Enhanced Phagocytosis through Inhibition of de Novo Ceramide Synthesis*

Vania Hinkovska-GalchevaDagger , Laurence BoxerDagger , Pamela J. MansfieldDagger , Alan D. Schreiber§, and James A. Shayman||

From the Dagger  Department of Pediatrics, Division of Hematology Oncology, the  Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, Michigan 48109 and the § Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, June 21, 2002, and in revised form, October 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fcgamma receptors are important mediators of the binding of IgG to and induction of phagocytosis in neutrophils. COS-1 cells provide a potentially useful model for studying these receptors because transfection with the Fcgamma RIIA renders these cells phagocytic. During Fcgamma RIIA-mediated phagocytosis in COS-1 cells, endogenous ceramide levels increased 52% by 20 min (p < 0.01). Phospholipase D activity increased by 62% (p < 0.01). Correspondingly, the phagocytic index increased by 3.7-fold by 20 min. Two inhibitors of ceramide formation were used to assess the consequences of reduced ceramide generation. L-Cycloserine, an inhibitor that blocks serine palmitoyltransferase activity, lowered both sphingosine and ceramide levels. Under these conditions, the phagocytic index increased 100% in the presence of 2 mM L-cycloserine. The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B1. When cells were treated with 5-50 µM fumonisin B1, the cellular level of ceramide decreased in a concentration-dependent manner, while simultaneously the phagocytic index increased by 52%. Concomitantly, three indirect measures of Fcgamma RIIA activity were altered with the fall in ceramide levels. Syk phosphorylation, phospholipase D activity, and mitogen-activated protein (MAP) kinase phosphorylation were increased at 30 min. When Syk phosphorylation was blocked with piceatannol and cells were similarly challenged, phosphatidylinositol 3-kinase activation was blocked, but no changes in either ceramide accumulation or MAP kinase activation were observed. Ceramide formation and MAP kinase activation are therefore not dependent on Syk kinase activity in this system. These results indicate that COS-1 cells provide a useful model for the recapitulation of sphingolipid signaling in the study of phagocytosis. Ceramide formed by de novo synthesis may represent an important mechanism in the regulation of phagocytosis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An important function of PMN1 and tissue macrophages is the phagocytosis of IgG-coated cells. Receptors for the constant region of IgG, the Fcgamma receptors, enable these cells to detect and destroy IgG-coated microorganisms during infection and IgG-coated blood cells in autoimmune disorders. There are three major classes of Fcgamma receptors, designated Fcgamma RI, Fcgamma RII, and Fcgamma RIII. The various isoforms of Fcgamma Rs have highly conserved extracellular portions, but their cytoplasmic domains are heterogeneous. This heterogeneity suggests that all Fcgamma Rs may not be involved in phagocytosis. One of the main problems for understanding the Fcgamma R requirements for phagocytosis has been that multiple isoforms are expressed on each type of phagocytic cell. The expression of Fcgamma R in a cell line that does not have endogenous Fcgamma Rs provides a model for defining the molecular signals associated with phagocytosis. Fcgamma receptor-transfected COS-1 established that a single class of human Fcgamma receptor can induce phagocytosis of IgG-sensitized red blood cells (1).

Fcgamma RIIA, which is restricted to cells of megakaryocytic and myeloid lines, mediates several functions. Stimulation of Fcgamma RIIA leads to antibody-dependent cellular cytotoxicity, superoxide production, phagocytosis in monocytes (2, 3), and platelet activation (4, 5). Fcgamma RIIA expression is increased by interferon-gamma and granulocyte-macrophage colony-stimulating factor and is decreased by glucocorticoids (6, 7). Thus COS-1 cells transfected with Fcgamma RIIA provide a potentially useful model for the study of phagocytosis.

The tyrosine kinase Syk plays a critical role in the phagocytic pathway mediated by Fcgamma receptors. A cytoplasmic amino acid motif, known as ITAM, is present on Fcgamma RIIA, Fcgamma RI/gamma , and Fcgamma RIIIA/gamma and is essential for phagocytosis. Cross-linking of the Fc receptor induces binding of Src family protein tyrosine kinase to the ITAM, leading to an activation of the Src family protein tyrosine kinases and ITAM tyrosine phosphorylation (8, 9). This serves to recruit Syk, an SH2-domain-containing tyrosine kinase, which when activated phosphorylates multiple substrates, including neighboring ITAMs. Syk is recruited from a cytosolic pool. It is also possible that a small number of preformed Syk-ITAM complexes exist in resting cells (10).

