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The Journal of Immunology, 2001, 166: 4831-4834.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: The Wiskott-Aldrich Syndrome Protein Is Required for Efficient Phagocytosis of Apoptotic Cells1

Yann Leverrier2,*,{dagger}, Roberto Lorenzi2,{ddagger},§, Michael P. Blundell§, Paul Brickell{ddagger}, Christine Kinnon§, Anne J. Ridley*,{dagger} and Adrian J. Thrasher3,§

* Ludwig Institute for Cancer Research, Royal Free and University College Medical School Branch, London, United Kingdom; {dagger} Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; {ddagger} Molecular Haematology Unit, Institute of Child Health, University College London, London, United Kingdom; and § Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Phagocytosis of apoptotic cells by macrophages and dendritic cells is necessary for clearance of proinflammatory debris and for presentation of viral, tumor, and self Ags. While a number of receptors involved in the cognate recognition of apoptotic cells by phagocytes have been identified, the signaling events that result in internalization remain poorly understood. Here we demonstrate that clearance of apoptotic cells is accompanied by recruitment of the Wiskott-Aldrich syndrome (WAS) protein to the phagocytic cup and that it’s absence results in delayed phagocytosis both in vitro and in vivo. Therefore, we propose that WAS protein plays an important and nonredundant role in the safe removal of apoptotic cells and that deficiency contributes significantly to the immune dysregulation of WAS. The efficiency of apoptotic cell clearance may be a key determinant in the suppression of tissue inflammation and prevention of autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Cells that die by apoptosis are efficiently cleared by specialized phagocytes (1). This process is important for normal turnover or remodeling of tissues and is crucial for the disposal of proinflammatory molecules that are released from dying cells (2). Phagocytosis of apoptotic cells by macrophages has also been shown to result in the active production of anti-inflammatory cytokines such as TGF-{beta} and suppression of proinflammatory cytokines such as TNF-{alpha} (3, 4). The Wiskott Aldrich syndrome protein (WASp)4 is only expressed in hemopoietic cells but is a member of a family of proteins including N-WASP and SCAR/WAVE that are able to transduce signals from membrane receptors to the actin cytoskeleton. WASp can be activated by the Rho family GTPase Cdc42 to stimulate actin polymerization through the Arp2/3 complex, and the lack of functional WASp has been shown to lead to defects of immune cell polarization, signaling, and cell motility (5, 6). Recently, both Cdc42 and Rac1 have been shown to regulate actin reorganization during Fc{gamma}R-mediated phagocytosis by promoting pseudopod extension and phagosome closure, respectively (7, 8, 9, 10, 11, 12). For optimal efficiency, this process is also dependent on WASp, which actively relocates to the region of the evolving phagocytic cup, and is required for recruitment of the Arp2/3 complex (13, 14). Complement receptor-mediated phagocytosis may be less dependent on WASp, but requires the participation of RhoA, and is also dependent on recruitment of the Arp2/3 complex to the cup (13). In contrast, the signaling events that occur during phagocytosis of apoptotic cells are much less clearly defined. In this study, we have observed the phagocytosis of apoptotic cells by macrophages derived from WASp-deficient mice and show that WASp is necessary for the efficient uptake of apoptotic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture

Baf-3 cells were maintained in DMEM (Life Technologies, Rockville, MD) containing 6% heat-inactivated FBS (Sigma, St. Louis, MO) and 5% WEHI-3B cell-conditioned medium as a source of IL-3 (15). Femoral bone marrow cells were isolated from 6- to 8-wk-old female 129/Sv wild-type mice and age- and sex-matched 129/Sv WASp-deficient mice (16). A total of 11 x 106 cells were plated on 10-cm bacteriological plastic plates (Falcon; Becton Dickinson, Mountain View, CA) in macrophage medium consisting of RPMI 1640 (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1x nonessential amino acids (Life Technologies), 0.2 mM 2-ME (Sigma), 10% heat-inactivated FBS (Sigma) supplemented with 10% L cell-conditioned medium as a source of CSF-1. After 3 days, nonadherent cells were collected, seeded at 105 cells/ml in bacteriological plates, and grown for at least 6 more days before use. Twenty-four hours before phagocytosis assay, 5 x 104 macrophages were seeded on 13-mm glass coverslips in four-well multidishes (Nunc, Naperville, IL) in macrophage medium.

