Adaptor protein SLAT modulates Fcgamma receptor-mediated phagocytosis in murine macrophages

Abstract

SLAT (SWAP-70-like adaptor protein of T cells) is an adaptor protein expressed in cells of the hematopoietic system. SLAT interacts with and alters the function of small GTPase Rac1 in fibroblasts. In these nonhematopoietic models, the SLAT-Rac interaction leads to changes in F-actin and causes cytoskeletal reorganization. In T cells, SLAT expression regulates the development of T helper cells through Cdc42- and Rac1-mediated activation of the NF-AT transcription factor. Here we show that SLAT is expressed in macrophages. Overexpression of SLAT in a macrophage cell line inhibits the IgG Fcgamma receptor-mediated phagocytic ability of THP1 cells. In bone marrow-derived macrophages, SLAT protein is recruited to the early phagosomes formed via Fcgamma receptor engagement. SLAT recruitment to the phagosome was most efficient when the macrophages express at least one isoform of Rac (Rac1 or Rac2), because SLAT recruitment was reduced in macrophages of Rac-deficient mice. Macrophages derived from animals lacking SLAT show an elevation in the rate of Fcgamma receptor-mediated phagocytosis. The absence of SLAT is associated with an increase in the amount of F-actin formed around these phagosomes as well as an increase in the amount of Rac1 protein recruited to the phagosome. Our results suggest that SLAT acts as a gatekeeper for the amount of Rac recruited to the phagosomes formed by Fcgamma receptor engagement and thus is able to regulate F-actin re-organization and consequently phagocytosis.

FIGURE 1.

SLAT is expressed in murine macrophages. A, SLAT protein expression in whole cell lysate of 25,000 cells of each cell type: peritoneal macrophages, bone marrow-derived macrophages, murine macrophage cell line RAW 264.1, human monocytic cell line THP1, human embryonic kidney cells, and human T cell line Jurkat. The immunoblot was stripped of anti-SLAT sera and re-probed with anti-GAPDH antibody. B, cDNA from HEK, BMM, and RAW 264 cells were analyzed for SLAT and GAPDH by 30 or 35 cycles of PCR as described under “Materials and Methods.” C, quantification of SLAT protein expression with respect to GAPDH expression in a given cell type. Data represented as the ratio of band intensity of SLAT to band intensity of GAPDH.

FIGURE 2.

SLAT overexpression in human monocytic cell line THP1 reduces phagocytosis. THP1 monocytes were transduced with SLAT or vector only and sorted to isolate the transduced cells. The SLAT- or vector-transduced THP1 cells were incubated with mouse anti-sheep RBC-opsonized sheep RBCs. The THP1 cells were analyzed for their phagocytic ability as described under “Materials and Methods.” A, percent phagocytic; for p values, ** is <0.05. B, phagocytic index; for p values, ** is <0.05. Data represent the average of three independent experiments.

FIGURE 3.

SLAT is recruited to the phagosome in FcγR-stimulated macrophages. A, bone marrow-derived macrophages from wild-type mice were stimulated with human IgG-coated latex beads. The cells were fixed, permeabilized, and stained with antibodies to SLAT or GAPDH as indicated; NRS and normal rabbit IgG (NRIg) were used as negative controls. All cells were stained with phalloidin to detect F-actin. Representative optical slice from a confocal micrograph of BMM after 3 min of ingestion of human IgG-coated latex beads, stained with the indicated antibodies and with phalloidin. B, representative four serial optical slices from a confocal micrograph showing recruitment of SLAT on the phagosome along with F-actin indicated with arrows. C, mean fluorescence intensity of SLAT and F-actin stain around 15 phagosomes. Data represent average from three independent experiments.

FIGURE 4.

Accelerated phagocytosis in macrophages of SLAT^–/– mice. Bone marrow-derived macrophages from wild-type (open bars) or SLAT^–/– mice (black bars) were prepared and provided IgG-opsonized sheep RBCs. Phagocytosis was quantitated as described under “Materials and Methods.” A, percent phagocytic cells; B, phagocytic index. For p values, * is <0.01 and ** is <0.05. Data represent average of four independent experiments.

FIGURE 5.

Phagosomes in macrophages of SLAT^–/– mice contain higher F-actin amounts. Bone marrow-derived macrophages from the indicated mouse strains were prepared and provided IgG-coated latex beads. The cells were fixed and stained with phalloidin. A shows a single optical slice from a confocal image of a representative field stained for F-actin after 3, 10, and 30 min of incubation with latex beads. Arrows indicate the F-actin formed around the phagocytosed latex beads. B, quantification of pixel intensity for F-actin stain around the phagosomes formed in macrophages from wild-type and SLAT knock-out mice from the experiment shown in A. For p values, * is <0.001. Average mean fluorescence intensity values around the phagosomes were plotted from four independent experiments.

FIGURE 6.

Elevated levels of Rac1 recruitment to the phagosomes of SLAT-deficient macrophages. A, lysates of bone marrow-derived macrophages from wild-type and Rac-deficient mice were probed with antibodies to Rac1. B, bone marrow-derived macrophages from wild-type and SLAT^–/– mice were incubated for 3 min with IgG-coated latex beads, fixed, and stained with antibodies to Rac1. The images are a single optical slice of a representative field co-stained for Rac1 protein and F-actin. Arrows indicate the actin-rich phagosomes containing the latex bead and co-localized Rac1 protein. C, quantification of pixel intensity for Rac1 and F-actin levels around the phagosomes. For p values, * is <0.05, and ** is <0.01. Average mean fluorescence intensity values around the phagosomes were plotted from three independent experiments. D, PAK-PBD domain was used to pull down Rac1 from lysates of bone marrow-derived macrophages from wild-type and SLAT^–/– mice, stimulated with IgG-coated latex beads. The levels of Rac1 in the pulldown were determined by probing with antibodies to Rac1; band intensity was quantified using ImageJ software, and the numbers below the blot represent fold increase in active levels.

FIGURE 7.

SLAT recruitment to the phagosome requires Rac. A, bone marrow-derived macrophages from wild-type and the indicated Rac^–/– strains of mice were stimulated with IgG-coated latex beads for 3 min and then fixed and stained with phalloidin and antibodies to SLAT. A single representative optical slice of the confocal images is shown. The boxed images at the right are digitally enhanced to ×300 magnification to show the amount of SLAT at the phagosome, indicated by the bright phalloidin staining. B, quantification of pixel intensity for SLAT and F-actin levels around 15 representative phagosomes. p value is as follows: * is <0.001; ** is 0.001, and # is <0.0001. Average mean fluorescence intensity values around the phagosomes were plotted from three independent experiments.

FIGURE 8.

Elevated migration of macrophages from SLAT^–/– mice to a Rac-dependent chemokine. Bone marrow-derived macrophages were generated from the indicated strains of mice. An equal number of macrophages were placed in Transwells (5-μm pore size), and the cells were stimulated with fMLP or M-CSF chemokines. At 30 or 60 min, the membranes were removed, and the number of macrophages that had migrated through were quantitated as described under “Materials and Methods.” The graphs show the average number of migrated cells at a given time point. For the p value, * is <0.05. Data represent seven independent experiments for SLAT-deficient macrophages and three independent experiments for Rac-deficient macrophages.