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(Journal of Leukocyte Biology. 2001;70:649-658.)
© 2001 by Society for Leukocyte Biology

Phosphatidylinositol 3-kinase and ERK are required for NF-{kappa}B activation but not for phagocytosis

Erick García-García, Gabriela Sánchez-Mejorada and Carlos Rosales

Immunology Department, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City

Correspondence: Dr. Carlos Rosales, Department of Immunology, Instituto de Investigaciones Biomédicas—UNAM, Apto. Postal 70228, Cd. Universitaria, México D.F.-04510, Mexico. E-mail: carosal{at}servidor.unam.mx


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular events that transduce signals from Fc receptors to the various cellular responses are still poorly defined. We have investigated the role of phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase (ERK) in gene activation and phagocytosis in monocytes. In the THP-1 monocytic cell line, cross-linking of Fc receptors by immune complexes results in activation of the transcription factor NF-{kappa}B, via activation of ERK. Activation of both ERK and NF-{kappa}B was blocked by wortmannin and LY294002, specific inhibitors of PI 3-K. Wortmannin also inhibited the Fc receptor-mediated increase in the cytosolic calcium concentration, but it did not block immunoglobulin G (IgG)-mediated phagocytosis. In addition, the ERK inhibitor PD98059 did not block phagocytosis of IgG-coated erythrocytes. Both the increase in the cytosolic calcium concentration and phagocytosis depend on an active actin cytoskeleton, as indicated by the total lack of both responses after treatment with cytochalasin B. In contrast, cytochalasin B did not affect Fc receptor-mediated activation of NF-{kappa}B. These results identify PI 3-K and ERK as important signaling molecules in the Fc receptor signal transduction pathway of monocytes, which leads to the nucleus for gene activation. These results also suggest that, in contrast to other cell types, unstimulated monocytes do not require PI 3-K and ERK for phagocytosis.

Key Words: monocytes • signal transduction • Fc receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cross-linking of receptors for the Fc portion of immunoglobulin G (IgG) molecules (Fc{gamma}R) on many cells of the immune system triggers various functions such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, generation of the respiratory burst, and production of inflammatory mediators and cytokines [1 , 2 ].

Three classes of Fc{gamma}R have been identified, Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), and Fc{gamma}RIII (CD16). They are coded for by different genes and differ in their relative avidity for IgG, molecular structure, and cellular distribution [3 ]. Activation of Fc{gamma}R as well as other immunoreceptors [such as T cell receptor (TCR), B cell receptor (BCR), and Fc{varepsilon}RI] results in common molecular events involving activation of Src family kinases followed by activation of Syk family kinases [4 , 5 ]. The particular kinases involved in signal transduction depend on the particular immunoreceptor tyrosine-based activation motif (ITAM) present on the cytoplasmic portion of each receptor [6 ].

After Fc{gamma}R aggregation, and activation of protein tyrosine kinases, several substrates are phosphorylated, and other enzymes are also activated. Among them, phospholipase (PL) C{gamma}1 and -{gamma}2, phosphatidylinositol 3-kinase (PI 3-K), and paxillin, a cytoskeletal protein, have all been reported [7 8 9 ].

One of the major cellular responses initiated by Fc{gamma}R cross-linking, especially in myelomonocytic and natural killer (NK) cells, is the activation of genes encoding cytokines important for the regulation of the inflammatory process, such as interleukin (IL)-1, IL-8, and tumor necrosis factor (TNF) [10 11 12 13 14 15 16 17 ]. Activation of the nuclear factor NF-{kappa}B is important for expression of these cytokines, because the genes encoding them contain binding sites for NF-{kappa}B in their 5' regulatory sequences [18 , 19 ]. The signaling pathway from Fc{gamma}R to the nucleus is not completely known, but it shares elements with the biochemical cascade used by other receptors known to activate gene transcription. In particular, ERK kinase (MEK) and extracellular signal-regulated kinase (ERK), also known as mitogen-activated protein kinase (MAPK), are required for Fc{gamma}R-mediated activation of the nuclear factor NF-{kappa}B [17 ]. Besides taking part in gene activation, ERK has an important role in other Fc{gamma}R-mediated functions in various cell types [20 21 22 23 24 25 26 ]. However, the participation of this molecule in phagocytosis is not clearly defined. There are reports indicating that ERK is needed for phagocytosis of IgG-opsonized particles by polymorphonuclear neutrophils (PMN) [24 , 25 ]. But there are also reports indicating that phagocytosis can proceed independently of ERK [23 , 26 ]. Thus, it seems that ERK may be involved in phagocytosis in some cases but not in others.

PI 3-K, a lipid kinase that phosphorylates phosphoinositides at the 3' position of the inositol ring, is a signaling molecule that controls numerous cellular properties and activities [27 ]. Activation of Fc{gamma}R also results in PI 3-K activation [28 29 30 31 ]. Because PI 3-K has been reported to be required for NF-{kappa}B activation in several systems [32 33 34 35 ], we decided to confirm that PI 3-K is also responsible for ERK and NF-{kappa}B activation after stimulation of Fc{gamma}R. In addition, we explored whether PI 3-K and ERK are involved in Fc{gamma}R-mediated phagocytosis by monocytic cells.