The induction of Syk phosphorylation by cross-linking of Fcgamma RII therefore suggests that Syk plays an important role in phagocytic signaling through Fcgamma RIIA (8, 11, 12). The essential role for Syk in phagocytosis signal transduction is supported by the observation that Syk is a necessary component in ITAM-dependent activation of actin assembly (13). Down-modulation of Syk expression in monocytes using Syk antisense oligonucleotides also results in the decreased ability of these cells to ingest IgG-coated particles (14). When expressed in COS-1 cells, Syk kinase enhances the phagocytic signal induced by Fcgamma RIIA and increases the number of cells able to mediate phagocytosis (12).

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. These not only include glycerolipids but sphingolipids as well. Sphingolipids are composed of all lipids carrying a long chain sphingoid amine. Although the role of sphingolipids in membrane structure and organization has been appreciated for some time, members of this diverse group of lipids have recently emerged as a novel class of signaling molecules that also affect phagocytosis.

We observed previously (15-17) that ceramide is formed in PMN following ingestion of EIgG and that ceramide plays a negative modulatory role in both phagolysosome formation and oxidant release. Sphingosine is known to inhibit phagocytosis by blocking the action of protein kinase C beta  or protein kinase C delta  as well as inhibiting the formation of diacylglycerol by phosphatidic phosphohydrolase (18, 19). Sphingolipids are interconvertible molecules. Thus when using exogenously added sphingolipids, it is often difficult to ascertain which metabolite is responsible for a specific biological response (20, 21). Inhibitors of sphingolipid metabolism provide an alternative method for altering the intracellular concentration of sphingolipids. In contrast to short chain sphingolipid analogs, these inhibitors alter the levels of endogenous sphingolipids (22, 23). The first step in sphingolipid synthesis is the condensation of a fatty acyl-CoA, usually palmitoyl-CoA, with serine in a reaction catalyzed by serine palmitoyltransferase (3-ketosphingosine synthase). L-Cycloserine and beta -chloro-L-alanine block sphingosine and ceramide biosynthesis by inhibiting serine palmitoyltransferase activity both in vitro and in vivo (24, 25). The next step, the formation of ceramides resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase, is inhibited by the fungal toxin fumonisin B1. These inhibitors will provide the first step in determining if de novo synthesis of either sphingosine or ceramide is required for phagocytosis.

In the present study we report that COS-1 cells transfected with Fcgamma IIA receptor replicate many of the events associated with phagocytosis in PMNs. We also observe that phagocytosis is associated with the formation of ceramide through de novo synthesis and that inhibition of ceramide synthesis increases phagocytosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All phospholipids, imidazole, fumonisin B1, L-cycloserine, diethylenetriaminepentaacetic acid, n-octyl-beta -D-glucopyranoside, and proteinase inhibitors pepstatin and leupeptin were obtained from Sigma. sn-1,2-Diacylglycerol kinase from Escherichia coli and dithiothreitol were purchased from Calbiochem. Silica Gel 60 thin layer chromatography plates were purchased from VWR (Chicago, IL); [gamma -32P]ATP was obtained from ICN Pharmaceuticals, Inc. (Irvine, CA), 1-O-[3H]Octadecyl-sn-glycero-3-phosphocholine, Western blotting detection reagents, and horseradish peroxidase-conjugated sheep anti-mouse antibody were from Amersham Biosciences, and [3H]acetic anhydride was purchased from American Radiolabeled Chemicals (St. Louis, MO). Polyclonal and monoclonal antibodies against Syk were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phosphotyrosine antibody 4G10 and antibody against phosphatidylinositol 3-kinase 85 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibodies against ERK1 and ERK2, recognizing the phosphorylated and nonphosphorylated forms of p44 and p42, were obtained from New England Biolabs (Beverly, MA). Dulbecco's modified Eagle's medium, trypsin EDTA, L-glutamine, penicillin/streptomycin, and geneticin (G118 sulfate) were from Invitrogen. Sheep erythrocytes were purchased from BioWhittaker (Walkersville, MD) and were opsonized with anti-sheep erythrocyte IgG (Chapel Organon Teknika, Durham, NC). Polyvinylidene difluoride membranes were from Schleicher & Schuell. Piceatannol (3,4,3',5'-tetra-hydroxytrans-stilbene) was obtained from Alexis (San Diego, CA).

Cell Cultures-- COS-1 cells stably transfected with Fcgamma RIIA receptor cDNA were maintained in Dulbecco's modified Eagle's medium containing glucose 4.5 mg/ml, glutamine 25 mg/ml, streptomycin 100 units/ml, penicillin (100 mg/ml), and 10% heat-inactivated fetal calf serum. After 24 h the medium was replaced with fresh medium (for control cells) or fresh medium containing inhibitors or agonists.

Sheep Erythrocytes-- Sheep red blood cells were sensitized with rabbit IgG anti-sheep erythrocyte antibody as described previously (16). Erythrocytes (109/ml) were incubated for 30 min at 37 °C with anti-sheep erythrocyte IgG (1:350), followed by incubation on ice for 30 min. Antibody-treated erythrocytes (EIgG) were washed three times and suspended in the same buffer at 2-3 × 109.

