Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kedzierska, K.
Right arrow Articles by Crowe, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kedzierska, K.
Right arrow Articles by Crowe, S. M.
(Journal of Leukocyte Biology. 2001;70:322-328.)
© 2001 by Society for Leukocyte Biology

Fc{gamma}R-mediated phagocytosis by human macrophages involves Hck, Syk, and Pyk2 and is augmented by GM-CSF

Katherine Kedzierska*,{dagger}, Nicholas J. Vardaxis{ddagger}, Anthony Jaworowski and Suzanne M. Crowe*,*,{dagger}

* AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, and National Centre for HIV Virology Research, Fairfield;
{dagger} Department of Medicine, Monash University, Prahran; and
{ddagger} Department of Medical Laboratory Science, Royal Melbourne Institute of Technology, Bundoora, Australia

Correspondence: Suzanne Crowe, AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Yarra Bend Rd., Fairfield, 3078 Melbourne, Australia. E-mail: crowe{at}burnet.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The receptors for the constant region of immunoglobulin G (Fc{gamma}R) are widely expressed on cells of hemopoietic lineage and plays an important role in host defense. We investigated the signaling pathways during Fc{gamma}R-mediated phagocytosis in human monocyte-derived macrophages (MDMs) and examined the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on these events. Fc{gamma}R-mediated phagocytosis resulted in enhanced tyrosine phosphorylation of a wide range of cellular proteins and activation of tyrosine kinases Hck, Syk, and Pyk2, as well as the multidomain adapter protein paxillin. Stimulation of MDMs with GM-CSF augmented Fc{gamma}R-mediated phagocytosis and increased the levels of tyrosine phosphorylation in phagocytosing MDM cultures, indicating tyrosine kinase-mediated activation. GM-CSF treatment of MDMs without a phagocytic stimulus did not activate Syk, suggesting that GM-CSF may act either distally to Syk in the Fc{gamma}R-mediated signaling cascade or on a parallel pathway activated by the Fc{gamma}R. This study shows that early signaling events during Fc{gamma}R-mediated phagocytosis in human MDMs involve activation of Syk, Hck, and paxillin. It also provides the first evidence for Pyk2 activation during phagocytosis and a baseline for further studies on the effect of GM-CSF on Fc{gamma}R-mediated phagocytosis.

Key Words: paxillin • monocyte-derived macrophage • tyrosine kinase • signaling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The receptors for the constant region of immunoglobulin (Ig) G (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIIIA) are the major means by which cells of the monocyte/macrophage lineage recognize IgG-opsonized particles and promote host defenses, including phagocytosis, antibody-dependent cellular cytotoxicity, and cytokine and chemokine secretion. The major Fc{gamma} receptors expressed on monocytes are the high-affinity Fc{gamma}RI (CD64) and a low-affinity Fc{gamma}RII (CD32), whereas macrophages also express Fc{gamma}RIIIA (CD16) [reviewed in reference 1]. Most studies delineating the specific signaling events during Fc{gamma}R-mediated phagocytosis have been performed using murine macrophages [2 3 4 5 6 7 ] or cell lines transfected with Fc{gamma}R [8 9 10 ], with only two studies done in human macrophages [11 ] or monocytes [12 ]. Several signal transduction pathways utilized by macrophages activated during Fc{gamma}R-mediated phagocytosis have been described [reviewed in 13], including requirements for isoforms of protein kinase C [12 ], phosphatidylinositol 3-kinase [14 , 15 ], and the Rho family of GTPases [16 , 17 ]. These signaling events are initiated after clustering of Fc{gamma}R via activation of tyrosine kinases of the Src family [18 , 19 ]. Src kinase activation results in the rapid and transient phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on either the ligand-binding subunit in the cytoplasmic domain of Fc{gamma} RII or the associated {gamma} chain of Fc{gamma}RI and RIIIA [10 , 20 , 21 ]. Phosphorylated ITAM motifs may represent docking sites for Syk that allow its subsequent activation [22 , 23 ]. A requirement for Syk in Fc{gamma}R-mediated phagocytosis was firstly demonstrated in human monocytes by using antisense oligodeoxynucleotides to eliminate Syk mRNA [24 ]. Gene knockout studies subsequently confirmed an absolute and specific requirement for Syk in Fc{gamma}R-mediated phagocytosis by murine macrophages [3 ]. Macrophages derived from the fetal livers of Syk-deficient mice had defective Fc{gamma}R-mediated phagocytosis and actin assembly but unimpaired phagocytosis of Escherichia coli, yeasts, or latex particles [3 ].

Activated Syk is thought to promote phosphorylation and localized accumulation of a number of cytoskeletal substrates [reviewed in 25], including the actin-binding proteins paxillin, vinculin, talin, and {alpha}-actinin [6 , 26 , 27 ]. Paxillin, a multidomain adapter protein, is thought to interact with a variety of proteins, such as focal adhesion kinase (FAK), Pyk2, and Vav, and in this way it organizes focal adhesion complexes and cytoskeletal rearrangement [reviewed in 28]. Human monocytes and macrophages do not express FAK [29 ], but they activate and phosphorylate another member of the FAK family, Pyk2. This kinase is 45% identical in amino acid sequence to FAK [30 ] and is shown to be involved in cytoskeletal engagement upon adherence and subsequent calcium or PKC costimulatory activation in human monocytes [29 ]. A role for Pyk2 in phagocytosis has not yet been reported.

