Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages

Shigeru Tsuboi,Jennifer Meerloo

doi:10.1074/jbc.m705999200

Abstract

Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP.WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients.

Highlights

Suffer from severe bleeding, eczema, recurrent infections, autoimmune diseases, and an increased risk of lymphoreticular malignancy [1,2,3]
A general role in filopodia formation is consistent with the requirement of WASPinteracting protein (WIP) in pathogenic settings, notably the intracellular motility of vaccinia virus and Shigella, which involves the formation of an actin comet-tail mediated by the N-WASP1⁄7WIP complex [40], and the transendothelial migration of macrophages, which involves the formation of the podosomes mediated by the WASP1⁄7WIP complex [35, 41]
Formation of the phagocytic cup was reduced in the macrophages expressing EGFP-WB compared with EGFP (Fig. 7B, right panel). These results indicate that the phagocytic cup formation was impaired when Wikott-Aldrich syndrome protein (WASP) binding to WIP was blocked in cells, suggesting that the complex formation of WASP with WIP is necessary for the phagocytic cup formation (Fig. 7)

Summary

EXPERIMENTAL PROCEDURES

Reagents—Recombinant human macrophage-colony stimulating factor-1 was purchased from R & D Systems. Monocytes cultured for 2 days were harvested and transfected with the WASP constructs using Human Monocyte Nucleofector kit and Amaxa Nucleofector (Amaxa Inc.) according to the manufacturer’s instructions. Assay for the Phagocytic Cup Formation—Latex beads (3 ␮m diameter) (Sigma-Aldrich) were opsonized with 0.5 mg/ml human IgG (Sigma-Aldrich) for 16 h at room temperature, washed with phosphate-buffered saline extensively, and suspended in RPMI1640 containing 1% FCS and 10 mM 3-methyladenine (3-MA) (Sigma-Aldrich). PMA-differentiated THP-1 cells or primary macrophages grown on coverslips in 6-well culture plates were incubated in prewarmed serum-free medium containing 10 mM 3-MA for 30 min at 37 °C. Assay for Phagocytosis—To assay phagocytosis, we measured the phagocytic uptake of IgG-opsonized fluorescence dye-conjugated latex beads (TransFluoSpheres; excitation at 488 nm/emission at 560 nm; Invitrogen) by THP-1 cells in the absence of 3-MA using a flow cytometer, FACSort (BD Biosciences). Confidence (95%) was set a priori as the desired level of statistical significance

RESULTS

DISCUSSION

Shigeru Tsuboi and Jennifer Meerloo