Phagocytosis Assays-- Phagocytosis assays were conducted as described previously (26). For studies with fumonisin B1 or cycloserine, cells were incubated 24 or 6 h, respectively, prior to the experiment. Fumonisin B1 was applied to cultured cells dissolved in Me2SO, and L-cycloserine was dissolved in water.

Assay for Ceramide Formation-- Lipids were extracted by the method of Van Veldhoven and Bell (27). Total cellular ceramide was assayed by the method of Preiss et al. (28).

Phosphatidic Acid and Phosphatidylethanol Formation-- COS-1 cells were cultured at 1.7 × 106/ml in 10 ml of Dulbecco's modified Eagle's media and incubated with fumonisin B1 (24 h) or L-cycloserine (6 h) in the concentrations shown in the figure legends. Cells were labeled with 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (10-8 mol/liter) for 30 min at 37 °C. The labeled cells were washed with phosphate-buffered saline and incubated in 5 ml of phosphate-buffered saline containing 1 mmol/liter Ca2+ and 1 mmol/liter Mg2+. Ethanol (200 mmol/liter) was added for 5 min at 37 °C. COS-1 cell phagocytosis was initiated as outlined above. Thirty minutes after the addition of opsonized targets, the cells were harvested with trypsin-EDTA; the EIgG not internalized were lysed, and COS-1 cells resuspended in phosphate-buffered saline. The lipids were extracted, and 3H-labeled phosphatidylethanol and phosphatidic acid were assayed as described previously (29).

Assay for Sphingosine Formation-- Sphingosine was quantitated by acetylation with [3H]acetic anhydride to form C2-[3H]ceramide as described by Yatomi et al. (30). Samples were spotted on high performance thin layer chromatography plates and developed in a chloroform/methanol/acetic acid (92:2:8, v/v) solvent. Sphingosine levels were calculated by interpolation from sphingosine standards that were run through the same procedure.

Immunoprecipitation-- For the Syk phosphorylation studies, COS-1 cells were lysed in buffer containing 1% Triton X-100 along with 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml each of leupeptin and aprotinin. Lysates were precleared with protein A-Sepharose for 30 min and incubated overnight at 4 °C with anti-Syk antibody. Protein A-Sepharose was added to each sample and incubated for 2 h with rotation at 4 °C. The beads were washed briefly three times with lysis buffer and twice with buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4. Absorbed proteins were solubilized in sample buffer and separated on 10% SDS-PAGE minigels. Proteins were transferred to PVDF for 2 h at 100 V and immunoblotted with 4G10 antiphosphotyrosine antibody. The membrane was washed three times with 0.2% Tween 20 in 50 mM Tris (pH 8.0) and 100 mM NaCl and then incubated with a secondary antibody (horseradish peroxidase-conjugated) in wash buffer containing 5% nonfat dry milk. Phosphorylated bands were visualized by using ECL. The PVDF membranes were stripped with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris (pH 6.5) at 50 °C and reprobed with the appropriate antibody to demonstrate equivalent amounts of immunoprecipitated protein. Immunoprecipitations for the measurement of phosphatidylinositol 3-kinase activity were performed as follows. Lysates were precleared with protein A-Sepharose and incubated with anti-phosphatidylinositol 3-kinase p85 subunit antibody overnight. The kinase activity was measured using phosphatidylinositol and 5 µCi per sample of [gamma -32P]ATP as substrates.

MAP kinase phosphorylation experiments were performed as follows. The COS-1 cells were cultured in medium containing 0.25% fetal bovine serum to reduce the basal levels of phosphorylation. One hour prior to phagocytosis, the media were aspirated, and the cells were maintained in phosphate-buffered saline containing 1 mM calcium chloride and 1 mM magnesium chloride followed by the addition of EIgG. COS-1 cells were lysed in 50 µl of lysis buffer. The cleared lysates were combined with sample buffer, boiled for 5 min, and run on 10% SDS-PAGE minigels. The proteins were transferred to PVDF membranes. The membranes were probed with antibody against phosphorylated p44/42 in blocking buffer, washed three times, and then incubated with a secondary antibody consisting of horseradish peroxidase-conjugated goat anti-rabbit antibody in wash buffer containing 5% nonfat dry milk.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study the role of ceramide in the phagocytic response of COS-1 cells transfected with Fcgamma RIIA was investigated. It was first determined whether the COS-1 cell model recapitulates the changes in ceramide content observed in PMNs. In the PMN ceramide acts as a negative regulator of phagocytosis (16). COS-1 cells transfected with Fcgamma RIIA were challenged with EIgG. The ceramide mass and phagocytic index were measured (Fig. 1). In the absence of phagocytic targets ceramide formation did not occur, but following phagocytosis, ceramide levels increased significantly in a time-dependent manner. When COS-1 cells were challenged with EIgG (1:200) endogenous ceramide levels increased 52% over control levels by 20 min (p < 0.01) and remained elevated at 30 min. Correspondingly, the phagocytic index increased by 3.7-fold by 20 min.