Here we demonstrate that Fc{gamma}R-mediated internalization of IgG-opsonized targets by human monocyte-derived macrophages (MDMs) was dependent on protein tyrosine phosphorylation, with transient activation of the protein tyrosine kinases Hck, Syk and Pyk2, and redistribution of the multidomain adaptor protein paxillin between Triton-soluble and Triton-insoluble cell fractions. In addition, acute stimulation of MDMs with granulocyte-macrophage colony-stimulating factor (GM-CSF) augmented phagocytosis of IgG-opsonized targets and concomitantly tyrosine phosphorylation of cellular proteins in an additive manner but did not activate Syk. These observations suggest that GM-CSF might either stimulate phagocytic pathways activated by the Fc{gamma}R downstream of Syk kinase or might do so independently of Syk activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of monocytes
Human monocytes were isolated from buffy coats of HIV-, hepatitis B virus-, and human T-cell-lymphotropic virus-seronegative blood donors (supplied by the Red Cross Blood Bank, Melbourne) by Ficoll-Paque density gradient centrifugation and plastic adherence as previously described [31 ]. Immediately after isolation, cell viability was greater than 95% as assessed by trypan blue exclusion, and the purity of monocytes was greater than 95% as determined by immunofluorescent staining with anti-CD14 monoclonal antibody (mAb) (Becton Dickinson, Mountain View, CA) and analysis by flow cytometry (FACStarPlus; Becton Dickinson). Monocytes were cultured in supplemented Iscove’s medium [Iscove’s modified Dulbecco medium (Cytosystem, Castle Hill, Australia) supplemented with 10% heat-inactivated human AB+ serum, 2 mM L-glutamine, and 24µg/mL of gentamicin] in suspension in polytetrafluorethylene (Teflon) pots (Savillex, Minnetonka, MN) at an initial concentration of 106 cells/mL. Monocytes were cultured for 5 to 7 days prior to phagocytosis assay. All the reagents and culture supernatants were tested for lipopolysaccharide levels using the Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD).

Opsonization of target particles
Target particles were opsonized immediately prior to the phagocytosis assay. Sheep red blood cells (E; ICN-Cappel, Aurora, OH) were washed three times in phosphate-buffered saline (PBS; Trace Biosciences, Castle Hill, Australia), and opsonized with a subagglutinating titer (1:300) of rabbit anti-E antibody (ICN-Cappel, Aurora, OH) for 30 min at room temperature. Cells were washed five times in cold PBS and resuspended at a concentration of 1 x 108/mL in Iscove’s medium. Latex beads (3 µm in diameter; Sigma, St Louis, MO) were coated with bovine serum albumin (BSA) by incubation in 1 mL of PBS containing 10 mg of BSA (Sigma) overnight at 4°C, followed by five washes with cold PBS. Beads were opsonized with 10% rabbit anti-BSA antiserum (ICN-Cappel, Aurora, OH) for 30 min at room temperature, washed three times in PBS, and resuspended in PBS at a concentration of 4 x 108 cells/mL.

Phagocytosis assay using IgG-opsonized E
On days 5–7 after isolation, MDMs were plated onto 96-well plates (Costar, Cambridge, MA) at 5 x 104 cells per well in 100 µL of supplemented Iscove’s medium and allowed to adhere for 2 h at 37°C in a 5% CO2 humidified incubator. IgG-opsonized or unopsonized E was added to adhered MDMs at an E/MDM ratio of 10:1. The plate was centrifuged at 100 g for 5 min at 4°C and then placed at 37°C for phagocytosis to proceed. Phagocytosis was terminated after 30 min by placing the plates on ice and washing cells with ice-cold PBS. Phagocytosis of erythrocytes was quantified by a colorimetric assay [32 ]. Briefly, after unbound E were removed by washing with PBS, non-phagocytosed erythrocytes attached to MDMs were lysed with 0.2% NaCl for 3 min, followed by three washes with warm Iscove’s medium. Phagocytosed erythrocytes were measured by reaction of hemoglobin with 2,7-diaminoflurene (Sigma) after lysis of MDMs in 0.2 M Tris-HCl buffer containing 6 M urea. Absorbance was determined at 620 nm in a plate reader (Labsystems Multiskan, Helsinki, Finland) and compared to a standard curve generated using known numbers of E (ranging from 4x103 to 5x105). In selected experiments MDMs were incubated with the protein tyrosine kinase inhibitor Genistein (Calbiochem, Croydon, Australia) at concentrations ranging from 0.1 to 30 µM at 37°C for 30 min prior to phagocytosis assay.

Phagocytosis assays using IgG-opsonized beads
MDMs (2x106 cells) were dispensed into 4 mL polypropylene tubes (Becton Dickinson, Paramus, NJ), washed twice in calcium- and magnesium-free PBS (PBS-CMF; 500 g for 5 min), and cooled on ice for 20 min in 100 µL of PBS-CMF. MDMs were incubated with or without IgG-opsonized beads at 37°C in a shaking-water bath with a target/MDM ratio of 10:1. At specified time points, phagocytosis was arrested by plunging the tubes into ice and washing the MDMs in ice-cold PBS-CMF, followed by centrifugation at 20,000 g for 30 s. For immunoblotting and immunoprecipitation experiments, washed cells were lysed in 100 µL of Triton lysis buffer containing 25 mM Tris-HCl (pH 7.5), 0.14 M NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with phosphatase inhibitors [50 mM NaF, 1 mM sodium orthovanadate (Sigma), 40 mM ß-glycerophosphate (Sigma)], and the following protease inhibitors: 1 mM pefabloc, 1µM pepstatin, and 1 µM leupeptin (Boehringer-Mannheim, Mannheim, Germany).

