Lanier, F. Colucci. 2003. NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nat. Immunol. 4:565.[Medline]
  7. Lanier, L. L., B. Corliss, J. H. Phillips. 1997. Arousal and inhibition of human NK cells. Immunol. Rev. 155:145.[Medline]
  8. Sivori, S., S. Parolini, M. Falco, E. Marcenaro, R. Biassoni, C. Bottino, L. Moretta, A. Moretta. 2000. 2B4 functions as a co-receptor in human NK cell activation. Eur. J. Immunol. 30:787.[Medline]
  9. Burshtyn, D. N., E. O. Long. 1997. Regulation through inhibitory receptors: lessons from natural killer cells. Trends Cell Biol. 7:473.[Medline]
  10. Long, E. O., D. F. Barber, D. N. Burshtyn, M. Faure, M. Peterson, S. Rajagopalan, V. Renard, M. Sandusky, C. C. Stebbins, N. Wagtmann, C. Watzl. 2001. Inhibition of natural killer cell activation signals by killer cell immunoglobulin-like receptors (CD158). Immunol. Rev. 181:223.[Medline]
  11. Billadeau, D. D., P. J. Leibson. 2002. ITAMs versus ITIMs: striking a balance during cell regulation. J. Clin. Invest. 109:161.[Medline]
  12. Colucci, F., E. Schweighoffer, E. Tomasello, M. Turner, J. R. Ortaldo, E. Vivier, V. L. Tybulewicz, J. P. Di Santo. 2002. Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases. Nat. Immunol. 3:288.[Medline]
  13. Davignon, D., E. Martz, T. Reynolds, K. Kurzinger, T. A. Springer. 1981. Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc. Natl. Acad. Sci. USA 78:4535.[Abstract/Free Full Text]
  14. Helander, T. S., T. Timonen. 1998. Adhesion in NK cell function. Curr. Top. Microbiol. Immunol. 230:89.[Medline]
  15. Kohl, S., T. A. Springer, F. C. Schmalstieg, L. S. Loo, D. C. Anderson. 1984. Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency. J. Immunol. 133:2972.[Abstract]
  16. Krensky, A. M., S. J. Mentzer, C. Clayberger, D. C. Anderson, F. C. Schmalstieg, S. J. Burakoff, T. A. Springer. 1985. Heritable lymphocyte function-associated antigen-1 deficiency: abnormalities of cytotoxicity and proliferation associated with abnormal expression of LFA-1. J. Immunol. 135:3102.[Abstract]
  17. Timonen, T., M. Patarroyo, C. G. Gahmberg. 1988. CD11a-c/CD18 and GP84 (LB-2) adhesion molecules on human large granular lymphocytes and their participation in natural killing. J. Immunol. 141:1041.[Abstract]
  18. Liu, J. H., S. Wei, D. K. Blanchard, J. Y. Djeu. 1994. Restoration of lytic function in a human natural killer cell line by gene transfection. Cell. Immunol. 156:24.[Medline]
  19. Schmits, R., T. M. Kundig, D. M. Baker, G. Shumaker, J. J. Simard, G. Duncan, A. Wakeham, A. Shahinian, A. van der Heiden, M. F. Bachmann, et al 1996. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J. Exp. Med. 183:1415.[Abstract/Free Full Text]
  20. Shier, P., K. Ngo, W. P. Fung-Leung. 1999. Defective CD8+ T cell activation and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling. J. Immunol. 163:4826.[Abstract/Free Full Text]
  21. Shibuya, K., L. L. Lanier, J. H. Phillips, H. D. Ochs, K. Shimizu, E. Nakayama, H. Nakauchi, A. Shibuya. 1999. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity 11:615.[Medline]
  22. Sims, T. N., M. L. Dustin. 2002. The immunological synapse: integrins take the stage. Immunol. Rev. 186:100.[Medline]
  23. Hynes, R. O.. 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110:673.[Medline]
  24. Kim, M., C. V. Carman, T. A. Springer. 2003. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301:1720.[Abstract/Free Full Text]
  25. Perez, O. D., D. Mitchell, G. C. Jager, S. South, C. Murriel, J. McBride, L. A. Herzenberg, S. Kinoshita, G. P. Nolan. 2003. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat. Immunol. 4:1083.[Medline]
  26. Sánchez-Martín, L., N. Sánchez-Sánchez, M. D. Gutiérrez-López, A. I. Rojo, M. Vicente-Manzanares, M. J. Pérez-Alvarez, P. Sánchez-Mateos, X. R. Bustelo, A. Cuadrado, F. Sánchez-Madrid, et al 2004. Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1. J. Biol. Chem. 279:16194.[Abstract/Free Full Text]