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Fig. 1.   Ceramide formation and phagocytic index during COS-1 cell phagocytosis. COS-1 cells (1.7 × 106/ml) transfected with the Fcgamma IIA receptor were incubated with EIgG (108/ml) at 37 °C for the indicated times. EIgG not internalized were lysed, and COS-1 cells were resuspended in phosphate-buffered saline. Ceramide was extracted and measured as described under "Experimental Procedures." Values represent the mean ± S.D., n = 5. **, p < 0.01; ***, p < 0.001 compared with the zero time point.

Phospholipase D activation is a principal signal in PMN activation and has been linked to the uptake of complement and EIgG-opsonized particles. Furthermore, phospholipase D has been identified as an intracellular target of ceramide action (15). Phospholipase D activity was measured as the formation of phosphatidylethanol. A time-dependent increase in phosphatidylethanol was observed in EIgG-stimulated COS-1 cells (62% at 30 min, Fig. 2). A corresponding increase in the phagocytic index was also observed under these conditions suggesting that the presence of ethanol did not impair phagocytic activity (Fig. 2).


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Fig. 2.   Phosphatidylethanol formation and phagocytic index during EIgG-induced phagocytosis. COS-1 cells were labeled with 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine. At the indicated time points cells were removed with trypsin; EIgG not internalized were removed by lysis, and the lipids were extracted and analyzed as described under "Experimental Procedures." Values represent the mean ± S.D. for n = 6 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, significantly different from zero time point.

The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B1. The cellular level of ceramide decreased in a concentration-dependent manner to 28% of control levels in cells treated with 5-50 µM fumonisin B1 for 24 h (Fig. 3A). A parallel decrement in ceramide levels was observed in fumonisin B1-treated COS-1 cells with EIgG exposure. Simultaneously the phagocytic index increased by 52% at 30 min (Fig. 3B). The dose dependence of ceramide depletion during treatment of cells with fumonisin B1 closely paralleled the phagocytic index. These results, therefore, suggest an inverse relationship between ceramide formation and phagocytosis.


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Fig. 3.   A and B, effect of fumonisin B1 on ceramide depletion and phagocytic index in COS-1 cells. COS-1 cells were treated with different concentrations of fumonisin B1. At the indicated time points, COS-1 cells were removed with trypsin; EIgG not internalized were removed by lysis, and the lipids were extracted and analyzed as described under "Experimental Procedures." Values represent the mean ± S.D. of 6 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 significantly different from zero time point.

Phospholipase D activity was measured during EIgG-stimulated phagocytosis to assess the consequences of reduced ceramide generation by fumonisin B1. During fumonisin B1 treatment of COS-1 cells, phospholipase D activity increased further by 62% (p < 0.02) (Fig. 4A). EIgG red blood cell stimulated ceramide levels were significantly decreased with fumonisin B1 treatment (Fig. 4B). Both the decrease in ceramide content and increase in phospholipase D activity correlated with increased phagocytosis as measured by the phagocytic index. The inclusion of ethanol in these experiments had no significant effect on the phagocytic index (Fig. 4C).


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Fig. 4.   A-C, effect of fumonisin on phosphatidylethanol and ceramide formation and phagocytic index during EIgG-induced phagocytosis. COS-1 cells were labeled with 1-O-[3H]octadecyl-sn-glycero-3-phosphorylcholine for 1 h followed by 20 min of incubation with EIgG (108/ml) at 37 °C. The cells were collected; EIgG not internalized were removed by lysis, and the lipids were extracted and analyzed as described under "Experimental Procedures." Values represent the mean ± S.D., n = 4. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

The activation of MAP kinase has been demonstrated previously (31) to be an early event preceding phagolysosome formation in PMNs. Ceramide also inhibits MAP kinase activation and phosphorylation in the PMN. When COS-1 cells were exposed to EIgG, a time-dependent increase in the phosphorylation of both p42 and p44 MAP kinase was observed (Fig. 5). Preincubation with fumonisin B1 resulted in an increase in MAP kinase phosphorylation in concert with an increase in the phagocytic index. The enhancement of MAP kinase activity by fumonisin B1 was concentration-dependent with a maximal change observed at 35 µM.