Quantification of IgG-opsonized SRBC phagocytosis

Before initiation of phagocytosis, SRBC were fluorescently labeled green and opsonized with IgG as follows. SRBC (Cappel, Thame, U.K.) were incubated at 37°C for 20 min in PBS containing 10 µM CFSE (Molecular Probes, Eugene, OR) and subagglutinating concentrations of rabbit IgG Abs to SRBC (Cappel). Reactions were stopped with cold PBS and IgG-SRBC resuspended in macrophage medium. For phagocytosis, IgG-SRBC were added to 7- to 9-day-old macrophages at a ratio of ~10 SRBC per cell and allowed to interact at 37°C for the indicated times. To remove unbound IgG-SRBC, coverslips were gently washed with PBS then fixed with 4% paraformaldehyde (BDH Chemical, Poole, U.K.) in PBS for at least 20 min. Fixed cells were stained for 20 min at room temperature with rhodamine-labeled anti-rabbit IgG (5 µg/ml, Southern Biotechnology Associates, Birmingham, AL) and a fluorescein-conjugated rat Ab against the macrophage cell surface marker F4/80 (2 µg/ml; Serotec, Oxford, U.K.). Slides were mounted with AF2 (Citifluor, Kent, U.K.) or mowiol (Calbiochem, La Jolla, CA) and examined by epifluorescence microscopy (Zeiss, Oberkochen, Germany). Using this procedure, the macrophages were immunodetected by the Ab against F4/80 and fluoresced green. The IgG-SRBC fluoresced intensely green due to the CFSE staining. The noninternalized IgG-SRBC also fluoresced red as their plasma membrane remained externally accessible to rhodamine-labeled anti-rabbit IgG. Phagocytosis was quantified by counting >100 macrophages from at least 5 randomly selected fields. IgG-SRBC were counted as phagocytosed if they only fluoresced green and bound if they fluoresced green and red. Results were expressed as phagocytic index (number of IgG-SRBC cells internalized per 100 macrophages). Statistical analysis consisted of a two-tailed Students’t test adjusted for two samples with unequal variance.

Quantification of apoptotic cell phagocytosis

Before the induction of apoptosis, Baf-3 cells were fluorescently labeled by culture for 15 min with 10 µM CFSE. Cells were washed in PBS containing 1 mM MgCl2 (PBS MgCl2) then incubated at 37°C for 20 min in PBS MgCl2 containing 1 mg/ml biotinamidocaproate N-hydroxysuccimide ester (Sigma) to biotinylate membrane proteins. Cells were resuspended at 5 x 105 cells/ml in culture medium without IL-3. After 24 h, the cells (>80% apoptotic) were washed once in macrophage medium and resuspended at 5 x 106 cells/ml. A total of 5 x 105 apoptotic Baf-3 cells were added to 5 x 104 macrophages (2–3 wk old) on coverslips for the indicated times. To remove unbound cells but preserve bound particles, coverslips were gently washed with PBS then fixed with 4% paraformaldehyde in PBS for at least 20 min. Fixed cells on coverslips were stained for 1 h at room temperature with rhodamine-conjugated streptavidin (10 µg/ml; Sigma) and a fluorescein-conjugated rat Ab against the macrophage cell surface marker F4/80 (2 µg/ml; Serotec) in PBS then washed twice in PBS for 5 min. Slides were mounted with mowiol (Calbiochem) and were examined by epifluorescence microscopy (Zeiss). Using this procedure, the macrophages were immunodetected by the Ab against F4/80 and fluoresced green. The apoptotic cells fluoresced intensely green due to the CFSE staining. The noninternalized apoptotic cells also fluoresced red as their plasma membrane was biotinylated and remained accessible to the rhodamine-conjugated streptavidin. Phagocytosis was quantified by counting >100 macrophages from at least five randomly selected fields. Apoptotic cells were counted as phagocytosed if they only fluoresced green, and bound if they fluoresced green and red. Results were expressed as phagocytic index (number of apoptotic cells internalized per 100 macrophages) and binding index (number of apoptotic cells bound per 100 macrophages).