  27. Jackson, M. R., E. S. Song, Y. Yang, P. A. Peterson. 1992. Empty and peptide-containing conformers of class I major histocompatibility complex molecules expressed in Drosophila melanogaster cells. Proc. Natl. Acad. Sci. USA 89:12117.[Abstract/Free Full Text]
  28. Barber, D. F., E. O. Long. 2003. Coexpression of CD58 or CD48 with intercellular adhesion molecule 1 on target cells enhances adhesion of resting NK cells. J. Immunol. 170:294.[Abstract/Free Full Text]
  29. Faure, M., D. F. Barber, S. M. Takahashi, T. Jin, E. O. Long. 2003. Spontaneous clustering and tyrosine phosphorylation of NK cell inhibitory receptor induced by ligand binding. J. Immunol. 170:6107.[Abstract/Free Full Text]
  30. Malnati, M. S., P. Lusso, E. Ciccone, A. Moretta, L. Moretta, E. O. Long. 1993. Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements. J. Exp. Med. 178:961.[Abstract/Free Full Text]
  31. Baker, B. M., R. V. Turner, S. J. Gagnon, D. C. Wiley, W. E. Biddison. 2001. Identification of a crucial energetic footprint on the {alpha}1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors. J. Exp. Med. 193:551.[Abstract/Free Full Text]
  32. Burshtyn, D. N., J. Shin, C. Stebbins, E. O. Long. 2000. Adhesion to target cells is disrupted by the killer cell inhibitory receptor. Curr. Biol. 10:777.[Medline]
  33. Godoy-Ramirez, K., K. Franck, H. Gaines. 2000. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry. J. Immunol. Methods 239:35.[Medline]
  34. Dustin, M. L., T. A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619.[Medline]
  35. Ciccone, E., D. Pende, O. Viale, A. Than, C. Di Donato, A. M. Orengo, R. Biassoni, S. Verdiani, A. Amoroso, A. Moretta, L. Moretta. 1992. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2). J. Exp. Med. 176:963.[Abstract/Free Full Text]
  36. Wagtmann, N., S. Rajagopalan, C. C. Winter, M. Peruzzi, E. O. Long. 1995. Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity 3:801.[Medline]
  37. Rajagopalan, S., E. O. Long. 1997. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity. J. Exp. Med. 185:1523.[Abstract/Free Full Text]
  38. Billadeau, D. D., K. M. Brumbaugh, C. J. Dick, R. A. Schoon, X. R. Bustelo, P. J. Leibson. 1998. The Vav-Rac1 pathway in cytotoxic lymphocytes regulates the generation of cell-mediated killing. J. Exp. Med. 188:549.[Abstract/Free Full Text]
  39. Wei, S., D. L. Gilvary, B. C. Corliss, S. Sebti, J. Sun, D. B. Straus, P. J. Leibson, J. A. Trapani, A. D. Hamilton, M. J. Weber, J. Y. Djeu. 2000. Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway. J. Immunol. 165:3811.[Abstract/Free Full Text]
  40. Cai, Z., A. Brunmark, M. R. Jackson, D. Loh, P. A. Peterson, J. Sprent. 1996. Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells. Proc. Natl. Acad. Sci. USA 93:14736.[Abstract/Free Full Text]
  41. Cai, Z., A. B. Brunmark, A. T. Luxembourg, K. C. Garcia, M. Degano, L. Teyton, I. Wilson, P. A. Peterson, J. Sprent, M. R. Jackson. 1998. Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells. Immunol. Rev. 165:249.[Medline]
  42. Falco, M., R. Biassoni, C. Bottino, M. Vitale, S. Sivori, R. Augugliaro, L. Moretta, A. Moretta. 1999. Identification and molecular cloning of p75/AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells. J. Exp. Med. 190:793.[Abstract/Free Full Text]
  43. Galandrini, R., R. De Maria, M. Piccoli, A. Santoni. 1994. CD44 triggering enhances human NK cell cytotoxic functions. J. Immunol. 153:4399.[Abstract]
  44. Matsumoto, G., M. P. Nghiem, N. Nozaki, R. Schmits, J. M. Penninger. 1998. Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity. J. Immunol. 160:5781.[Abstract/Free Full Text]
  45. Cantoni, C., M. Ponassi, R. Biassoni, R. Conte, A. Spallarossa, A. Moretta, L. Moretta, M. Bolognesi, D. Bordo. 2003. The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity. Structure 11:725.[Medline]
  46. Roth, J., A. Kempf, G. Reuter, R. Schauer, W. J. Gehring. 1992. Occurrence of sialic acids in Drosophila melanogaster. Science 256:673.[Abstract/Free Full Text]
