Impaired NK-cell migration in WAS/XLT patients: role of Cdc42/WASp pathway in the control of chemokine-induced beta2 integrin high-affinity state

Abstract

We analyzed the involvement of Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton remodeling, in the control of natural killer (NK)-cell migration. NK cells derived from patients with Wiskott-Aldrich syndrome/X-linked thrombocytopenia (WAS/XLT), carrying different mutations in the WASP coding gene, displayed reduced migration through intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or endothelial cells in response to CXCL12/stromal cell-derived factor-1 and CX3CL1/fractalkine. Inhibition of WAS/XLT NK-cell migration was associated with reduced ability of these cells to up-regulate the expression of CD18 activation neoepitope and to adhere to ICAM-1 or VCAM-1 following chemokine stimulation. Moreover, chemokine receptor or beta1 or beta2 integrin engagement on NK cells rapidly resulted in Cdc42 activation and WASp tyrosine phosphorylation as well as in WASp association with Fyn and Pyk-2 tyrosine kinases. NK-cell pretreatment with wiskostatin, to prevent Cdc42/WASp association, impaired chemokine-induced NK-cell migration through ICAM-1 and beta2 integrin activation-dependent neoepitope expression. These results show that the Cdc42/WASp pathway plays a crucial role in the regulation of NK-cell migration by acting as a critical component of the chemokine-induced inside-out signaling that regulates lymphocyte function-associated antigen-1 function and suggest that after integrin or chemokine receptor engagement WASp function is regulated by the coordinate action of both Cdc42 and tyrosine kinases.

Figure 1

NK-cell migration in response to chemokines is impaired in patients with XLT and patients with WAS. (A-B) Cultured NK cells from healthy donors or patients with WAS/XLT (W27, W28, W29, W19, W35) or highly purified freshly isolated W2 NK cells were assayed for their ability to migrate through ICAM-1 (5 μg/mL)–, or VCAM-1 (5 μg/mL)–precoated polycarbonate filters using CX3CL1/fractalkine (1nM) or CXCL12/SDF-1 (10nM) as chemoattractants. (C) Freshly isolated PBMCs from healthy donors or patients with WAS/XLT were allowed to migrate through a monolayer of TNFα (10 ng/mL)–activated endothelial cells on transwell filters in response to CXCL12/SDF-1 (1nM) or CX3CL1/fractalkine (1nM). After 1 hour of incubation, the number of migrated PBMCs was evaluated by FACS; the percentage of CD56⁺CD3⁻ NK cells in input and migrated cells was assessed by immunofluorescence and FACS analysis. The percentage of migrated NK cells was calculated as follows: number of migrated NK cells/number of input NK cells × 100. Data from patients with WAS/XLT are expressed as the mean ± SD of the percentage of migrated cells obtained from 2 independent determinations. Control (Cntl) value represent the mean ± SD of the percentage of NK-cell migration obtained from 6 (A-B) or 4 healthy donors (C). ND indicates not determined. Statistical analysis performed by Student t test comparing the mean percentage of XLT/WAS NK-cell migration with that of control donors indicates that the inhibition of migration observed in all patients with WAS/XLT except for W35 is statistically significant (P < .02).

Figure 2

Integrin or chemokine receptor engagement on human NK cells results in Cdc42 activation. Human NK cells were left untreated (−) or stimulated with GAM cross-linked anti-α4 (HP2/1), anti-β1 (TS2/16), anti-β2 (TS1/18), or anti-CD56 (C218) mAb (A) or with VCAM-1–, ICAM-1– or BSA-coated beads (B) or with CXCL12/SDF-1 or CX3CL1/fractalkine (C) for the indicated time periods at 37°C. Cell lysates were incubated with GST-PAK fusion protein, and bound active GTP-Cdc42 molecules were evaluated by Western blotting with anti-Cdc42 mAb (top). Cell lysates probed for total Cdc42 are shown as loading control (bottom). Sizes are indicated in kilodaltons. These results represent 1 of 3 independent experiments.

Figure 3

Integrin or chemokine receptor engagement on human NK cells enhances WASp tyrosine phosphorylation status. Human NK cells were left untreated (-) or incubated with GAM cross-linked anti-β1 (TS2/16), anti-β2 (TS1/18), anti-α4 (HP2/1), or anti-CD56 (C218) mAb (A) or stimulated with CXCL12/SDF-1, CX3CL1/fractalkine (B) for the indicated time periods at 37°C. Cell lysates were then immunoprecipitated with anti-WASp mAb. The resulting immunocomplexes were resolved by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and sequentially immunoblotted with anti-pTyr mAb (top) and anti-WASp antiserum (bottom). Sizes are indicated in kilodaltons. These results represent 1 of 3 independent experiments.

Figure 4

β1 or β2 integrin ligation on human NK cells enhances WASp association with Pyk2 and Fyn tyrosine kinases. Human NK cells were left untreated (−) or stimulated with GAM cross-linked anti-β1 (TS2/16), anti-β2 (TS1/18), or anti-CD56 (C218) mAb for the indicated time periods at 37°C. Cell lysates were then immunoprecipitated with anti-Fyn (A) or with anti-Pyk2 mAb (B). The resulting immunocomplexes were resolved by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and sequentially immunoblotted with anti-pTyr mAb (top), anti-WASp antiserum (middle), anti-Fyn antiserum, or anti-Pyk2 mAb as loading controls (bottom). Sizes are indicated in kilodaltons. These results represent 1 of 3 independent experiments.

Figure 5

Wiskostatin inhibits chemokine-induced integrin-dependent NK-cell migration. NK cells were preincubated for 1 hour with different concentration of Wiskostatin or vehicle (DMSO) and then assayed for their ability to migrate through ICAM-1 (5 μg/mL)–precoated polycarbonate filters using CXCL12/SDF1 (10nM) or CX3CL1/fractalkine (1nM) as chemoattractants. Data are expressed as the mean ± SD of the percentage of migrated cells obtained from 3 independent determinations. Statistical significance was evaluated by Student t test, *P < .02.

Figure 6

The ability of chemokine to induce LFA-1 high-affinity state is impaired in NK cells from patients with WAS. Freshly isolated peripheral blood NK cells from healthy donors or patients with WAS were left untreated or stimulated with CXCL12/SDF-1 (10nM) or CX3CL1/fractalkine (1nM) and simultaneously stained with the high-affinity reporter mAb 327C for 10 minutes at 37°C. Cells were then analyzed by flow cytometry. The ratio between the mean fluorescence intensity of stimulated (open profile) versus unstimulated (black profile) cells is shown. The results shown for healthy control are representative of 6 individual experiments.

Figure 7

Wiskostatin inhibits chemokine-induced LFA-1 high-affinity state. Freshly isolated peripheral blood NK cells were preincubated for 1 hour with wiskostatin (10μM) or vehicle (DMSO) and then stimulated with CX3CL1/fractalkine (1nM) or CXCL12/SDF-1 (10nM) and simultaneously stained with the high-affinity reporter mAb 327C for the indicated time periods at 37°C. Stained cells were then analyzed by flow cytometry. The ratio between the mean fluorescence intensity of stimulated (open profile) versus unstimulated (black profile) cells is shown. These results represent 1 of 3 independent experiments.