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Fig. 5.   Effect of fumonisin B1 on MAP kinase activation in COS-1 cells during EIgG-stimulated phagocytosis. COS-1 cells (3.2 × 106/dish) were cultured in medium containing 0.25% fetal bovine serum. One hour before phagocytosis, the medium was aspirated, and the cells were maintained in phosphate-buffered saline followed by the addition of EIgG (1 × 108/ml) for 30 min at 37 °C. Unstimulated control cells were incubated in parallel for equal times with no addition or in the presence of 10 or 25 µM fumonisin B1. In the upper panel the membranes were probed with anti-MAP kinase antibody recognizing the phosphorylated forms of ERK1 and ERK2. In the lower panel, membranes were stripped and reprobed with an antibody that recognizes the nonphosphorylated forms of ERK1 and ERK2.

The tyrosine phosphorylation of Syk kinase is activated by the ligation of Fcgamma R and has also been implicated in the regulation of phagocytosis. To evaluate whether enhanced Syk phosphorylation occurs during ingestion of EIgG in COS-1 cells after fumonisin B1 treatment, anti-phosphotyrosine immunoblotting was performed on lysates of EigG-stimulated COS-1 cells. Syk phosphorylation increased 4.9-fold with increased concentrations of fumonisin B1 and increased phagocytosis (Fig. 6). There was no tyrosine phosphorylation of Syk when cells were treated with fumonisin B1 alone and not challenged with EIgG.


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Fig. 6.   Effect of fumonisin B1 on Syk phosphorylation in COS-1 cells transfected with Fcgamma IIA receptor. The COS-1 cells (3.2 × 106/dish) were treated with different concentrations of fumonisin B1 for 24 h and then were activated with EIgG, and at 30 min phagocytosis was terminated. The samples were immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. Tyrosine phosphorylation was detected by Western blotting. The blot was re-probed with anti-Syk antibody to compare protein loading. 1st to 4th lanes indicate cells treated without EIgG stimulation; 5th to 8th lanes indicate cells with EIgG.

The first step in sphingolipid synthesis is the condensation of palmitoyl-CoA and serine in a reaction catalyzed by serine palmitoyltransferase. L-Cycloserine blocks sphingosine and ceramide biosynthesis by inhibiting serine palmitoyltransferase activity. Cells treated for 6 h with 0.25 to 2 mM L-cycloserine showed a decline in sphingosine and ceramide levels (Fig. 7). In the presence of L-cycloserine, cellular levels of sphingosine decreased by almost 70%. The depletion of ceramide levels by 50% was also observed. Simultaneously, the phagocytic index increased more than 100%. The peak effect on phagocytosis was observed when ceramide and not sphingosine was maximally lowered.


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Fig. 7.   A and B, effect of L-cycloserine on ceramide and sphingosine formation and phagocytic index during EIgG-induced phagocytosis. COS-1 cells were treated for 6 h with L-cycloserine and then EIgG were added for 30 min at 37 °C. At the indicated time points cells were collected, and EIgG that were not internalized were removed by lysis. Ceramide (squares) and sphingosine (triangles) were determined as described under "Experimental Procedures." Values represent the mean ± S.D., n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, significantly different from zero time point.

When cells were treated with fumonisin B1, a significant decrease in ceramide was observed, but no significant change in sphingosine levels was seen. The effects of exogenously added sphingosine on the reconstitution of ceramide and the impact on phagocytosis and tyrosine phosphorylation of Syk were evaluated. In COS-1 cells treated with fumonisin B1 alone (25-50 µM) ceramide levels declined and the phagocytic index increased as observed previously. Treatment with sphingosine (0.5, 1, and 5 µM) resulted in increased ceramide levels and a decrease in the phagocytic index. However, the effect of sphingosine was blocked by the concomitant addition of fumonisin B1 (Fig. 8A). In samples that were immunoprecipitated with anti-Syk antibody and run on 10% SDS-PAGE followed by protein transfer to PVDF membranes, the protein phosphorylation detected by Western blot showed decreased Syk phosphorylation with increased ceramide levels (Fig. 8B). These data are consistent with the interpretation that ceramide and not sphingosine is primarily responsible for the inhibition of phagocytosis.


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Fig. 8.   A, effect of fumonisin and sphingosine treatment on ceramide content and phagocytic index of COS-1 cells stably transfected with Fcgamma RIIA. Cells were treated with fumonisin B1 (25 or 50 µM) and then sphingosine (0.5, 1, and 5 µM) for 30 min. COS-1 cells were removed with trypsin; EIgG not internalized were removed by lysis. Samples were extracted, and ceramide recovery was analyzed as described under "Experimental Procedures." Immunoprecipitation was then performed (see Fig. 9). Values represent the mean ± S.D. for 3 experiments. **, p < 0.01; ***, p < 0.001. B, effect of fumonisin B1 and sphingosine on Syk phosphorylation in FCgamma IIA receptor-transfected COS-1 cells. The COS-1 cells (3.2 × 106/dish) were treated with different concentrations of fumonisin B1 for 24 h. Cells were then incubated with sphingosine at the indicated concentrations for 30 min. Cells were then activated with EIgG for 30 min. The samples were immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. Tyrosine phosphorylation was detected by Western blot. The blot was reprobed with anti-Syk antibody to show equal loading.