We previously reported that stimulation of the THP-1 monocytic cell line with insoluble immune complexes (IIC) results in activation of the nuclear factor NF-{kappa}B by ERK [17 ]. In the present study, we found that the signaling pathway leading to this response involves PI 3-K, since both ERK activation and NF-{kappa}B activation were blocked by wortmannin and LY294002, specific inhibitors of PI 3-K. In contrast, we found that wortmannin and LY294002 did not affect IgG-mediated phagocytosis in THP-1 cells. In addition, PD98059, a specific inhibitor of MEK/ERK, did not inhibit IgG-mediated phagocytosis. As expected, phagocytosis and the increase in the cytosolic calcium concentration ([Ca2+]i) both required an active actin cytoskeleton, as indicated by their complete inhibition after treatment with cytochalasin B. Surprisingly, cytochalasin B did not affect Fc receptor-mediated activation of NF-{kappa}B.

These results suggest that PI 3-K and ERK are important signaling molecules in the Fc receptor signal transduction pathway leading to the nucleus for gene activation and that these molecules function independently of an actin cytoskeleton. In contrast, phagocytosis proceeds independently of PI 3-K and MAPK in these monocytic cells but requires a functional actin cytoskeleton.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The human monocytic cell line THP-1 was maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), 20 µM glutamine, 50 U/mL of penicillin, and 50 µg/mL of streptomycin.

Primary monocytes were obtained from heparinized venous blood from healthy adult donors and were purified by standard techniques as previously described [36 37 38 ].

Plasmids and reagents
The following antibodies were used: anti-pan-ERK monoclonal antibody (catalog no. E171120; Transduction Laboratories, Lexington, KY); horseradish peroxidase (HRP)-conjugated F(ab')2 goat anti-mouse IgG (catalog no. 55559; Cappel, Aurora, OH); and anti-MEK-1 rabbit polyclonal IgG (catalog no. sc-436), anti-phosphorylated ERK (ERK*) rabbit polyclonal IgG (catalog no.sc-7383), and anti-PI 3-K p110ß rabbit polyclonal IgG (catalog no. sc-7189) (all from Santa Cruz Biotechnology, Santa Cruz, CA). Wortmannin, LY294002, Fura-2/acetoxymethl ester (AM), and 1,2-bis-5-methyl-amino-phenoxyethane-N1N1N'-tetraacetoxymethyl acetate (MAPT/AM) were from Calbiochem (San Diego, CA). PD98059 was from New England Biolabs (Beverly, MA). Plasmid 3XMHC-luc was a generous gift from Dr. John Westwick and Dr. David A. Brenner of the University of North Carolina at Chapel Hill. 3XMHC-luc contains NF-{kappa}B-responsive elements upstream of the luciferase (luc) reporter gene. This plasmid directs the expression of luciferase in response to activation of the nuclear factor NF-{kappa}B. The PI 3-K active mutant p110{alpha}K227E [39 ] cloned into the pSG5 vector (Stratagene, La Jolla, CA) was a gift from Dr. Julian Downward of The Imperial Cancer Research Fund, London, United Kingdom. Kinase-defective ERK (K- ERK) was purified from bacteria as previously described [40 , 41 ]. The cytokine monocyte chemoattractant protein (MCP)-4 was donated by Dr. Eduardo García of the Instituto de Investigaciones Biomédicas, UNAM, Mexico City, Mexico. All other chemicals were from Sigma Chemical Company (St. Louis, MO).

IIC
IIC were prepared as previously described [17 , 42 ]. Briefly, 300 µL of rabbit anti-horse ferritin or anti-albumin sera were mixed with 30 µL of horse ferritin type I or bovine serum albumin (BSA) (100 mg/mL), respectively, in Eppendorf tubes and incubated at 37°C for 60 min, followed by 12 h on ice. IIC were separated by centrifugation at 20,000 g, washed three times with sterile phosphate-buffered saline (PBS), resuspended in 750 µL of PBS, and kept sterile at 4°C until use.

Cell lysates
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl, 5 mM EDTA, 50 mM HEPES, 0.5% sodium deoxycholate, 1% Nonidet P-40 (NP-40), 10 mM 2-mercaptoethanol (pH 7.5)] containing 1 mM sodium vanadate, 1 mM p-nitrophenyl-phosphate, 2 mM phenylmethylsulfonyl fluoride (PMSF), 50 µg/mL of aprotinin A, 25 µg/mL of leupeptin, and 25 µg/mL of pepstatin, for 15 min at 4°C. Cell lysates were then cleared by centrifugation at 20,000 g for 5 min and kept cold on ice.

Western blotting
Total cell lysates or immunoprecipitates of ERK or MEK were resolved by sodium dodecyl sulfate (SDS)–12% polyacrylamide gel electrophoresis (PAGE). For PI 3-K, SDS–10% polyacrylamide gels were used. Proteins were then electrotransferred onto polyvinylidine fluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer [1% BSA, 5% nonfat dry milk (Carnation; Nestle Food Co., Glendale, CA), and 0.1% Tween 20 in PBS] overnight at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer for 1 h at room temperature: anti-ERK (MAPK) monoclonal antibody at 0.075 µg/mL, anti-MEK monoclonal antibody at 0.025 µg/mL, or anti-PI 3-K at 0.05 µg/mL. Membranes were washed with PBS six times, for 5 min each time, and incubated with a 1/3,000 dilution of HRP-conjugated F(ab')2 goat anti-mouse IgG for 1 h at room temperature. After six more washes with PBS, antibody-reactive proteins were detected using a chemiluminescence substrate (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Detection of activated ERK
ERK was immunoprecipitated from THP-1 cell lysates (1.5x107 cell equivalents) with 1 µg of anti-pan-ERK monoclonal antibody. The antibody was first incubated with 20 µL of protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) for 2 h at 4°C and then mixed with the cell lysate for another 2 h at 4°C. Sepharose beads were then washed once with cold RIPA buffer and four more times with cold washing buffer [0.25 M Tris-HCl (pH 7.5), 0.1 M NaCl]. Immunoprecipitates were resolved on SDS–12% PAGE gels and Western blotted with anti-ERK* rabbit polyclonal IgG at 75 ng/mL.