GM-CSF stimulation
MDMs were stimulated with GM-CSF at 100 ng/mL (kindly provided by A. Lopez, Hanson Centre, Adelaide, Australia), immediately prior to addition of phagocytic targets. These phagocytic assays always commenced within 2 min of adding GM-CSF. Lysates were analyzed either for phagocytosis by a colorimetric assay, for tyrosine-phosphorylated proteins by immunoblotting, or for Syk phosphorylation by immunoprecipitation.

Immunoblotting and immunoprecipitation
Cell extracts containing equal amounts of proteins as determined by DC protein assay (Bio-Rad Laboratories, Hercules, CA) were boiled in sodium dodecyl sulfate (SDS) sample buffer [10 mM Tris (pH 8.0), 2 mM EDTA, 1% SDS, 5% ß-mercaptoethanol, 5% glycerol], resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and blocked for 2 h in either 3% BSA or 5% nonfat milk. The blots were probed with a recombinant antibody directed against phosphotyrosine (RC20; Transduction Laboratories, Lexington, KY) or paxillin (Transduction Laboratories) overnight at 4°C, followed by secondary antibody conjugated with horseradish peroxidase (Amersham Pharmacia, Buckinghamshire, England), and developed for enhanced chemiluminescence according to manufacturer’s instructions (Amersham Pharmacia). Alternatively, to determine the involvement of various proteins in Fc{gamma}R-mediated phagocytosis, cell lysates were immunoprecipitated with either a rabbit antibody against Hck (gift from H. C. Cheng, School of Biochemistry, Melbourne University), anti-Syk mAb (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-paxillin mAb and then probed with anti-phosphotyrosine antibody (PY20; Transduction Laboratories) or conversely, immunoprecipitated with anti-phosphotyrosine mAb and then probed with anti-Pyk2 (Transduction Laboratories) or anti-Syk mAb. Proteins were immunoprecipitated from extracts with antibodies overnight at 4°C, collected with 15 µL of protein G-Sepharose beads (1 h of incubation at 4°C) (Pharmacia Biotech, Uppsala, Sweden), washed five times in Triton lysis buffer, boiled in SDS sample buffer, resolved by 10% SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting as described above. To verify the equal protein, input blots were reprobed with relevant antibodies.

Immunofluorescence microscopy
Cells fixed with 1 mL of 3% Ultrapure formaldehyde (Polysciences, Warrington, PA) for 20 min were washed twice with cold 0.1 M glycine in PBS-CMF and permeabilized with 0.1% Triton X-100 (Merck, Kilsyth, Australia) for 1 min. After two washes with 1% fetal bovine serum in PBS-CMF, cells were stained for intracellular proteins with mouse anti-phosphotyrosine mAb (PY20) or isotype-matched control IgG1 (MOPC 21; Bionetics, Charleston, SC) for 30 min on ice. After two washes with cold PBS-CMF, cells were incubated with biotin-conjugated anti-mouse Ig (Silenus, Melbourne, Australia) for 30 min on ice, followed by two further washes with cold PBS-CMF. Subsequently, MDMs were incubated with Texas Red-conjugated streptavidin (Amersham Pharmacia) for 30 min on ice, washed once with cold PBS-CMF, fixed with 200 µL of 1% formaldehyde, and cytocentrifuged onto glass slides. Protein tyrosine phosphorylation in both resting and phagocytosing MDMs was analyzed by confocal laser microscopy (Bio-Rad MRC500). For some samples, cells were also stained with phalloidin conjugated to Alexa 488 (Molecular Probes, Eugene, OR) to determine colocalization of F-actin and phosphotyrosine.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enhanced protein tyrosine phosphorylation during Fc{gamma}R-mediated phagocytosis by MDMs
Two minutes after the addition of IgG-opsonized latex beads, increased tyrosine phosphorylation of a number of human MDM cytoplasmic proteins was evident by immunoblot analysis, with phosphorylation reaching a peak between 5 and 15 min (Fig. 1a ). Phagocytosis of IgG-opsonized E was inhibited by the protein tyrosine kinase inhibitor genistein in a dose-dependent manner (Fig. 1b) , suggesting that tyrosine kinase activity was required for Fc{gamma}R-mediated phagocytosis. Phagocytosis of unopsonized E by MDM cultured in the presence or absence of genistein was below the detection level of the assay.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 1. Fc{gamma}R-mediated phagocytosis and protein tyrosine phosphorylation. (a) MDMs incubated with IgG-opsonized latex beads (2x107) for the indicated times were lysed in Triton X-100 buffer, and samples of lysate containing 50 µg of protein were resolved by SDS-polyacrylamide gel electrophoresis, then probed with antiphosphotyrosine conjugated to horseradish peroxidase (RC20). Results shown are representative of five experiments using MDMs prepared from different donors. (b) Inhibition of phagocytosis by genistein. Phagocytosis of IgG-opsonized E (•) or E ({circ}) was measured in the presence of the indicated concentrations of genistein via colorimetric assay as described in Materials and Methods. Data represent means (±SD) of triplicate determinations.

 
The majority of antiphosphotyrosine immunoreactivity in MDMs was localized around the internalized IgG-opsonized latex beads (Fig. 2b ) colocalized with sites of actin polymerization (Fig. 2d) . This finding is consistent with binding of tyrosine-phosphorylated proteins to a cytoskeleton which has been rearranged around the phagosome. Within 2 min of initiation of phagocytosis, paxillin, a multidomain adapter protein involved in organizing focal adhesion complexes, was also found to be preferentially localized around the phagosome (Fig. 2c) . Control experiments verified a lack of nonspecific staining with the isotype-matched antibody (Fig. 2a) .