Microinjection and immunofluorescence microscopy

Wild-type macrophages seeded on 13-mm glass coverslips were injected into the nucleus with 50 ng/ml expression vector (p63dCMV-WASP) encoding human WASp (kindly provided by Dr Giles Cory, Ludwig Institute for Cancer Research, London, U.K.) and returned to the incubator for 3 h to allow expression of WASp. A total of 5 x 105 apoptotic Baf-3 cells with cell surface proteins biotinylated (see above) were added for 30 min, washed with PBS, and fixed. After permeablization, specimens were then incubated for 60 min with 20 µg/ml polyclonal rabbit anti-WASp Ab (H-250; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS 20% goat serum followed by 30 min with fluorescein-conjugated goat anti-rabbit Ab (10 µg/ml; Southern Biotechnology Associates), rhodamine-conjugated phalloidin (0.1 µg/ml; Sigma) and Cy5-conjugated streptavidin (4 µg/ml; Jackson ImmunoResearch, West Grove, PA). Images of cells (single sections) were obtained using a Zeiss LSM 510 confocal laser-scanning microscope.

In vivo clearance of apoptotic cells by peritoneal macrophages

The method for determination of apoptotic cell clearance by peritoneal macrophages in vivo was kindly provided by Prof. Mark Walport (17). Apoptosis was induced in Jurkat T cells by incubation with polyclonal rabbit IgM anti-Fas Ab (CH-11; TCS Biologicals, Buckingham, U.K.), 30 ng/ml for 5 h at 37°C, which resulted in ~50% apoptotic cells.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Recently, we have demonstrated that FcR{gamma}-mediated phagocytosis is impaired in human monocytes derived from patients with severe Wiskott-Aldrich syndrome (WAS), indicating that WASp is necessary for efficient removal of IgG-opsonized particles (14). To confirm that murine cells show a similar dependence on WASp, we investigated the phagocytosis of IgG-opsonized targets by primary macrophages derived in vitro from wild-type and WASp-deficient mice. For this, macrophages were incubated for varying lengths of time with SRBC preopsonized with IgG (IgG-SRBC), and phagocytosis was quantified as described in Materials and Methods. Internalization of IgG-SRBCs was clearly reduced in WASp-deficient cells compared with wild type (Fig. 1Go), though binding of targets to the macrophage cell surface was similar in both groups (Fig. 1Go, inset). Therefore, WASp-deficient murine macrophages mirror the phagocytic defect observed in human WAS cells.



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  		FIGURE 1. Reduced IgG-mediated phagocytosis in WASp-deficient macrophages. Mouse macrophages were cultured for the indicated time with SRBC preopsonized with IgG (IgG-SRBC). Results are expressed as the mean ± SD of the phagocytic index (n = 6 data points pooled from three independent experiments). The differences between the phagocytic index of wild-type and WASp-deficient macrophages are statistically significant (*, p < 0.05) or very significant (**, p < 0.01). Inset, Relative phagocytic index (•) or binding index ({circ}) are calculated as the percentage of the ratio between the value for WASp-deficient and the value for wild-type macrophages at each time point.

 
We then investigated the requirement for WASp during apoptotic cell phagocytosis. Wild-type and WASp-deficient macrophages were cultured for varying lengths of time with apoptotic Baf-3 cells, and phagocytosis was quantified using epifluorescence methodology. As for IgG-opsonized targets, ingestion of apoptotic cells was impaired in WASp-deficient macrophages and remained significantly below the level achieved by normal macrophages after 120 min (Fig. 2Go). This was not due to impaired binding, as WASp-deficient macrophages and wild-type macrophages bound apoptotic cells to similar extents (Fig. 2Go, inset). Therefore, WASp is required for both FcR{gamma}-mediated and apoptotic cell phagocytosis. It is notable that the rate of apoptotic cell uptake by macrophages is slower than that of IgG-SRBC. This may reflect a lower frequency of productive attachment between apoptotic cells and macrophages compared with IgG-SRBC and macrophages, as the receptors and ligands involved in these interactions differ.



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  		FIGURE 2. Reduced apoptotic cell phagocytosis in WASp-deficient macrophages. Macrophages were cultured for the indicated time with apoptotic cells. Quantification of the phagocytosis was conducted as described in Materials and Methods. Results expressed as the mean ± SD (n = 3 independent experiments) are reported as percent of the phagocytic index of wild-type macrophages after 120 min. Inset, Relative binding index being the binding index of WASp-deficient macrophages divided by the binding index of wild-type macrophages for each time point. The differences between relative phagocytic index of wild-type and WASp-deficient macrophages are statistically significant (*, p < 0.05) or very significant (**, p < 0.01).