MEK activity assay
MEK was immunoprecipitated from THP-1 cell lysates (1.5x107 cell equivalents) with 0.5 µg of anti-MEK antibody. The antibody was first incubated with 20 µL of protein A-Sepharose (Pharmacia Biotech) for 2 h at 4°C and then mixed with the cell lysate for another 2 h at 4°C. Sepharose beads were then washed once with cold RIPA buffer and four more times with cold washing buffer [0.25 M Tris-HCl (pH 7.5), 0.1 M NaCl]. Immunoprecipitates were resuspended in 40 µL of kinase assay buffer [10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 25 µM ATP] containing 5 µCi of [{gamma}-32P]ATP (0.11 TBq/mmol; 2 mCi/mL) (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and 3 µg of K- ERK as a substrate for MEK. Mixtures were incubated at room temperature for 30 min. The reaction was stopped by adding 20 µL of 3x SDS sample buffer and boiling for 5 min. Proteins were resolved by SDS–12% PAGE. The phosphorylated proteins were analyzed by autoradiography. To evaluate the amount of protein immunoprecipitated, an aliquot of the sample was separated and Western blotted with anti-MEK antibodies.

PI 3-K activity assay
PI 3-K was immunoprecipitated from THP-1 cell lysates (1.5x107 cell equivalents), and its activity was determined as previously described [41 ].

Transfections and Fc{gamma}R stimulation
THP-1 monocytic cells were transiently transfected using a diethylaminoethyl (DEAE)-dextran method as previously described [43 ]. Briefly, 106 cells in 0.5 mL of serum-free RPMI-1640 medium were transfected with 5 µg of plasmid DNA by incubating cells with 200 µg/mL of DEAE-dextran (Pharmacia Biotech) for 60 min and, after one wash, with 0.1 mM chloroquine for another hour at 37°C. Transfection efficiencies were evaluated, as previously described [17 , 43 ], by transfecting the cells with the plasmid pGL3 control (Promega, Madison, WI), which constitutively expresses luciferase from the simian virus 40 (SV40) promoter, or with plasmid pEGFP-N1 (Clontech, Palo Alto, CA), which expresses the green fluorescence protein (GFP) from the cytomegalovirus promoter. Efficiency was estimated from the number of cells presenting green fluorescence at 24 h after transfection.

For Fc{gamma}R stimulation, cells were resuspended in 4 mL of serum-free RPMI-1640 medium 24 h after transfection and mixed with 40 µL of IIC. After a 5-h incubation at 37°C, cells were collected and lysed with 60 µL of lysis buffer [0.1 M Tris-HCl (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA].

Luciferase activity
Luciferase enzymatic activity was determined in cell lysates using a Monolighy 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). Briefly, 50 µL of cell lysate were mixed with 100 µL of assay buffer [30 mM triglycine (pH 7.8), 3 mM ATP, 15 mM MgSO4, 10 mM dithiothreitol] and 100 µL of the luciferase substrate (250 µM D-luciferin, pH 6.5). Light measurements were performed for 20 s in each assay.

Opsonization of sheep erythrocytes
Sheep red blood cells were purchased from Erikar, S. A. (Mexico City, Mexico). IgG-opsonized erythrocytes (EIgG) were prepared as follows. Erythrocytes were washed three times in Alsever’s solution [0.1 M dextrose, 40 mM citric acid, 10 mM NaCl (pH 6.1)] and adjusted to a concentration of 109 cells/mL. One milliliter of this suspension was mixed with 0.9 mL of Alsever’s solution and 100 µL of a 1/1,000 dilution of rabbit serum anti-sheep erythrocytes. This mixture was incubated for 10 min at 37°C. Unbound antibody was removed by washing the cells several times with Alsever’s solution.

Phagocytosis
Phagocytosis of EIgG by THP-1 cells in the fluid phase was performed basically as described previously for neutrophils [44 ]. THP-1 cells (2.5x105) in 100 µL of PBS containing 1% human serum albumin (Red Cross Blood Bank, Geneva, Switzerland), 2 mM Ca2+, and 1.5 mM Mg2+ were mixed with 15 µL of an EIgG suspension (5x108 cells/mL) and incubated for 1 h at 37°C. After this time, EIgG that were not internalized were lysed by addition of 1 mL of a freshly prepared ice-cold 0.83% NH4Cl solution. Cell suspensions were maintained at room temperature for 3 min with light mixing, then centrifuged at 2,940 g for 1 min in a microcentrifuge, and cells were brought back to isotonicity by decanting and resuspending the pellet in 1 mL of ice-cold PBS. Cells were then centrifuged under the same conditions, and after complete removal of supernatant, they were resuspended in approximately 3 µL of normal human serum diluted twofold with PBS. The resulting suspension was lightly spread over a glass slide and left to air dry. Slides were then stained with Wright stain (Sigma) for 2 min, washed with PBS for approximately 3 min, and finally washed with abundant distilled water. Phagocytosis was scored by light microscopy, counting cells at high magnification, and reported as the phagocytic index (PI); that is, the number of EIgG ingested by 100 THP-1 cells. More than 100 cells in each field were counted each time. In assays involving inhibition of PI 3-K, MEK, and calcium transients, THP-1 cells were preincubated with 30 nM wortmannin or 50 µM LY294002, 30 µM PD98059, or 125 µM MAPT/AM, respectively.