COS-1 cells were incubated with N-acetylsphingosine (C2-ceramide), N-acetyldihydrosphingosine, and sphingosine to evaluate further the role of ceramide in Syk phosphorylation. Syk phosphorylation decreased with increased C2-ceramide and sphingosine concentrations, whereas C2-dihydroceramide had no impact on tyrosine phosphorylation (Fig. 9).


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Fig. 9.   Effect of C2-ceramide, C2-dihydroceramide, and sphingosine on Syk phosphorylation in COS-1 cells transfected with FCgamma IIA. The COS-1 cells (3.2 × 106/dish) were treated with N-acetylsphingosine (10 and 20 µM), N-acetyldihydrosphingosine (10 µM), and sphingosine (10 and 20 µM) for 30 min, and then phagocytosis proceeded for 30 min. The samples were immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. Tyrosine phosphorylation was detected by Western blot. The blot was reprobed with anti-Syk antibody to show equal loading.

In order to ascertain the potential role of Syk kinase in phagocytosis, ceramide formation, and MAP kinase activation, a selective inhibitor of Syk kinase was employed. Preincubation of cells with piceatannol (100 µM) for 30 min resulted in a marked inhibition of both Syk phosphorylation (Fig. 10A) and phagocytosis (Fig. 10B) in response to EIgG. Cells were next incubated with or without piceatannol (100 µM) for 30 min and then with EIgG for different times. In the absence of piceatannol, phosphatidylinositol 3-kinase activity increased over time as measured by the appearance of phosphatidylinositol 3-phosphate. Inhibition of Syk kinase, however, blocked the phosphatidylinositol 3-kinase activity (Fig. 11A). By contrast piceatannol had no effect on MAP kinase activation (Fig. 11B) or ceramide formation (Fig. 11C).


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Fig. 10.   Effect of piceatannol on Syk phosphorylation and phagocytic index in COS-1 cells transfected with Fcgamma IIA receptor during EIgG-stimulated phagocytosis. The COS-1 cells (3.2 × 106/dish) were cultured in medium containing 0.25% fetal bovine serum. One hour before phagocytosis media were aspirated, cells were kept in phosphate-buffered saline with 1 mM Ca2+ and 1 mM Mg2+, followed by the addition of piceatannol (100 µM) for 30 min. Control cells were incubated in parallel for equal times with no addition of piceatannol. Following the incubation, COS-1 cells underwent phagocytosis with EIgG (1 × 108/ml) for different times at 37 °C. A, the samples were immunoprecipitated with anti-Syk antibody and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. Tyrosine phosphorylation was detected by Western blotting. The blot was reprobed with anti-Syk antibody to compare protein loading. B, phagocytic index was determined as described under "Experimental Procedures." Values represent the mean ± S.D. of n = 6. **, p < 0.01; ***, p < 0.001 compared with the zero time point.


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Fig. 11.   Effect of piceatannol on phosphatidylinositol 3-kinase, MAP kinase, and ceramide formation and phagocytic index during EIgG-stimulated phagocytosis. The COS-1 cells (3.2 × 106/dish) were cultured in medium containing 0.25% fetal bovine serum. One hour before phagocytosis the media were aspirated, and the cells were kept in phosphate-buffered saline with 1 mM Ca2+ and 1 mM Mg2+, followed by the addition of piceatannol (100 µM) for 30 min. Control cells were incubated in parallel for equal times with no addition of piceatannol. Following the incubation COS-1 cells underwent phagocytosis with EIgG (1 × 108/ml) for different times at 37 °C. EIgG not internalized were lysed, and COS-1 cells were resuspended in phosphate-buffered saline. Phosphatidylinositol 3-kinase activity (A), MAP kinase activity (B), and ceramide levels (C) were measured as described under "Experimental Procedures." Values represent the mean ± S.D. of n = 3 (ceramide). The MAP kinase and phosphatidylinositol 3-kinase data are representative of 3 or more experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study the effects of de novo synthesis of ceramides and sphingoid bases on Fcgamma RIIA-mediated phagocytosis in COS-1 cells were studied. Four significant findings were observed. First, Fcgamma RIIA-transfected COS-1 cells demonstrated phagocytosis and associated increases in ceramide content. Phagocytosis was accompanied by phospholipase D and MAP kinase activation. Therefore these changes recapitulate those previously observed in PMNs. Second, the increase in COS-1 cell ceramide levels was the result of de novo synthesis, a result not previously observed in PMNs. The rise in ceramide was blocked by inhibitors of ceramide synthase, fumonisin B1, and of 3-ketodihydrosphingosine synthase, L-cycloserine. Third, inhibition of ceramide formation resulted in an increase in phagocytosis as measured by the phagocytotic index. This inhibition was associated with corresponding changes in Syk, MAP kinase, phosphatidylinositol 3-kinase, and phospholipase D activities as well. Fourth, inhibition of Syk kinase impaired phosphatidylinositol 3-kinase activation and phagocytosis but had no effect on ceramide generation or MAP kinase activation. These data suggest that de novo ceramide formation is not dependent on Syk kinase but that ceramide inhibits Syk kinase activity.