 
Recently, deficiencies of classical complement components C4 and C1q have been shown to result in impairment of apoptotic cell clearance in vivo (17). We used this approach to determine whether WASp-deficient mice manifest similar defects. We first confirmed that the cell composition and kinetics of thioglycollate-induced sterile peritonitis was similar in both WASp-deficient and control mice, and that mutant cells expressed equivalent levels of F4/80 and Mac1, indicating that that there was no defect in cell differentiation (data not shown). Mice were then injected i.p. with apoptotic human Jurkat T cells 4 days after induction of sterile peritonitis. Consistently, phagocytosis of apoptotic cells (determined as the percentage of macrophages with internalized apoptotic cells) in WASp-deficient mice was impaired, indicating that the defects observed in vitro also operate in vivo (Fig. 3Go).



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  		FIGURE 3. Reduced phagocytic clearance of apoptotic Jurkat T cells by inflammatory macrophages in vivo. A, Representative cytospin preparation of peritoneal lavage cells recovered 30 min after injection of control Sv/129 mice with 3 x 107 apoptotic Jurkat cells. Phagocytosis was scored on cytospins of peritoneal macrophages stained with Diff-Quik (Dade Behring, Dundingen, Switzerland), and was expressed as percentage of macrophages containing apoptotic bodies. Between 100 and 200 macrophages were scored for each mouse, and scores were verified blindly by an independent observer. Arrowheads indicate apoptotic bodies ingested by macrophages. B, Scatter plot showing the percentage of peritoneal macrophages with phagocytosed apoptotic bodies 30 min after i.p. inoculation of 1–3 x 107 apoptotic or nonapoptotic Jurkat cells and 4 days after induction of sterile peritonitis. Compared with age-, sex-, and strain-matched control mice (Sv/129, n = 9) (•), WASp-deficient mice (WASp, n = 10) ({circ}) showed a significant inhibition of phagocytosis, p < 0.001, Students t test. Horizontal bars represent the mean percentage of cells containing apoptotic bodies.

 
Apoptotic cell uptake by normal macrophages is accompanied by polymerization of actin and the formation of an actin cup (18). To determine the role of WASp in this process, we examined its localization during formation of the actin cup around apoptotic Baf-3 cells. In the absence of Ab sensitive enough to visualize endogenous murine WASp, wild-type macrophages were microinjected with a plasmid vector encoding human WASp. In injected cells, apoptotic cell uptake was accompanied by the formation of an actin cup (Fig. 4GoA). WASp clearly colocalized with F-actin in the contact area between the macrophage and the apoptotic cell (Fig. 4GoB). In the absence of actin cup formation, detectable relocalization of WASp to sites of apoptotic cell-macrophage interaction was not observed (data not shown).



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  		FIGURE 4. WASp colocalizes with F-actin at the site of apoptotic ingestion. Wild-type macrophages microinjected with a plasmid encoding human WASp were cultured with biotinylated apoptotic cells for 30 min. F-actin was detected using rhodamine-conjugated phalloidin (A). WASp was detected with rabbit anti-WASp Ab followed by fluorescein-conjugated anti-rabbit Ab (B). Apoptotic cells were detected using Cy5-conjugated streptavidin (C). Arrow heads indicate colocalization of the actin cup, WASp, and an apoptotic cell. Bar = 10 µm

 
Here we have shown that deficiency of WASp results in impairment of apoptotic cell phagocytosis and that, normally, WASp is localized to the phagocytic cup. Cdc42 is known to activate WASp, and indeed we have observed that Cdc42 is required for uptake of apoptotic cells (18). It is likely that interaction between WASp and the Arp2/3 complex contributes significantly to the actin polymerization necessary for this process to occur (19). Furthermore, the fact that several different types of receptors are implicated in the uptake of apoptotic cells suggests that signals activated by these receptors all converge on a nonredundant, WASp-dependent, common pathway for optimal initiation of localized actin polymerization (4, 20, 21, 22, 23). Interestingly, cells deficient in WASp are capable of forming an actin cup and of phagocytosing apoptotic cells, although the process is much less efficient than in wild-type cells (data not shown). Therefore, in the absence of WASp, other related molecules such as SCARs/WAVEs presumably contribute to the recruitment of the Arp2/3 complex and subsequent actin polymerization, as suggested for CR3-mediated phagocytosis (13). Consistent with this, we have observed that Rac, which can activate SCARs/WAVEs, as well as Cdc42, is required for apoptotic cell uptake (18, 24).