Fluorescent calcium measurements
THP-1 cells were loaded with Fura-2/AM (Calbiochem) as previously described [44 , 45 ]. Briefly, THP-1 cells at 108/ml in PBS, containing 1.5 mM Ca2+ and 1.5 mM Mg2+, were incubated with 10 µM Fura-2/AM at 37°C for 5 min. The cell suspension was diluted 10-fold and kept at 37°C for another 20 min. After one wash with PBS + Ca2+ + Mg2+, the cells were resuspended in fresh PBS + Ca2+ + Mg2+ and incubated for 15 min at 37°C to allow total cleavage of the acetoxymethyl ester from the internalized dye. Finally, THP-1 cells were washed, resuspended at 2.5 x 106/mL, and kept on ice until use. Fluorescence changes of a 2-mL stirred THP-1 cell suspension kept at 37°C were monitored with an Aminco-Bowman® Series 2 spectrofluorimeter (SLM Instruments, Rochester, NY) by use of 340- and 380-nm excitation wavelengths, and a 510-nm emission wavelength. Calcium concentrations were calculated as described by Grynkiewiez et. al. [46 ].

To prevent all calcium transients, THP-1 cells were also loaded with MAPT/AM (an intracellular calcium chelator) by following a protocol similar to that for Fura-2, using a concentration of 125 µM. Simultaneous loading of MAPT/AM and Fura-2 had no effect on the loading of Fura-2 [47 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PI 3-K is required for ERK activation
We have previously reported that Fc{gamma}R-dependent ERK activation is important for activation of the nuclear factor NF-{kappa}B and for cytokine production [17 ]. Because PI 3-K is involved in the activation of NF-{kappa}B in several other systems [32 33 34 ], we investigated whether PI 3-K was required for ERK activation after Fc{gamma}R stimulation. The activation state of ERK was directly assessed in THP-1 cells and primary monocytes after stimulation with IIC in the presence of the PI 3-K inhibitors wortmannin and LY294002. Use of these drugs prevented the increase in ERK activity induced by Fc{gamma}R stimulation (Fig. 1 ). This result clearly indicates that PI 3-K is an upstream element in the Fc{gamma}R signaling pathway leading to ERK activation. MEK has been known for some years to be the enzyme responsible for ERK activation. To confirm that PI 3-K is indeed responsible for MEK activation in Fc{gamma}R signaling in monocytes, we directly evaluated the activity of MEK after Fc{gamma}R stimulation. Fc{gamma}R cross-linking by IIC promoted a strong increment in MEK activity (Fig. 2 ). This activation was inhibited by wortmannin treatment (Fig. 2) . In order to confirm that PI 3-K was indeed activated in response to Fc{gamma}R stimulation, and that wortmannin was indeed inhibiting this enzyme, we directly measured the activity of PI 3-K after stimulation of THP-1 cells with IIC. As expected, PI 3-K activity was augmented after Fc{gamma}R cross-linking and was completely blocked by the specific inhibitor wortmannin (Fig. 3 ) The other PI 3-K inhibitor, LY294002, gave similar results (data not shown). These data support the idea that PI 3-K is required for activation of the MEK-ERK signaling pathway in monocytic cells.



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  		Figure 1. PI 3-K is an upstream element in Fc{gamma}R-mediated ERK activation. A total of 1.5 x 107 THP-1 cells (A) or primary monocytes (B) in 5 mL of serum-free medium were stimulated for 1 min in the absence (Control) or presence (IIC) of 40 µL of IIC. Some cells were also treated with 30 nM wortmannin (IIC+Wort) or 50 µM LY294002 (IIC+LY) for 30 min prior to IIC stimulation. After stimulation, cell lysates were prepared, ERK was immunoprecipitated, and blots were then resolved by SDS-PAGE. Upper panels are Western blots of phosphorylated active ERK (ERK*). Lower panels are Western blots of ERK showing equivalent amounts of protein immunoprecipitated in each determination. Data are representative of three separate experiments.

 


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  		Figure 2. Fc{gamma}R cross-linking induces MEK activation in a PI 3-K-dependent manner. A total of 1.5 x 107 cells in 5 mL of serum-free medium were stimulated for 1 min in the absence (Control) or presence (IIC) of 40 µL of IIC. Some cells were also treated with 30 nM wortmannin (IIC+Wort) for 30 min prior to IIC stimulation. After stimulation, cell lysates were prepared, and MEK was immunoprecipitated. The upper panel shows MEK activity determined by an in vitro kinase assay using a kinase-defective ERK (K- ERK) as the substrate. The lower panel is a Western blot of MEK showing equivalent amounts of protein immunoprecipitated in each determination. Data are representative of three separate experiments.

 


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  		Figure 3. Inhibition of Fc{gamma}R-induced PI 3-K activity. A total of 1.5 x 107 cells in 5 mL of serum-free medium were either stimulated with 40 µL of IIC without pretreatment (IIC), pretreated with 30 nM wortmannin for 30 min before stimulation with IIC (IIC+Wort), or left untreated (Control). The upper panel shows PI 3-K activity measured by immune complex kinase assays from cell lysates. The lower panel is a Western blot of PI 3-K showing equivalent amounts of protein immunoprecipitated in each determination. PI3P, phosphatidylinositol 3-phosphate. Data are representative of three separate experiments.