View larger version (80K):
[in this window]
[in a new window]
  		Figure 2. Colocalization of tyrosine-phosphorylated proteins and polymerized actin around phagocytic cups in MDMs ingesting IgG-opsonized latex beads. MDMs were incubated with IgG-opsonized latex beads (3 µm diameter) for 2 min, fixed with 3% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with (a) an isotype-matched control (MOPC 21); (b, d) mouse anti-phosphotyrosine mAb; or (c) mouse anti-paxillin mAb, followed by biotinylated anti-mouse Ig and Texas Red streptavidin conjugate; (d) MDMs were double stained for phosphotyrosine first as above and then with Alexa 488-labeled phalloidin. Areas of the cell staining with both antibodies appear yellow. MDMs were examined by confocal laser microscopy. Bar = 3 µm.

 
Activation of Hck, Syk, and Pyk2 tyrosine kinases
Having demonstrated a requirement for tyrosine kinase activity during Fc{gamma}R-mediated phagocytosis by MDMs, we sought to identify the proteins which were tyrosine phosphorylated in the experiments depicted in Fig. 1 . Based on estimated size, we investigated several tyrosine kinases that could potentially be activated during this process. Enhanced tyrosine phosphorylation of Hck, Syk, and Pyk2 was observed within 2–5 min of Fc{gamma}R-mediated phagocytosis (Fig. 3 ). The input of Syk, Hck, and Pyk2 proteins was standardized according to Lowry’s determinations. Protein levels are shown by reprobing the blots with relevant antibodies [Fig. 3a and 3b (lower panels)].



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Activation of Hck, Syk, and Pyk2 tyrosine kinases during Fc{gamma}R-mediated phagocytosis in human MDMs. Cells (2x106) were incubated with IgG-opsonized latex beads (2x107) in polypropylene tubes in a shaking water bath for the indicated times (0 to 30 min) at 37°C and then lysed in Triton X-100 buffer. Samples of lysate containing 100 µg of protein were immunoprecipitated with (a) anti-Hck or (b) anti-Syk; resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-PY-horseradish peroxidase; or immunoprecipitated with (c) anti-PY mAb, resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Syk or anti-Pyk2 mAb. The data are representative of three experiments using MDMs from different donors. Protein levels are shown by re-probing the blots with (a) anti-Hck mAb (lower panel) or (b) anti-Syk mAb (lower panel).

 
Redistribution of paxillin
Within 2 to 5 min of Fc{gamma}R-mediated phagocytosis, there was an increase in paxillin phosphorylation which had declined by 30 min (Fig. 4a ). Immunoblot analysis of Triton-soluble lysates probed with anti-paxillin antibody showed that the total level of extracted paxillin levels changed in a corresponding fashion (Fig. 4b) . Total extracted paxillin levels did not vary during phagocytosis when MDMs were lysed in SDS sample buffer (total cell extract) (Fig. 4c) . Taken together, these results suggest that in different phases of Fc{gamma}R-mediated phagocytosis, paxillin is distributed predominantly in either the cytoskeletal (detergent-insoluble) fraction or cytoplasmic (detergent-soluble) fraction.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 4. Redistribution of paxillin between cytoplasm and cytoskeleton during Fc{gamma}R-mediated phagocytosis in MDMs. Cells were incubated with IgG-opsonized latex beads (2x107) in a shaking water bath for the indicated times (0 to 30 min) at 37°C and lysed in either Triton X-100 or SDS sample buffer. (a) Triton X-100 lysates containing 100 µg of protein were immunoprecipitated with anti-paxillin mAb, resolved by SDS-polyacrylamide gel electrophoresis, and probed with anti-PY mAb. (b) Triton X-100 lysates (cytoplasmic fraction) or (c) SDS lysates (whole-cell extracts) containing 50 µg of protein were resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-paxillin mAb. The data shown are representative of three experiments.