Sphingolipids have been studied previously in signaling events in PMNs where they have been implicated as endogenous modulators of leukocyte function. Ceramide has been observed to inhibit both the respiratory burst in freshly isolated PMNs (15) and the phagocytosis of opsonized erythrocytes (16). Ceramide is formed as a late response to formyl peptide in adherent PMNs. The time course of ceramide generation is consistent with the down-regulation of oxidant formation, attaining significance 90 min following fMet-Leu-Phe exposure. Ceramide levels rise during phagocytosis in PMNs exposed to EIgG. Significant changes in ceramide are observed 30 min following EIgG exposure. Ceramide accumulation correlates with the termination of both oxidant formation and the phagocytic response. The inhibitory effects of endogenous ceramide are reproduced by the addition of short chain ceramides, specifically N-acetylsphingosine. By contrast, tumor necrosis factor-alpha , a well characterized agonist known to stimulate ceramide formation, has been reported to stimulate sphingomyelin hydrolysis in human PMNs within minutes. In this model tumor necrosis factor-alpha primes PMNs at low concentrations and inhibits oxidant formation at higher concentrations. In each system ceramide changes have been correlated with the activation of a neutral sphingomyelinase. Cell fractionation studies indicate that both the sphingomyelinase and ceramide formed are localized to the plasma membrane (17).

Although freshly isolated human PMNs provide an important model for the study of phagocytosis and oxidant formation, the use of these cells for the study of sphingolipid metabolism is problematic. PMNs cannot be cultured for prolonged periods without losing much of their functional responsiveness over the course of hours following isolation. As a result, PMNs are not amenable to study with the use of many of the inhibitors of sphingolipid synthesis commonly employed, most of which require a prolonged period of preincubation. Short chain sphingolipid analogs have been used extensively for evaluating phagocytotic signaling. The use of these analogs is predicated on the assumption that they are metabolically inert. However, short chain ceramides are metabolized to their free base, and sphingosine is converted to ceramide. Inhibitors of sphingolipid metabolism provide an alternative approach to using sphingolipid analogs for ascertaining the role of ceramides and sphingolipid bases in phagocytosing COS-1 cells. In contrast to short chain sphingolipid analogs, these inhibitors alter the levels of endogenous sphingolipids.

During cell activation, ceramide is frequently derived from the hydrolysis of sphingomyelin by the action of a neutral or acidic sphingomyelinase, resulting in the generation of ceramide and phosphorylcholine. Ceramide can also be formed as a result of the N-acylation of sphingosine or through de novo biosynthesis. The de novo biosynthesis of ceramide is initiated by the condensation of serine and palmitoyl-CoA, which results in the formation of 3-ketosphinganine, which is subsequently reduced to dihydrosphingosine. Dihydroceramide is formed by the addition of a fatty acid in amide linkage. Ceramides formed through this pathway usually serve as precursors for complex sphingolipids such as galactosylceramide and glucosylceramide. The accumulation of ceramide resulting from de novo synthesis was first reported by Kolesnick and colleagues (32) and resulted from anthracycline exposure.

Phospholipase D activation is a principal signal in PMN activation and has been associated with the uptake of both complement and IgG-opsonized particles (33-35). Furthermore, phospholipase D has been identified as an intracellular target of ceramide during phagocytosis in PMNs (15, 16) and in fibroblasts (36). We reported previously (16) that N-acetylsphingosine blocked both phagocytosis and activation of phospholipase D in PMNs. In the present study we observed a fumonisin B1-dependent depletion of ceramide with an associated increase in phospholipase D activity, consistent with the regulation of phospholipase D by ceramide (Fig. 4). During L-cycloserine treatment of cells, we were unable to detect changes in phospholipase D activity, but the levels of sphingosine and ceramide decreased after 6 h of treatment of the cells, and the phagocytic index increased even more compared with cells treated with fumonisin B1. Sphingosine and ceramide are known to have different effects on cellular processes. Sphingosine is known to inhibit phosphatidic acid phosphohydrolase and protein kinase C activation and activation of phospholipase D. Sphingosine also alters levels of Ca2+, inositol 1,4,5-trisphosphate, and cAMP. An independent role for sphingosine in cell signaling remains to be proven because it is actively metabolized to other products. The mitogenic effect of sphingosine on Swiss 3T3 fibroblast is clearly independent of protein kinase C (37, 38), and inhibition of the respiratory burst of human neutrophils is due to a combination of inhibition of phosphatidic acid phosphohydrolase and protein kinase C (39). At the same time that ceramide is activating protein phosphatases and kinases and protein kinase Czeta , ceramide inhibits phospholipase D activity (40).