Our findings have clear implications for the efficiency of viral or tumor Ag presentation in WAS, which is associated with complex immunodeficiency, susceptibility to EBV-driven lymphoproliferative disease, and, in over 40% of patients, autoimmune disease (25). The parallels between our findings and similar studies on C1q-deficient mouse and human cells are also striking (17). C1q is a classical pathway complement component that has been shown to bind to the surface of apoptotic cells, and its deficiency in humans is strongly associated with the development of systemic lupus erythematosus (26). Therefore, it has been suggested that impaired clearance of apoptotic cells (which are a major source of autoantigen) is responsible for the development of systemic lupus erythematosus (17, 26, 27). We propose that impaired phagocytosis of apoptotic cells by macrophages (and possibly dendritic cells) in WAS results in delayed tissue clearance of proinflammatory debris and breakdown of normal mechanisms that suppress inflammation and maintain peripheral immunological tolerance. This may explain the susceptibility of WAS patients to autoimmune disease and is further evidence for an association between impaired phagocytosis of apoptotic cells and development of sterile tissue inflammation.


   Acknowledgments
 
We are grateful to Fred Alt and Scott Snapper for providing the WASp-deficient mice and to Giles Cory for providing p63dCMV-WASp.


   Footnotes
 
1 This work was supported by the Ludwig Institute for Cancer Research and by grants from the Primary Immunodeficiency Association (to R.L. and C.K.) and the Wellcome Trust (to A.J.T.). Y.L. is a recipient of a Medical Research Council postdoctoral fellowship. Back

2 Y.V. and R.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Adrian Thrasher, Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, U.K. Back

4 Abbreviations used in this paper: WASp, Wiskott-Aldrich syndrome protein; WAS, Wiskott-Aldrich syndrome. Back

Received for publication November 28, 2000. Accepted for publication February 26, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
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T. S. Strom, S. J. Turner, S. Andreansky, H. Liu, P. C. Doherty, D. K. Srivastava, J. M. Cunningham, and A. W. Nienhuis
Defects in T-cell-mediated immunity to influenza virus in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene transfer into repopulating hematopoietic cells
Blood, November 1, 2003; 102(9): 3108 - 3116.
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Y. Leverrier, K. Okkenhaug, C. Sawyer, A. Bilancio, B. Vanhaesebroeck, and A. J. Ridley
Class I Phosphoinositide 3-Kinase p110{beta} Is Required for Apoptotic Cell and Fc{gamma} Receptor-mediated Phagocytosis by Macrophages
J. Biol. Chem., October 3, 2003; 278(40): 38437 - 38442.
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S. Fais and W. Malorni
Leukocyte uropod formation and membrane/cytoskeleton linkage in immune interactions
J. Leukoc. Biol., May 1, 2003; 73(5): 556 - 563.
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E. Garcia-Garcia and C. Rosales
Signal transduction during Fc receptor-mediated phagocytosis
J. Leukoc. Biol., December 1, 2002; 72(6): 1092 - 1108.
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G. O. C. Cory, R. Garg, R. Cramer, and A. J. Ridley
Phosphorylation of Tyrosine 291 Enhances the Ability of WASp to Stimulate Actin Polymerization and Filopodium Formation
J. Biol. Chem., November 15, 2002; 277(47): 45115 - 45121.
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N. N. Gorgani, B. A. Smith, D. H. Kono, and A. N. Theofilopoulos
Histidine-Rich Glycoprotein Binds to DNA and Fc{gamma}RI and Potentiates the Ingestion of Apoptotic Cells by Macrophages
J. Immunol., November 1, 2002; 169(9): 4745 - 4751.
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H. Falet, K. M. Hoffmeister, R. Neujahr, and J. H. Hartwig
Normal Arp2/3 complex activation in platelets lacking WASp
Blood, August 28, 2002; 100(6): 2113 - 2122.
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S. Somersan and N. Bhardwaj
Tethering and tickling: a new role for the phosphatidylserine receptor
J. Cell Biol., November 12, 2001; 155(4): 501 - 504.
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