  47. Iizuka, K., O. V. Naidenko, B. F. Plougastel, D. H. Fremont, W. M. Yokoyama. 2003. Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nat. Immunol. 4:801.[Medline]
  48. Carlyle, J. R., A. M. Jamieson, S. Gasser, C. S. Clingan, H. Arase, D. H. Raulet. 2004. Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors. Proc. Natl. Acad. Sci. USA 101:3527.[Abstract/Free Full Text]
  49. Porter, J. C., M. Bracke, A. Smith, D. Davies, N. Hogg. 2002. Signaling through integrin LFA-1 leads to filamentous actin polymerization and remodeling, resulting in enhanced T cell adhesion. J. Immunol. 168:6330.[Abstract/Free Full Text]
  50. Riteau, B., D. F. Barber, E. O. Long. 2003. Vav1 phosphorylation is induced by {beta}2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J. Exp. Med. 198:469.[Abstract/Free Full Text]
  51. Gismondi, A., J. Jacobelli, R. Strippoli, F. Mainiero, A. Soriani, L. Cifaldi, M. Piccoli, L. Frati, A. Santoni. 2003. Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration. J. Immunol. 170:3065.[Abstract/Free Full Text]
  52. Perez, O. D., D. Mitchell, G. C. Jager, and G. P. Nolan. LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56+CD8+ natural killer cells. Blood. In press.
  53. Watzl, C., E. O. Long. 2003. Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts. J. Exp. Med. 197:77.[Abstract/Free Full Text]
  54. Miranti, C. K., L. Leng, P. Maschberger, J. S. Brugge, S. J. Shattil. 1998. Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr. Biol. 8:1289.[Medline]
  55. Melero, I., M. A. Balboa, J. L. Alonso, E. Yague, J. P. Pivel, F. Sanchez-Madrid, M. López-Botet. 1993. Signaling through the LFA-1 leukocyte integrin actively regulates intercellular adhesion and tumor necrosis factor-{alpha} production in natural killer cells. Eur. J. Immunol. 23:1859.[Medline]
  56. Somersalo, K., O. Carpén, E. Saksela, C. G. Gahmberg, P. Nortamo, T. Timonen. 1995. Activation of natural killer cell migration by leukocyte integrin-binding peptide from intracellular adhesion molecule-2 (ICAM-2). J. Biol. Chem. 270:8629.[Abstract/Free Full Text]
  57. Poggi, A., F. Spada, P. Costa, E. Tomasello, V. Revello, N. Pella, M. R. Zocchi, L. Moretta. 1996. Dissection of lymphocyte function-associated antigen 1-dependent adhesion and signal transduction in human natural killer cells shown by the use of cholera or pertussis toxin. Eur. J. Immunol. 26:967.[Medline]
  58. Porter, J. C., N. Hogg. 1998. Integrins take partners: cross-talk between integrins and other membrane receptors. Trends Cell Biol. 8:390.[Medline]
  59. Stebbins, C. C., C. Watzl, D. D. Billadeau, P. J. Leibson, D. N. Burshtyn, E. O. Long. 2003. Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity. Mol. Cell. Biol. 23:6291.[Abstract/Free Full Text]
  60. Helander, T. S., O. Carpén, O. Turunen, P. E. Kovanen, A. Vaheri, T. Timonen. 1996. ICAM-2 redistributed by ezrin as a target for killer cells. Nature 382:265.[Medline]
  61. Shen, J., S. S. T-To, L. Schrieber, N. J. King. 1997. Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines. J. Virol. 71:9323.[Abstract]
  62. Frick, A. G., T. D. Joseph, L. Pang, A. M. Rabe, J. W. I. St. Geme, D. C. Look. 2000. Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells. J. Immunol. 164:4185.[Abstract/Free Full Text]
  63. Ishido, S., J. K. Choi, B. S. Lee, C. Y. Wang, M. DeMaria, R. P. Johnson, G. B. Cohen, J. U. Jung. 2000. Inhibition of natural killer cell-mediated cytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity 13:365.[Medline]
  64. Takagi, J., B. M. Petre, T. Walz, T. A. Springer. 2002. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110:599.[Medline]
  65. Mócsai, A., M. J. Zhou, F. Y. Meng, V. L. Tybulewicz, C. A. Lowell. 2002. Syk is required for integrin signaling in neutrophils. Immunity 16:547.[Medline]



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