In our previous work we evaluated the effects of C2-ceramides and sphingoid bases on PEt and PA formation during phagocytosis. N-Acetylsphingosine inhibited both PEt and PA formation, but N-acetyldihydrosphingosine did not have a significant effect on either PEt or PA formation. In contrast, the sphingoid bases markedly stimulated both PEt and PA formation (16). Thus, even though both C2-ceramides and sphingoid bases inhibit phagocytosis they act on different signaling pathways. Even though C2-ceramide completely inhibited the phagocytosis of EIgG and phospholipase D activation, treatment of these cells with 1% ethanol only partially (50%) inhibited phagocytosis. These findings suggest that other components of the signaling pathways, in addition to phospholipase D, are susceptible to ceramide-mediated inhibition.

Fcgamma RIIA mediates several functions. Stimulation of monocyte Fcgamma RII leads to antibody cellular cytotoxicity, superoxide production, and phagocytosis (2, 3). Transfecting COS-1 cells with Fcgamma RIIA in the absence of other Fcgamma R renders them capable of phagocytosis of IgG-coated erythrocytes (41). Tyrosine phosphorylation is one of the earliest responses in PMN activation and is required for Fcgamma R-mediated phagocytosis by macrophages (11, 42). Two classes of protein tyrosine kinases, Src and Syk families, have been found to play a role in Fcgamma R signaling. Syk belongs to the ZAP-70 kinase family. Fcgamma R-transfected COS-1 cells, although phagocytic, presented lower activity levels of Syk than macrophages (43), suggesting that there was another element present in leukocytes that was important for phagocytosis. Because Syk is exclusively present in leukocytes, it was a good candidate to be this component. COS-1 cells transfected with Fcgamma RIIA and treated with fumonisin showed 4.9 times increased Syk phosphorylation and increased phagocytosis. There was no phosphorylation of Syk in control cells pretreated with fumonisin B1 in the absence of EIgG challenge. Treating the cells with fumonisin B1 led to a depletion of ceramide content, which was restored by incubation with sphingosine for 30 min (Fig. 8B). Sphingosine treatment led to a decrease in the phagocytic index. Protein phosphorylation detected by Western blot showed decreased Syk phosphorylation most directly correlated with increased ceramide levels.

To evaluate the role of Syk kinase activation on downstream signaling, the inhibitor piceatannol was used. Inhibition of Syk kinase completely blocked EigG-mediated phagocytosis and phosphatidylinositol 3-kinase activity, but no effects on ceramide formation or MAP kinase activation were observed. Presumably a domain on the Fcgamma RIIA receptor not associated with ITAM mediates these signals. Because most systems in which de novo ceramide formation occurs are not associated with agonist receptor interactions, this COS-1 cell system may provide a useful model for delineating the downstream signals associated with ceramide formation.

Our studies have shown that cross-linking Fcgamma receptors with EIgG results in increased tyrosine phosphorylation and activation of Syk in COS-1 cells. High levels of ceramide and sphingosine inhibited phagocytosis and Syk activation, thus blocking activation of phagocytosis. Inhibitors of sphingolipid metabolism provide an alternative approach for using sphingolipid analogs for assessing the role of ceramides and sphingoid bases in phagocytosis. They demonstrate that most of the ceramide formed during Fcgamma RIIA activation is through de novo ceramide synthesis and may provide a novel mechanism in the regulation of phagocytosis in COS-1 cells. COS-1 transfection with the Fcgamma RIIA renders these cells phagocytic and recapitulates the changes in ceramide content observed in PMNs.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI20065 (to L. A. B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Nephrology Division, Dept. of Internal Medicine, University of Michigan, Box 0676, Rm. 1560 MSRB II, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0676. Tel.: 734-763-0992; Fax: 734-763-0982; E-mail: jshayman@umich.edu.

Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M206199200

    ABBREVIATIONS

The abbreviations used are: PMN, polymorphonuclear leukocytes; EIgG, opsonized red blood cells; ITAM, immunoreceptor tyrosine-based activation motif; MAP kinase, mitogen-activated protein kinase; PA, phosphatidic acid; PEt, phosphatidylethanol; PVDF, polyvinylidene difluoride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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