 
Stimulation of Fc{gamma}R-mediated phagocytosis by GM-CSF
Stimulation of MDMs with GM-CSF (100 ng/mL) augmented the level of Fc{gamma}R-mediated phagocytosis by 20–30% (Fig. 5a ). GM-CSF stimulated tyrosine phosphorylation of MDM cellular proteins, and this effect was further augmented during phagocytosis of IgG-opsonized latex beads (Fig. 5b) . We next determined whether GM-CSF directly activated Syk, because in murine macrophages this kinase is a well-established critical intermediate in Fc{gamma} receptor signaling [3 ]. Incubation of MDM with GM-CSF for 2 to 5 min did not alter the extent of tyrosine phosphorylation of Syk above the levels observed during Fc{gamma}R-mediated phagocytosis (Fig. 5c) . In addition, control MDMs which had not been exposed to target particles did not display detectable Syk phosphorylation in either GM-CSF-treated or untreated samples. The level of Syk protein was equal for all the samples (Fig. 5c , lower panel). Syk coimmunoprecipitated from Triton lysates with several other phosphoproteins in a reproducible pattern. This pattern may represent a stable complex of Syk with Fc{gamma} receptors and/or other signaling molecules located around the phagosome because a similar pattern was observed in experiments when MDM lysates were immunoprecipitated with anti {gamma}-chain antibody (data not shown). Taken together, these data suggest that GM-CSF augments the process of Fc{gamma}R-mediated phagocytosis by triggering tyrosine phosphorylation of a number of cellular proteins and most probably by acting downstream of Syk (and, by extension, downstream of Src) in the Fc{gamma}R-mediated cascade or via a pathway parallel to these events.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Effect of GM-CSF on Fc{gamma}R-mediated phagocytosis, tyrosine phosphorylation and Syk activation. MDMs were treated with GM-CSF (100 ng/mL), where indicated, immediately prior to addition of target particles. (a) MDMs (5x104 per well) plated onto 96-well plates were incubated with IgG-opsonized E or E for 20 min, and phagocytosis was determined as described in Materials and Methods. Data are representative of 3 donors and are means ± SD. MDMs (2x106) were incubated with IgG-opsonized latex beads (2x107) for the indicated times, and Triton X-100 buffer lysates were analyzed for (b) tyrosine-phosphorylated proteins by probing with horseradish peroxidase-conjugated anti-PY antibody (representative of five experiments using MDMs prepared from different donors); or (c) Syk tyrosine phosphorylation by immunoprecipitation of Syk and immunoblotting with horseradish peroxidase-conjugated anti-PY antibody (representative of five MDM donors). Input level of Syk was determined by reprobing the blots with Syk mAb.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data show that Fc{gamma}R-mediated phagocytosis by human macrophages was mediated by tyrosine phosphorylation of a number of cellular proteins which localized around the phagocytic cup and that phagocytosis was blocked by a protein tyrosine kinase inhibitor, genistein. Both Hck (a macrophage-specific Src family kinase) and Syk were phosphorylated after engagement of human MDM Fc{gamma} receptors. Here, we also provide the first evidence for activation of Pyk2 and redistribution of the phosphorylated form of paxillin between the cytoplasm and cytoskeleton during Fc{gamma}R-mediated phagocytosis. In addition, we show augmentation of Fc{gamma}R-mediated phagocytosis by GM-CSF, concomitant with increased tyrosine phosphorylation of a number of cellular proteins, which did not however include Syk.

A role for Hck in Fc{gamma}R signaling has been suggested, based on data showing that macrophages prepared from mice deficient in three Src family tyrosine kinases, Hck, Fgr, and Fyn, exhibit poor signaling downstream of Fc{gamma}R (i.e., Syk activation) as well as reduced levels of Fc{gamma}R-induced functional responses such as phagocytosis, actin cup formation, and respiratory burst [3 , 33 ]. A specific requirement for Hck in phagocytosis has also been demonstrated using Hck knock-out mice, which fail to internalize IgG-opsonized E, while other hemopoietic functions remain intact [34 ]. This study also suggested that some functions of Hck are subsumed through compensatory increases in the activity of another Src kinase, Lyn, although Lyn could not substitute for Hck in phagocytosis. Both Hck and Lyn have been shown also to coimmunoprecipitate with Fc{gamma}R after cross-linking of receptors on THP-1 monocytic cell lines [35 ] and human monocytes [36 ]. In the present work we demonstrate activation of Hck during Fc{gamma}R-mediated phagocytosis in human macrophages.

Phosphorylation of ITAM creates docking sites for Syk, a kinase shown by a number of studies to play a critical role in coupling phagocytosis-promoting Fc{gamma}Rs to the actin-based cytoskeleton [reviewed in 25]. A study using stem-loop Syk antisense oligonucleotides in human monocytes [24 ], as well as transfection studies using COS and DT40 cell lines and gene knockout studies, has demonstrated that Syk activation is absolutely required for Fc{gamma}R-mediated phagocytosis, actin assembly, and Fc{gamma}R-mediated transport to lysosomes [2 , 3 , 8 , 22 ]. Furthermore, Syk has also been found to be a part of the activated Fc{gamma}R complex after cross-linking of receptors in U937 and THP-1 cell lines [37 , 38 ]. Our data confirm that Syk is phosphorylated in human MDMs after Fc{gamma}R-mediated phagocytosis.

Activation of Syk is thought to result in the phosphorylation of various substrates located in the submembranous region beneath phagocytosed particles, which are required for actin polymerization and cytoskeletal rearrangement [8 ]. Paxillin is a potential downstream effector of Syk, because this protein has been shown to interact with a variety of proteins involved in growth control and cytoskeletal reorganization [reviewed in 28]. The abundance of binding motifs in paxillin for structural and regulatory proteins has led to a suggestion that it is important in recruiting signaling molecules at sites of actin rearrangement and in facilitating their interaction during phagocytosis [39 ]. Paxillin has been previously shown to be tyrosine phosphorylated in murine peritoneal macrophages during Fc{gamma}R-mediated phagocytosis [6 ] and to colocalize with phagosomes [6 , 26 ]. Here, we confirm and extend those observations by showing that the phosphorylated form of paxillin is redistributed between cytoplasm and cytoskeleton during phagocytosis in human macrophages.

Paxillin phosphorylation after cell adhesion has been postulated to involve the kinase p125 FAK, shown previously to bind to paxillin LD motifs [28 ]. The involvement of FAK in Fc{gamma}R-mediated phagocytosis remains controversial, with one study showing no enhancement of tyrosine phosphorylation of FAK during phagocytosis in murine macrophages [6 ] and another report demonstrating the expression and phosphorylation of FAK as a result of Fc{gamma}R cross-linking in human monocytes [40 ]. However, it has been reported that human monocytes/macrophages isolated under stringent conditions and free of platelet contamination do not express FAK [29; A. Jaworowski, unpublished results). We therefore determined whether Pyk2, a kinase with 45% sequence identity to FAK [41 ], known to be expressed in human monocytes/macrophages [29 ] and to bind to paxillin [42 ], is activated during Fc{gamma}R-mediated phagocytosis. Our results demonstrated that tyrosine phosphorylation of Pyk2 was increased during Fc{gamma}R-mediated phagocytosis. This is the first report of stimulation of Pyk2 tyrosine phosphorylation in response to phagocytosis, although this kinase has been implicated in other processes involving reorganization of the cytoskeleton, such as locomotion and adhesion [29 , 43 , 44 ].