 
PI 3-K is required for NF-{kappa}B activation
Because PI 3-K has been shown to be required for NF-{kappa}B activation in other signaling systems [32 33 34 35 ], and because our data indicated that PI 3-K was needed for ERK activation; it was likely that PI 3-K was also required for activation of this nuclear factor in response to Fc{gamma}R cross-linking in monocytic cells. To test this idea, THP-1 monocytic cells were transfected with an NF-{kappa}B-responsive plasmid that directs the expression of luciferase upon activation of this nuclear factor. Stimulation of THP-1 cells with IIC caused a strong activation of NF-{kappa}B (Fig. 4 ). This NF-{kappa}B activation was ~4-fold over basal activity. To make sure that the Fc{gamma}R-mediated stimulation of NF-{kappa}B was not due to lipopolysaccharide (LPS) contamination, THP-1 cells were transfected with the NF-{kappa}B-responsive plasmid and then stimulated by two types of IIC in the presence or absence of polymyxin B, an inhibitor of LPS [48 ]. Fc{gamma}R ligation by IIC prepared either with ferritin and anti-ferritin antibodies or with albumin and anti-albumin antibodies increased NF-{kappa}B activity to similar levels (Fig. 4) . The presence of 10 µg/mL of polymyxin B did not alter this response, indicating that there was no LPS contamination. When these cells were stimulated by IIC in the presence of wortmannin and LY294002, NF-{kappa}B activation was significantly reduced (Fig. 5A ). This result clearly indicated that PI 3-K is required for Fc{gamma}R-mediated NF-{kappa}B activation. To confirm the role of PI 3-K in NF-{kappa}B activation, we expressed a constitutively active form of this enzyme (p110{alpha}K227E) in our THP-1 cells, expecting it to have an enhancing effect on the NF-{kappa}B response. Cotransfection of the plasmid coding for the p110{alpha}K227E mutant version of PI 3-K [39 ] with the NF-{kappa}B reporter plasmid resulted in increased Fc{gamma}R-mediated activation of this nuclear factor (Fig. 5B) . Taken together, these data support the idea that, in monocytic cells, Fc{gamma}R-mediated gene induction requires PI 3-K for NF-{kappa}B activation, via the MEK-ERK signaling pathway.



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  		Figure 4. Fc{gamma}R stimulation by IIC activates NF-{kappa}B. A total of 106 THP-1 monocytes were transiently transfected with the NF-{kappa}B-responsive plasmid 3XMHC-luc. Twenty-four hours after transfection, cells were placed in 4 mL of serum-free medium and either left untreated (Medium) or stimulated with either 40 µL of IIC of ferritin (IIC-ferr) or 40 µL of IIC of bovine serum albumin (IIC-BSA). Some cell cultures were treated with 10 µg/mL of polymyxin B (pol B), an inhibitor of LPS. After a 5-h incubation, cells were lysed, and luciferase activity, representing NF-{kappa}B activation, was determined in a luminometer. Data are means ± SE of three determinations.

 


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  		Figure 5. PI 3-K is necessary for NF-{kappa}B activation. (A) A total of 3 x 106 THP-1 monocytes were transiently transfected with the NF-{kappa}B-responsive plasmid 3XMHC-luc. Twenty-four hours after transfection, cells were placed in 4 mL of serum-free medium and either left untreated (Medium) or stimulated with 40 µL of IIC (IIC). Some cell cultures were treated with 30 nM wortmannin (Wort) or with 50 µM LY294002 (LY) for 30 min before stimulation with IIC. After a 5-h incubation, cells were lysed, and luciferase activity, representing NF-{kappa}B activation, was determined in a luminometer. Data are means ± SE of seven or nine determinations. (B) A total of 3 x 106 THP-1 cells were transiently cotransfected with the NF-{kappa}B-responsive plasmid 3XMHC-luc and a plasmid encoding the constitutively active PI 3-K mutant p110{alpha}K227E (p110*), or the empty pSG5 vector. Twenty-four hours after transfection, cells were placed in 4 mL of serum-free medium and either stimulated with 40 µL of IIC (IIC) or left untreated (Medium). Luciferase activity, representing NF-{kappa}B activation, was determined 5 h later. Data are means ± SE of five different determinations.

 
PI 3-K and ERK are not involved in Fc{gamma}R-mediated phagocytosis
PI 3-K and ERK are thus clearly important signaling molecules in Fc{gamma}R signaling to the nucleus. However, their involvement in other Fc{gamma}R-mediated functions, such as phagocytosis, does not seem to be a general rule. To further explore the participation of the PI 3-K-ERK signaling pathway in Fc{gamma}R-mediated phagocytosis by unstimulated monocytic cells, THP-1 cells were allowed to ingest EIgG after treatment with wortmannin and LY294002 (PI 3-K inhibitors) or with PD98059 (an ERK inhibitor). THP-1 cells were able to ingest nonopsonized erythrocytes (E) presenting a basal PI of ~15 (Fig. 6 ). In contrast, EIgG were ingested at a PI of ~30. This increment thus represents phagocytosis mediated by Fc receptors. Neither PD98059 nor wortmannin or LY294002 had any effect on this type of phagocytosis (Fig. 6) . These results indicate that, contrary to signaling to the nucleus, PI 3-K and ERK are not implicated in phagocytosis. PI 3-K has been demonstrated in other systems to regulate [Ca2+]i, mainly by stimulating PLC{gamma} activation and inositol triphosphate (IP3) production [49 ]. To test whether the calcium response induced by Fc{gamma}R stimulation in monocytic cells was also regulated by PI 3-K, we evaluated the rise in [Ca2+]i in the presence of wortmannin. Stimulation of Fc{gamma}R with IIC on THP-1 cells caused a rapid increase in [Ca2+]i to ~175 nM from a baseline of ~100 nM (Fig. 7 ). Treatment with wortmannin blocked this increase in [Ca2+]i (Fig. 7A) , whereas treatment with PD98059 did not have any effect (data not shown). Furthermore, rises in [Ca2+]i induced by the chemotactic protein MCP-4, which binds to a G protein-coupled serpentin receptor [50 , 51 ], were completely insensitive to wortmannin (Fig. 7B) and PD98059 (data not shown). The blockage of a [Ca2+]i rise by wortmannin suggests that phagocytosis should be [Ca2+]i independent in monocytes, since our results showed that phagocytosis proceeded independently of PI 3-K (Fig. 6) . MAPT/AM, an intracellular calcium chelator, completely blocked any change in [Ca2+]i after stimulation of Fc{gamma}R with IIC (Fig. 8A ). However, THP-1 cells treated with MAPT/AM exhibited normal phagocytosis (Fig. 8B) . These results showed that phagocytosis of EIgG by unstimulated THP-1 cells was, as expected, not dependent on the rise in [Ca2+]i.



