Triggering through CD16 or phorbol esters enhances adhesion of NK cells to laminin via very late antigen 6

Cell Activation Enhances VLA-6-mediated NK Cell Adhesion to LM subunit (50-70%). Although they do not express detectable levels of cx1 and cx2, a small amount of 0 was found in some donors (Fig. 1) . It has been reported that a6 subunit can associate with both (31 and ,Q4 (18) . We therefore analyzed the expression of (34 subunit on NK cells . As shown in Fig. 2 , no detectable levels of (34 were observed on CD56+ NK cells, whereas all of them expressed (31, and a6 was present on a subset . These data indicate that ot6 is present on NK cell surface as x6/31 (VLA-6), but not as c16O4 heterodimer. Biochemical Characterization of VLA-6 Expressed on Human NK Cells. Cell lysates from 1 zII_radiolabeled highly purified human NK cells were immunoprecipitated with anti-/31 and anti-cx6 mAb (Fig . 3 ) . As we have previously shown (17), immunoprecipitation with anti-/31 mAb resulted in a doublet migrating at 150 and 110 kD under nonreducing conditions, and at 150/130 kD under reducing conditions . Anti-c& mAb immunoprecipitated two proteins migrating at 140 and 110 kD under nonreducing conditions corresponding to the a6 and (31 subunits, respectively. Both proteins migrated at 130 kD under reducing conditions. Adhesion of NK Cells to LM Is Mediated by VLA-6. To determine whether VLA-6 present on NK cells is a functional Figure 3 . Immunochemical analysis of VLA-6 expressed on human NK cells. 125 1-labeled human NK cell lysate was immunoprecipitated with control rabbit anti-mouse Ig (lanes 1 and 5), AlA5 (anti-/31) mAb (lanes 2 and 6), control rabbit anti-rat Ig (lanes 3 and 7), or GoH3 (anti-a6) mAb (lanes 4 and 8), and analyzed by SDS-PAGE under nonreducing (NR) and reducing (R) conditions . 1253 Gismondi et al. receptor, we analyzed the ability of NK cells to bind to LMcoated surfaces . "Cr-labeled human NK cells were incubated on LM-coated plates for 2 h at 37°C in the presence of different doses of GoH3 (anti-cx6) mAb, or a saturating dose of anti-01 antiserum . 10-15% of highly purified NK cells bound specifically to different doses of LM (1-50 N.g/ml) and not to BSA (Fig. 4 A) , and this adhesion was completely blocked by anti-01 antiserum and, in a dose-dependent manner, by anti-a6 mAb . Control antibodies such as anti-MHC I (W6/32) mAb were not inhibitory (Fig. 4 B) . These data indicate that NK cells adhere to LM and that this adhesion is mediated by VLA-6 . Activation ofNK Cells by CD16-crosslinking or Phorbol Esters Increases Adhesion to LM We have investigated whether stimuli able to trigger several NK cell functions can modulate their adhesion to LM . "Cr-labeled NK cells were treated for different times at 37°C with TPA (10 ng/ml) or, in a more physiologically relevant manner, with anti-CD16 mAb. As shown in Fig . 5 A, activation of NK cells with TPA or by crosslinking of CD16 antigen with saturating doses of B73 .1 or 3G8, two mAb directed against different epitopes of this molecule, resulted in enhanced adhesion to LM. Similar results were observed when NK cells were stimulated with 3G8 F(ab')2 fragments . Enhanced adhesion was already observed 10 min after treatment and remained at the same levels over the stimulation period (30 min) (Fig. 5 B) . VLA-6 Mediates Activation-dependent Adhesion to LM To investigate whether increased adhesion to LM induced by NK cell activation was mediated by VLA-6, we performed the binding assay in the presence of anti-c16 or anti-01 antibodies. Both anti-a6 and anti-01 antibodies but not control