Fc{gamma}R phagocytosis by both human [45 ] and murine [46 ] macrophages has been found to be up-regulated by GM-CSF, a cytokine known to augment a number of macrophage effector functions [reviewed in 47, 48]. Since GM-CSF receptor does not possess an intrinsic tyrosine kinase catalytic domain, the activation of protein tyrosine kinases associated with the ß subunit of the receptor (Janus kinase, signal-transducing activator of transcription, and Src families) mediates GM-CSF-stimulated proliferation, differentiation, and gene expression [49 , 50 ]. Stimulation of human monocytes with GM-CSF for 48 h resulted in increased expression of Fc{gamma}R II and increased binding of IgG-opsonized particles [51 ]. In our experiments, short-term pretreatment of MDMs with GM-CSF (2–5 min) was unlikely to increase Fc{gamma}R expression and de novo receptor synthesis. Our observations and those of Rossman et al. suggest that GM-CSF may augment Fc{gamma}R-mediated phagocytosis by several mechanisms, i.e., by stimulating the level of Fc{gamma} receptor expression as well as the phosphorylation of intracellular tyrosine kinases. Because GM-CSF treatment of MDMs did not enhance phosphorylation of Syk, we postulate that GM-CSF exerts its stimulatory effect on Fc{gamma}R-phagocytosis by acting either distally to Syk kinase in the Fc{gamma}R-mediated signaling cascade or in a parallel pathway mediated via Fc{gamma} receptor.

Our study shows that the early signaling events during Fc{gamma}R-mediated phagocytosis in human MDMs were similar to those previously characterized in murine macrophages. In both species, key protein tyrosine kinases were activated during this process. In addition, we have demonstrated a novel role for the FAK-related kinase, Pyk2, in phagocytosis and have provided a baseline for further studies on the stimulatory role of GM-CSF on macrophage function. Because recent clinical studies have demonstrated the successful outcome of adjunctive GM-CSF treatment for opportunistic infections in immunosuppressed patients [52 , 53 ], an understanding of the mechanism underlying macrophage stimulation by GM-CSF is potentially of therapeutic benefit.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Australian National Council on AIDS and related Diseases through the Australian National Centres in HIV Virology Research and the Macfarlane Burnet Centre Research Fund. Katherine Kedzierska is a recipient of a National Health and Medical Research Council Postgraduate Scholarship.

The authors thank John Mills for his critical review of the manuscript.

Anthony Jaworowski and Suzanne M. Crowe contributed equally to this work.