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  		Figure 6. PI 3-K and ERK are not required for Fc{gamma}R-mediated phagocytosis. THP-1 cells were mixed with nonopsonized sheep erythrocytes (E) or EIgG for 1 h at 37°C to allow ingestion of erythrocyte targets. Cells were treated with either 50 nM wortmannin (Wort), 50 µM LY294002 (LY), 30 µM PD98059 (PD), or only the solvent [dimethyl sulfoxide (DMSO)] before being mixed with the erythrocyte targets. Data are reported in terms of the PI (erythrocytes ingested by 100 THP-1 cells). Data are means ± SE of 12 independent determinations.

 


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  		Figure 7. The Fc{gamma}R-mediated increase in [Ca2+]i is dependent on PI 3-K. Fura-2-loaded THP-1 cells were either incubated with 30 nM wortmannin for 30 min at 37°C or left untreated (Control). Cells were then stimulated with either 40 µL of IIC (A) or 200 ng/mL of MCP-4 (B). The increment of [Ca2+]i was calculated by measuring the variation in fluorescence of Fura-2. Calcium tracings are representative of three different experiments with similar results.

 


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  		Figure 8. The increase in [Ca2+]i is not required for EIgG phagocytosis. (A) THP-1 cells were loaded with Fura-2 in the absence (Control) or presence of MAPT/AM. Cells were then stimulated with 40 µL of IIC,and the [Ca2+]i was monitored as described in Figure 7 . Calcium tracings are representative of three different experiments with similar results. (B) Phagocytosis of nonopsonized sheep erythrocytes (E) or EIgG by unloaded or MAPT/AM-loaded THP-1 cells was scored after a 1-h incubation at 37°C. Phagocytosis is reported in terms of the PI (erythrocytes ingested by 100 THP-1 cells). Data are means ± SE of three independent experiments.

 
NF-{kappa}B activation does not require the actin cytoskeleton
It is well known that several Fc{gamma}R-mediated responses, such as increases in [Ca2+]i [52 ] and phagocytosis [53 ], require a functional actin cytoskeleton. To determine whether the PI 3-K–ERK–NF-{kappa}B signaling pathway activated by Fc{gamma}R cross-linking on THP-1 cells was also dependent on the actin cytoskeleton, THP-1 cells were treated with cytochalasin B (which disrupts actin filaments) and then stimulated with IIC. As expected, the increase in [Ca2+]i was inhibited by disruption of the cytoskeleton (data not shown). The [Ca2+]i rise induced by the chemoattractant MCP-4, which is known to use IP3 to release calcium independently of the cytoskeleton, was not affected at all by the cytochalasin B treatment (data not shown). In addition, phagocytosis (Fig. 9A ) was completely blocked in THP-1 cells treated with cytochalasin B. However, Fc{gamma}R-dependent NF-{kappa}B activation was not affected by the absence of a functional actin cytoskeleton (Fig. 9B) .



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  		Figure 9. The actin cytoskeleton is required for phagocytosis, but not for NF-{kappa}B activation. (A) Phagocytosis of nonopsonized sheep erythrocytes (E) or EIgG by THP-1 cells. In some experiments THP-1 cells were treated with 10 µg/mL of cytochalasin B (Cyt. B) for 15 min before being mixed with the erythrocyte targets. Data are reported in terms of the PI (erythrocytes ingested by 100 THP-1 cells). Data are means ± SE of six independent experiments. (B) NF-{kappa}B activation by IIC. THP-1 cells transiently transfected with the NF-{kappa}B reporter plasmid (3XMHC-luc) were placed in serum-free medium and either left untreated (Medium) or stimulated with IIC before (IIC) or after (IIC+Cyt. B) treatment with 10 µg/mL of cytochalasin B. Five hours later, cells were collected and cell lysates were prepared as described in Materials and Methods. Luciferase activity, representing NF-{kappa}B activation, was determined in a luminometer. Data are means ± SE of four determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have investigated the involvement of PI 3-K and ERK in two major Fc receptor-mediated responses, namely, gene activation and phagocytosis in monocytic cells. Stimulation of monocytes by IIC results in activation of the transcription factor NF-{kappa}B through a signaling pathway that involves PI 3-K and ERK. In contrast, phagocytosis of EIgG by unstimulated monocytes is independent of these two signaling molecules.

