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Case Reports
. 2002 Aug 20;99(17):11351-6.
doi: 10.1073/pnas.162376099. Epub 2002 Aug 12.

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

Jordan S Orange  1 , Narayanaswamy Ramesh, Eileen Remold-O'Donnell, Yoji Sasahara, Louise Koopman, Michael Byrne, Francisco A Bonilla, Fred S Rosen, Raif S Geha, Jack L Strominger
Affiliations
Case Reports

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

Jordan S Orange et al. Proc Natl Acad Sci U S A. .

Abstract

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

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Figures

Fig 1.
Fig 1.
WASp is expressed in normal but not in WAS NK cells. Total cell lysates were prepared, proteins were separated, and Western blotting was performed by using mAb 5A5. PBMC lysate from control donor 1 (P1) is shown for comparison with enriched NK cell lysates from control donor 1 (N1), control donor 2 (N2), control donor 3 (N3), and WAS patients 1 (W1) and 2 (W1). The arrowhead points to the band correlating with the expected size of WASp.
Fig 2.
Fig 2.
Deficiency of NK cell cytotoxicity in WAS and Cyt-D-treated NK cells. (A) Cytotoxic activity against K562 target cells by enriched NK cells from controls (□) and WAS patients 1 (○) and 2 (▵) is shown. Lysis mediated by WAS patient samples was different from controls (P < 0.005). Mean data for 10 controls and 3 individual donations for each WAS patient are shown ± SE. (B) K562 lytic units were calculated from individual curves shown in A. Mean data + SE are shown. Values for WAS patients 1 and 2 were less than controls (P < 0.01). (C) Normal enriched NK cells were incubated with either 10 μM Cyt-D (▿) or DMSO vehicle (⋄). Lytic activity against K562 target cells ± SE in four separate experiments is shown. Lysis by Cyt-D-treated cells was essentially eliminated as compared with DMSO control-treated cells (P < 0.001).
Fig 3.
Fig 3.
Quantification of F-actin, WASp, and perforin in the NK cell-activating IS. NK cell/K562 target cell conjugates were evaluated as detailed in Methods. Synaptic localization of F-actin (open bars), WASp (solid bars), and perforin (shaded bars) is shown. Means of five individual experiments for control donors, four each for DMSO and Cyt D, and three for WAS patients are shown. Error bars show SE. Differences between Cyt-D and DMSO treatment as well as differences between WAS patients and control donors were statistically significant (P < 0.001).
Fig 4.
Fig 4.
Cellular localization of F-actin, WASp, and perforin in conjugates of normal NK cells and K562 target cells. NK cell (small cell) and K562 target cell (large cell) conjugates were visualized by three-color laser-scanning confocal microscopy. Images were captured by using Nomarski optics (Left) and fluorescent detectors localizing F-actin (blue), WASp (red), perforin (green), or an overlay of all fluorescent channels (Right in which white areas represent colocalization of all three fluorescent signals and purple areas represent colocalization of WASp and F-actin). Examples are shown for control donor NK cell/target cell conjugates with synaptic localization of perforin (A) and nonsynaptic localization of perforin (B). Three-dimensional supramolecular organization in Z sections of an NK cell/K562 cell interface with synaptic localization of all three signals is shown projected and rotated for two-dimensional viewing (C).
Fig 5.
Fig 5.
Effect of Cyt-D or WASP mutation on cellular localization of F-actin, WASp, and perforin in NK cells or NK cell/K562 target cell conjugates. NK cells (small cells) were imaged by three-color laser-scanning confocal microscopy alone or after conjugation with K562 target cells (large cells). Images using Nomarski optics (top row) and fluorescent detectors for F-actin (blue), WASp (red), or perforin (green) were acquired. An overlay of all fluorescent channels is shown (bottom row). The effect of DMSO vehicle (A) or Cyt-D (B) incubation on the accumulation of molecules in conjugates between control donor NK cells and K562 target cells is shown. Individual NK cells from controls (C) and WAS patient 2 (D) are compared. An NK cell from WAS patient 2 is shown in conjugation with a target cell (E). Faint or barely visible blue signal in WAS patient cells is consistent with low F-actin content in cells containing WASP mutation.
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