Received December 22, 2000; revised April 4, 2001; accepted April 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Daeron, M. (1997) Fc receptor biology Annu. Rev. Immunol. 15,203-234[Medline]
  2. Kiefer, F., Brumell, J., Al-Alawi, N., Latour, S., Cheng, A., Veillette, A., Grinstein, S., Pawson, T. (1998) The Syk protein tyrosine kinase is essential for Fc{gamma} receptor signaling in macrophages and neutrophils Mol. Cell. Biol. 18,4209-4220[Abstract/Free Full Text]
  3. Crowley, M. T., Costello, P. S., Fitzer-Attas, C. J., Turner, M., Meng, F., Lowell, C., Tybulewicz, V. L., DeFranco, A. L. (1997) A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages J. Exp. Med. 186,1027-1039[Abstract/Free Full Text]
  4. Strzelecka, A., Pyrzynska, B., Kwiatkowska, K., Sobota, A. (1997) Syk kinase, tyrosine-phosphorylated proteins and actin filaments accumulate at forming phagosomes during Fcgamma receptor-mediated phagocytosis Cell. Motil. Cytoskeleton 38,287-296[Medline]
  5. Aderem, A. (1995) The MARCKS family of protein kinase-C substrates Biochem. Soc. Trans. 23,587-591[Medline]
  6. Greenberg, S., Chang, P., Silverstein, S. C. (1994) Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages J. Biol. Chem. 269,3897-3902[Abstract/Free Full Text]
  7. Greenberg, S., Chang, P., Silverstein, S. C. (1993) Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages J. Exp. Med. 177,529-534[Abstract/Free Full Text]
  8. Cox, D., Chang, P., Kurosaki, T., Greenberg, S. (1996) Syk tyrosine kinase is required for immunoreceptor tyrosine activation motif-dependent actin assembly J. Biol. Chem. 271,16597-16602[Abstract/Free Full Text]
  9. Greenberg, S., Chang, P., Wang, D. C., Xavier, R., Seed, B. (1996) Clustered Syk tyrosine kinase domains trigger phagocytosis Proc. Natl. Acad. Sci. USA 93,1103-1107[Abstract/Free Full Text]
  10. Indik, Z. K., Hunter, S., Huang, M. M., Pan, X. Q., Chien, P., Kelly, C., Levinson, A. I., Kimberly, R. P., Schreiber, A. D. (1994) The high affinity Fc gamma receptor (CD64) induces phagocytosis in the absence of its cytoplasmic domain: the gamma subunit of Fc gamma RIIIA imparts phagocytic function to Fc gamma RI Exp. Hematol. 22,599-606[Medline]
  11. Kusner, D. J., Hall, C. F., Jackson, S. (1999) Fc gamma receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgG-opsonized particles J. Immunol. 162,2266-2274[Abstract/Free Full Text]
  12. Zheleznyak, A., Brown, E. J. (1992) Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation: evidence for protein kinase C translocation to phagosomes J. Biol. Chem. 267,12042-12048[Abstract/Free Full Text]
  13. Aderem, A., Underhill, D. (1999) Mechanisms of phagocytosis in macrophages Annu. Rev. Immunol. 17,593-623[Medline]
  14. Kanakaraj, P., Duckworth, B., Azzoni, L., Kamoun, M., Cantley, L. C., Perussia, B. (1994) Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA-ligand interaction J. Exp. Med. 179,551-558[Abstract/Free Full Text]
  15. Ninomiya, N., Hazeki, K., Fukui, Y., Seya, T., Okada, T., Hazeki, O., Ui, M. (1994) Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling J. Biol. Chem. 269,22732-22737[Abstract/Free Full Text]
  16. Hackam, D. J., Rotstein, O. D., Schreiber, A., Zhang, W., Grinstein, S. (1997) Rho is required for the initiation of calcium signaling and phagocytosis by Fcgamma receptors in macrophages J. Exp. Med. 186,955-966[Abstract/Free Full Text]
  17. Cox, D., Chang, P., Zhang, Q., Reddy, P. G., Bokoch, G. M., Greenberg, S. (1997) Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes J. Exp. Med. 186,1487-1494[Abstract/Free Full Text]
  18. Jouvin, M. H., Adamczewski, M., Numerof, R., Letourneur, O., Valle, A., Kinet, J. P. (1994) Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor J. Biol. Chem. 269,5918-5925[Abstract/Free Full Text]
  19. Sarmay, G., Pecht, I., Gergely, J. (1994) Protein-tyrosine kinase activity tightly associated with human type II Fc gamma receptors Proc. Natl. Acad. Sci. USA 91,4140-4144[Abstract/Free Full Text]
  20. Indik, Z. K., Park, J. G., Hunter, S., Mantaring, M., Schreiber, A. D. (1995) Molecular dissection of Fc gamma receptor-mediated phagocytosis Immunol. Lett. 44,133-138[Medline]
  21. Park, J. G., Murray, R. K., Chien, P., Darby, C., Schreiber, A. D. (1993) Conserved cytoplasmic tyrosine residues of the gamma subunit are required for a phagocytic signal mediated by Fc gamma RIIIA J. Clin. Invest. 92,2073-2079
  22. Bonnerot, C., Briken, V., Brachet, V., Lankar, D., Cassard, S., Jabri, B., Amigorena, S. (1998) Syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes EMBO J 17,4606-4616[Medline]
  23. Indik, Z. K., Park, J. G., Pan, X. Q., Schreiber, A. D. (1995) Induction of phagocytosis by a protein tyrosine kinase Blood 85,1175-1180[Abstract/Free Full Text]
  24. Matsuda, M., Park, J. G., Wang, D. C., Hunter, S., Chien, P., Schreiber, A. D. (1996) Abrogation of the Fc gamma receptor IIA-mediated phagocytosis signal by stem-loop Syk antisense oligonucleotides Mol. Biol. Cell. 7,1095-1106[Abstract]
  25. Greenberg, S. (1995) Signal transduction of phagocytosis Trends Cell. Biol. 5,93-99[Medline]
  26. Allen, L. A., Aderem, A. (1996) Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages J. Exp. Med. 184,627-637[Abstract/Free Full Text]
  27. Greenberg, S., Burridge, K., Silverstein, S. C. (1990) Colocalization of F-actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages J. Exp. Med. 172,1853-1856[Abstract/Free Full Text]
  28. Turner, C. E. (1998) Paxillin Int. J. Biochem. Cell. Biol. 30,955-959[Medline]
  29. Li, X., Hunter, D., Morris, J., Haskill, J. S., Earp, H. S. (1998) A calcium-dependent tyrosine kinase splice variant in human monocytes: activation by a two-stage process involving adherence and a subsequent intracellular signal J. Biol. Chem. 273,9361-9364[Abstract/Free Full Text]
  30. Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S., Pasztor, L. M., White, R. A., Groopman, J. E., Avraham, H. (1995) Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain J. Biol. Chem. 270,27742-27751[Abstract/Free Full Text]