One of the most important functions activated by immune complexes in myelomonocytic cells is the induction of genes and the production of inflammatory cytokines such as IL-1, IL-8, and TNF [10 11 12 13 14 15 16 17 , 54 ]. Transcriptional activation of these genes depends on the activation of diverse nuclear factors. Very little is known about the signal transduction pathway from Fc{gamma}R to activation of transcription factors in the cell nucleus. It has been observed that the 5' regulatory sequences of the cytokine genes induced by Fc{gamma}R cross-linking (IL-1, TNF) contain binding sites for the nuclear factor NF-{kappa}B [55 , 56 ]. We therefore reasoned that NF-{kappa}B activation would be an ideal way to monitor the Fc{gamma}R signaling pathway leading to gene induction. Stimulation of monocytic cells by IIC indeed caused NF-{kappa}B activation, as reported previously [17 ] and as indicated by luciferase production from the NF-{kappa}B-specific reporter plasmid (Fig. 4) . This response is clearly mediated by Fc receptors, because, as we reported previously, F(ab')2 fragments, immune complexes prepared with these fragments, or antigens alone were unable to stimulate the NF-{kappa}B-specific reporter plasmid [17 ]. Fc{gamma} receptors, and also the antigen receptors on T and B lymphocytes, all present a common feature that is important for signaling [57 ]. They all contain a conserved intracytoplasmic motif, know as ITAM [6 ], which contains phosphorylation sites important for signal transduction. Polyvalent ligands induce receptor cross-linking and activation of Src family- and Syk/ZAP-70 family-related kinases [4 , 50 , 58 ] which associate with the phosphorylated ITAM in the cytoplasmic tail of the receptor. After Fc{gamma}R aggregation, these activated kinases catalyze the phosphorylation of cellular substrates on tyrosine residues [17 , 59 ]. However, the nature of these substrates and other molecules involved in the signal transduction pathway has not been clearly identified.

We have previously reported that one of the signaling molecules important for activation of NF-{kappa}B upon Fc{gamma}R stimulation with IIC is ERK [17 ]. Another signaling molecule that has also been implicated in NF-{kappa}B activation by several receptor types is PI 3-K [32 33 34 35 ]. We therefore decided to confirm whether PI 3-K was also responsible for ERK and NF-{kappa}B activation after stimulation of Fc{gamma}R. Although PI 3-K has been reported to participate in several Fc{gamma}R-mediated responses [60 61 62 63 64 65 ], this is the first study confirming that PI 3-K is indeed responsible for Fc{gamma}R-dependent NF-{kappa}B activation in monocytic cells. In addition, we showed that PI 3-K leads to activation of the MEK-ERK signaling pathway that is needed for NF-{kappa}B activation [17 ]. The mechanism used by PI 3-K to activate MEK is not completely clear, but it may involve the protein kinase Akt (protein kinase B), a known target of PI 3-K [66 ]. Both of the PI 3-K inhibitors used were capable of complete inhibition of Fc{gamma}R-mediated ERK activation. However, these inhibitors did not completely block Fc{gamma}R-mediated NF-{kappa}B activation. This indicated that PI 3-K is necessary, but not sufficient, for full activation of this nuclear factor. It is possible that other signaling molecules are also needed for complete NF-{kappa}B activation. One likely candidate for such molecules is protein kinase C (PKC). Various isoforms of PKC have been shown to modulate the activity of NF-{kappa}B [67 68 69 70 71 ], and some of them have also been reported to be activated by Fc{gamma}R engagement [72 73 74 75 76 ]. Thus, complete activation of NF-{kappa}B would require both PI 3-K and PKC activities.

It is also well established that ERK is activated upon Fc{gamma}R cross-linking in various cell types [20 21 22 23 24 25 26 ]. However, its participation in phagocytosis, one of the most important Fc{gamma}R-initiated responses, is not clearly defined. Some reports indicate that ERK is needed for phagocytosis of IgG-opsonized particles [24 , 25 ], but others show that ERK is not required for this function [23 , 26 ]. Thus, it seems that ERK may be involved in phagocytosis in some cases but not in others. Similarly, PI 3-K has been reported to be an important molecule during Fc{gamma}R-mediated phagocytosis [61 , 77 78 79 ]. The role of PI 3-K in this function seems to be the modulation of the assembly of the submembranous actin filament system and membrane redistribution leading to particle internalization [80 , 81 ]. Because PI 3-K and ERK are both needed for NF-{kappa}B activation in monocytes, we decided to look at the participation of these signaling molecules in phagocytosis of EIgG by monocytic cells. Neither wortmannin or LY294002 nor PD98059 had any effect on EIgG phagocytosis. This suggested that Fc{gamma}R signaling to the nucleus was independent of the signaling pathway regulating phagocytosis.

An increase in [Ca2+]i is a well-known response in leukocytes after Fc{gamma}R stimulation [42 , 52 , 82 83 84 85 ]. PI 3-K is a key enzyme regulating the activation of PLC [49 ] and production of IP3 to release calcium from internal stores [86 ]. We therefore decided to explore the relationship between PI 3-K and the calcium response, in our THP-1 monocytic cells, after Fc{gamma}R stimulation by IIC. We found that wortmannin, but not PD98059, inhibited the Fc{gamma}R-induced increase in [Ca2+]i, indicating that a product of PI 3-K is involved in the release of calcium after Fc{gamma}R cross-linking. The specific inhibition of Fc{gamma}R signaling was made evident by stimulating the cells with MCP-4, which binds to a G-protein-coupled receptor [40 ]. The signaling cascade from this receptor is known to be independent of PI 3-K [50 , 51 ]. As indicated above, PI 3-K is known to be required for full activation of PLC{gamma} and IP3 formation. Thus, it may be possible that, in our system, wortmannin was inhibiting the rise in [Ca2+]i by this mechanism.

Our results also suggested that phagocytosis by these monocytic cells should be independent of [Ca2+]i, because wortmannin and LY294002 did not affect their levels of phagocytosis (Fig. 6) . To confirm this idea, we directly blocked the rise in [Ca2+]i by treatment with the intracellular chelator MAPT/AM. Under these conditions, THP-1 cells were able to phagocytose normally (Fig. 8) . These data further support the idea that, though PI 3-K and ERK are needed for gene activation, neither is required for Fc{gamma}R-mediated phagocytosis. However, several reports have suggested that PI 3-K is important for phagocytosis [61 , 64 , 65 ]. This is in opposition to our results with monocytic cells. An important difference between all these previous studies and our present report is the cell type involved. Neutrophils or macrophages were used in all these earlier studies. These cells are more efficient phagocytes than monocytes [53 , 80 ]. It is thus possible that in monocytic cells the phagocytic machinery is not very efficient because it is not coupled to PI 3-K, which, as has been suggested, is needed for membrane remodeling leading to particle internalization [80 , 81 ].

The idea that the particular molecular machinery used by a given receptor to modulate a certain response may change depending on the cell type and on the state of activation or differentiation of a cell is not really new. For example, in previous studies, neutrophils were shown to be able to phagocytose EIgG in a calcium-dependent manner when stimulated by the chemotactic peptide formyl-Met-Leu-Phe (fMLP) and in a calcium-independent manner when stimulated by phorbol esters [44 ]. Also, for Fc{gamma}RI, the release of calcium seems to involve a molecular switch from PLD to PLC upon cell differentiation [87 ]. More recently, it has been reported that, in cells with a monocytic phenotype, Fc{gamma}RI stimulation induced calcium-independent PKC activity, whereas, in cells with a macrophage phenotype, Fc{gamma}RI-induced PKC activity was calcium dependent [75 ]. Thus, it is likely that our THP-1 monocytic cells show calcium- and PI 3-K-independent Fc{gamma}R-mediated phagocytosis because they have been cultured in nondifferentiating (more monocytic) conditions. This concept is underscored by the fact that the monocytic cell line U937 showed Fc{gamma}R-mediated phagocytosis in a PI 3-K-dependent manner after it was differentiated to a macrophage phenotype by treatment with phorbol myristate acetate for 72 h [61 ]. We have preliminary data confirming that the signaling pathway for phagocytosis in THP-1 monocytes indeed changes from PI 3-K independent to PI 3-K dependent according to the state of differentiation of the cell.

Phagocytosis [53 ] and the increase in [Ca2+]i [52 ] have also been reported to require an active cytoskeleton. To assess whether the actin cytoskeleton was also needed for Fc{gamma}R signaling to the nucleus, we used the fungal metabolite cytochalasin B, which disrupts actin fibers. Cytochalasin B did not have any effect on Fc{gamma}R-mediated NF-{kappa}B activation in monocytic cells (Fig. 9) . However, when we evaluated phagocytosis and the rise in [Ca2+]i induced by immune complexes in the presence of cytochalasin B, both Fc{gamma}R-mediated responses were, as expected, completely abolished. Thus, these results indicate that Fc{gamma}R signaling to the nucleus is independent of an actin cytoskeleton.

Our studies stress the fact that different signals are generated upon immune complex stimulation. In the near future it will be of interest to elucidate which pathways are used by each type of Fc{gamma} receptor on different cell types. Also, it will be interesting to investigate which other molecules participate in the phagocytic process of monocytic cells, which thus far seems to be different from that of macrophages and macrophage-like cells. In addition, it will be interesting to assess whether the observed increase in [Ca2+]i, in these monocytic cells, is relevant for other Fc{gamma}R-mediated responses, such as the respiratory burst or degranulation, as suggested for neutrophils [88 ]. One more interesting issue that remains controversial is the role of IP3 in regulating changes in [Ca2+]i after Fc{gamma}R stimulation. Some studies have shown phosphoinositide turnover linked to a calcium response after Fc{gamma}R stimulation [89 , 90 ]. However, others have reported no IP3 production after Fc{gamma}R stimulation in neutrophils [42 ]. More recent studies have identified sphingosine-1-phosphate as a novel second messenger regulating the calcium response mediated by Fc{varepsilon}RI [84 ] and by Fc{gamma}RI [85 ]. The present report suggests that, in monocytic cells, the actual second messenger responsible for calcium mobilization could also be IP3, based on the dependence of the [Ca2+]i response on PI 3-K. However, in neutrophils, PI 3-K and sphingosine kinase seem to participate in a common signaling pathway for the release of calcium [31 ]. Elucidating what molecules are responsible for the Fc{gamma}R-mediated rise in [Ca2+]i in monocytes and other cell types will certainly be an active line of research in the near future.

In conclusion, this report shows for the first time that PI 3-K is required for NF-{kappa}B activation after Fc{gamma}R cross-linking. The data presented also suggest that the signal transduction pathway leading to the nucleus for gene activation, namely, PI 3-K->MEK->ERK->NF-{kappa}B, is independent of an actin cytoskeleton. In addition, the data suggest that, in these monocytic cells, unstimulated phagocytosis proceeds independently of PI 3-K and ERK but requires a functional actin cytoskeleton.


   ACKNOWLEDGEMENTS
 
This work was supported by grant IN201797 from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (UNAM), and by grant 31088-M from Consejo Nacional de Ciencia y Tecnología, Mexico. We thank Dr. John Westwick and Dr. David A. Brenner (University of North Carolina at Chapel Hill) for generously donating the 3XMHC-luc plasmid. We also thank Dr. Patricia H. Warne and Dr. Julian Downward (Imperial Cancer Research Fund) for the PI 3-K mutant p110{alpha}K227E. We also thank Sandra Margarita Morales and Teresa Romero for helping with the intracellular calcium measurements, and Nancy Mora Perez for technical assistance. E. G.-G. and G. S.-M. contributed equally to this work.

Received September 5, 2000; revised April 26, 2001; accepted April 26, 2001.


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 DISCUSSION
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