  31. Crowe, S. M., Mills, J., McGrath, M. S. (1987) Quantitative immunocytofluorographic analysis of CD4 surface antigen expression and HIV infection of human peripheral blood monocyte/macrophages AIDS Res. Hum. Retroviruses 3,135-145[Medline]
  32. Gebran, S. J., Romano, E. L., Pons, H. A., Cariani, L., Soyano, A. N. (1992) A modified colorimetric method for the measurement of phagocytosis and antibody-dependent cell cytotoxicity using 2,7-diaminofluorene J. Immunol. Methods 151,255-260[Medline]
  33. Fitzer-Attas, C. J., Lowry, M., Crowley, M. T., Finn, A. J., Meng, F., DeFranco, A. L., Lowell, C. A. (2000) Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn J. Exp. Med. 191,669-682[Abstract/Free Full Text]
  34. Lowell, C. A., Soriano, P., Varmus, H. E. (1994) Functional overlap in the src gene family: inactivation of hck and fgr impairs natural immunity Genes Dev 8,387-398[Abstract/Free Full Text]
  35. Ghazizadeh, S., Bolen, J., Fleit, H. (1994) Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells J. Biol. Chem. 269,8878-8884[Abstract/Free Full Text]
  36. Wang, A., Scholl, P., Geha, R. (1994) Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn J. Exp. Med. 180,1165-1170[Abstract/Free Full Text]
  37. Darby, C., Geahlen, R., Schreiber, A. (1994) Stimulation of macrophage Fc gamma RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein J. Immunol. 152,5429-5437[Abstract]
  38. Ghazizadeh, S., Bolen, J., Fleit, H. (1995) Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells Biochem. J. 305,669-674
  39. Turner, C., Brown, M., Perrotta, J., Riedy, M., Nikolopoulos, S., McDonald, A., Bagrodia, S., Thomas, S., Leventhal, P. (1999) Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal remodeling J. Cell Biol. 145,851-863[Abstract/Free Full Text]
  40. Pan, X. Q., Darby, C., Indik, Z. K., Schreiber, A. D. (1999) Activation of three classes of nonreceptor tyrosine kinases following Fc gamma receptor crosslinking in human monocytes Clin. Immunol. 90,55-64[Medline]
  41. Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., Sasaki, T. (1995) Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily J. Biol. Chem. 270,21206-21219[Abstract/Free Full Text]
  42. Hiregowdara, D., Avraham, H., Fu, Y., London, R., Avraham, S. (1997) Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin J. Biol. Chem. 272,10804-10810[Abstract/Free Full Text]
  43. Duong, L. T., Lakkakorpi, P. T., Nakamura, I., Machwate, M., Nagy, R. M., Rodan, G. A. (1998) Pyk2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha/beta integrin, and phosphorylated by Src kinase J. Clin. Invest. 102,881-892[Medline]
  44. Astier, A., Avraham, H., Manie, S. N., Groopman, J., Canty, T., Avraham, S., Freedman, A. S. (1997) The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas J. Biol. Chem. 272,228-232[Abstract/Free Full Text]
  45. Capsoni, F., Minonzio, F., Ongari, A. M., Rizzardi, P. G., Lazzarin, A., Zanussi, C. (1992) Monocyte-derived macrophage function in HIV-infected subjects: in vitro modulation by rIFN-gamma and rGM-CSF Clin. Immunol. Immunopathol. 62,176-182[Medline]
  46. Coleman, D. L., Chodakewitz, J. A., Bartiss, A. H., Mellors, J. W. (1988) Granulocyte-macrophage colony-stimulating factor enhances selective effector functions of tissue-derived macrophages Blood 72,573-578[Abstract/Free Full Text]
  47. Metcalf, D. (1985) The granulocyte-macrophage colony stimulating factors Science 229,16-21[Abstract/Free Full Text]
  48. Armitage, J. (1998) Emerging applications of recombinant human granulocyte-macrophage colony stimulating factor Blood 92,4491-4508[Free Full Text]
  49. Corey, S. J., Anderson, S. M. (1999) Src-related protein tyrosine kinases in hematopoiesis Blood 93,1-14[Free Full Text]
  50. Mui, A.-F., Wakao, H., O’Farrel, A.-M., Harada, N., Miyajima, A. (1995) Interleukin-1, granulocyte-macrophage colony stimulating factor and interleukin -5 transduce signal through two STAT5 homologs EMBO J 14,1166-1175[Medline]
  51. Rossman, M. D., Ruiz, P., Comber, P., Gomez, F., Rottem, M., Schreiber, A. D. (1993) Modulation of macrophage FC gamma receptors by rGM-CSF Exp. Hematol. 21,177-183[Medline]
  52. Kedzierska, K., Mak, J., Mijch, A., Cooke, I., Rainbird, M., Roberts, S., Paukovics, G., Jolley, D., Lopez, A., Crowe, S. M. (2000) Granulocyte-macrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type 1-infected monocytes/macrophages in vitro and in vivo J. Infect. Dis. 181,390-394[Medline]
  53. Vazquez, J. A., Gupta, S., Villanueva, A. (1998) Potential utility of recombinant human GM-CSF as adjunctive treatment of refractory oropharyngeal candidiasis in AIDS patients Eur. J. Clin. Microbiol. Infect. Dis. 17,781-783[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Tsuboi and J. Meerloo
Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages
J. Biol. Chem., November 23, 2007; 282(47): 34194 - 34203.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Leeansyah, B. D. Wines, S. M. Crowe, and A. Jaworowski
The Mechanism Underlying Defective Fc{gamma} Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages
J. Immunol., January 15, 2007; 178(2): 1096 - 1104.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
N. L. Webster, K. Kedzierska, R. Azzam, G. Paukovics, J. Wilson, S. M. Crowe, and A. Jaworowski
Phagocytosis stimulates mobilization and shedding of intracellular CD16A in human monocytes and macrophages: inhibition by HIV-1 infection
J. Leukoc. Biol., February 1, 2006; 79(2): 294 - 302.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. P. Scott, F. Zappacosta, E. Y. Kim, R. S. Annan, and W. T. Miller
Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1
J. Biol. Chem., July 26, 2002; 277(31): 28238 - 28246.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Kedzierska, P. Ellery, J. Mak, S. R. Lewin, S. M. Crowe, and A. Jaworowski
HIV-1 Down-Modulates {gamma} Signaling Chain of Fc{gamma}R in Human Macrophages: A Possible Mechanism for Inhibition of Phagocytosis
J. Immunol., March 15, 2002; 168(6): 2895 - 2903.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kedzierska, K.
Right arrow Articles by Crowe, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kedzierska, K.
Right arrow Articles by Crowe, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS