INTRODUCTION

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Antibodies (immunoglobulins) present two main functions in host defense: the binding to antigen via their antigen-combining sites and the mobilization of cellular defense mechanisms via their carboxyl-terminal Fc portion. Cross-linking of receptors for the Fc portion of immunoglobulin G molecules (FcR)¹ 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 (1-3).

Three classes of FcR have been identified, FcRI (CD64), FcRII (CD32), and FcRIII (CD16). They are coded for by different genes and differ in their relative avidity for IgG, molecular structure, and cellular distribution (4). Activation of FcR as well as other immunoreceptors (such as TCR, BCR, and FcRI) results in common molecular events involving activation of Src family kinases followed by activation of Syk family kinases (5-7). The particular kinases involved depend on the particular immunoreceptor tyrosine-based activation motif (ITAM) present on the cytoplasmic portion of each receptor (8, 9).

After FcR aggregation and activation of protein-tyrosine kinases (10), several substrates are phosphorylated and other enzymes are also activated. Among them, phospholipase C 1 and 2 (11-14), phosphatidylinositol 3-kinase (15, 16), and paxillin (17), a cytoskeletal protein, have all been reported.

One of the major cellular responses initiated by FcR cross-linking, specially in myelomonocytic and natural killer (NK) cells, is the activation of genes encoding cytokines important in inflammation, such as interleukin 1 (IL-1), IL-8, and tumor necrosis factor (TNF) (2, 18, 19). The signaling pathway from FcR to the nucleus is not known, but it probably shares elements with the biochemical cascade used by other receptors known to activate gene transcription. In particular, receptors with intrinsic tyrosine kinase activity have been shown to induce transcription of genes via activation of the Ras signaling pathway (20), which turns on sequentially Ras, Raf-1, MEK, and mitogen-activated protein kinase (MAPK) (21, 22). MAPK, also known as extracellular signal-regulated kinase (ERK) (23) phosphorylates and activates several transcription factors (24, 25).

Due to the fact that recent reports indicate that MAPK is activated after FcR cross-linking in various cell types (26-32), it has been assumed that the classical Ras pathway is activated upon FcR signaling. However, no direct proof that Ras is used in FcR signaling has been provided, except for a single report on NK cells (33).

Because activation of the transcription factor NF-B is required for IL-1 gene induction (34-36), we decided to investigate directly if FcR cross-linking on monocytic cells resulted in activation of this nuclear factor, and then we used this response as a final read-out to examine the involvement of the several elements of the Ras pathway in FcR signaling, leading to gene activation and cytokine production.

We found that stimulation of the THP-1 monocytic cell line with insoluble immune complexes results in production of IL-1 and also in activation of the nuclear factor NF-B. Moreover, activation of this nuclear factor is mediated by MAPK but activation of this kinase does not seem to involve the classical Ras pathway defined for other receptor tyrosine kinases (20, 37), since expression of dominant negative mutants of Ras and Raf did not have any effect on either MAPK activation or NF-B activation. In contrast, the MAPK kinase (MEK) specific inhibitor, PD98059, efficiently blocked activation of this nuclear factor to basal levels. These results indicate that MAPK is an important element in FcR -mediated induction of cytokine genes (e.g. IL-1) in monocytes, and strongly suggest that activation of MEK and subsequently of MAPK occurs via a pathway that is independent of Ras and Raf.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Plasmids and Reagents-- The following antibodies were used: Anti-pan ERK monoclonal antibody (catalog no. E171120, Transduction Laboratories, Lexington, KY), horseradish peroxidase-conjugated F(ab')₂ goat anti-mouse IgG (Cappel, Aurora, OH) The specific MEK (MAPK kinase) inhibitor PD98059 was from New England Biolabs, Inc. (Beverly, MA). The plasmids HIV-luc and E18pal-luc were a generous gift from Dr. John Westwick and Dr. David A. Brenner of the University of North Carolina, Chapel Hill, NC. HIV-luc contains NF-B-responsive elements within the human immunodeficiency virus long terminal repeat promoter placed upstream of the luciferase (luc) gene and directs the expression of luciferase in response to activation of the nuclear factor NF-B. E18pal-luc that activates luciferase transcription in response to the nuclear factor Ets. The plasmid encoding HA-MAPK was a gift from Mike Weber from the University of Virginia, Charlottesville, VA. Plasmids that direct the synthesis of normal or mutant forms of Ras and Raf were a gift from Dr. Channing Der from the University of North Carolina, Chapel Hill, NC. Ras constructs were all cloned in the retroviral vector pZIP. The Ras N17 (asparagine 17) mutant is a dominant negative form of this gene, while the Ras L61 (leucine 61) mutant is an active oncogenic form (38). The Raf 23-284 construct, which contains the amino-terminal domain of Raf-1 and acts as a dominant negative mutant (39), was cloned in the pCGN vector. To test the dominant negative mutants, cells were transfected with the Ras pathway-responsive reporter system GAL-Elk/5XGal-luc (40). The plasmid pEGFP-N1 (CLONTECH) containing the cDNA for the green fluorescent protein (GFP) was a gift of David García Díaz from the School of Medicine, University of Mexico, Mexico City. All other chemicals were from Sigma.

Insoluble Immune Complexes-- Insoluble immune complexes (IIC) were prepared by mixing 300 µl of rabbit anti-horse ferritin antibody (28 mg/ml) (Miles Laboratories Ltd., Slough, United Kingdom) and 30 µl of horse ferritin type I (100 mg/ml) (Sigma) in Eppendorf tubes and incubating at 37 °C for 60 min, followed by 12 h on ice. Insoluble immune complexes were separated by centrifugation at 20,000 × g and were washed three times with sterile PBS. IIC were resuspended in 750 µl of PBS and kept sterile at 4 °C until use.

Preparation of F(ab')₂ Fragments-- Anti-ferritin antibodies were subjected to pepsin digestion to prepare F(ab')₂ fragments. Briefly, 2.8 mg were dissolved in 0.1 M citrate buffer, pH 3.5, and pepsin (EC 3.4.23.1) (Sigma) was added at 25 µg/ml. The mixture was incubated at 37 °C for 4 h and then neutralized with 3 M Tris-HCl, pH = 8.6. Undigested antibody was separated in a protein A-Sepharose column. Purity of F(ab')₂ fragments was confirmed by SDS-PAGE.

Cell Culture-- The human monocytic THP-1 cell line was maintained in RPMI 1640 medium (Life Technologies, Inc.), supplemented with 10% heat inactivated fetal bovine serum (Life Technologies, Inc., Grand Island, NY), 20 µM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.

IL-1 Measurement-- THP-1 cells (1 × 10⁶) were stimulated with 40 µl insoluble immune complexes in 0.5 ml of RPMI 1640 complete medium for various times (0-48 h) at 37 °C. At the end of the incubation time, cells were centrifuged at 20,000 × g and the supernatant collected and immediately frozen at 80 °C. Interleukin 1 was measured in the supernatants with an ELISA kit (Amersham, Buckinghamshire, United Kingdom) according to the manufacturer's instructions. In some experiments, 30 µM PD98059 or 10 µM herbimycin A (Life Technologies, Inc.) was added 1 h before stimulation.

Transfections-- THP-1 monocytic cells were transiently transfected with a DEAE-dextran method as described previously (41). Briefly, 1 × 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 DEAE-dextran (Pharmacia Biotech, Uppsala Sweden) for 60 min and after one wash, with 0.1 mM chloroquine for another hour at 37 °C. Twenty-four hours after transfection, cells were resuspended in 4 ml of serum-free RPMI 1640 medium and stimulated with 40 µl of insoluble immune complexes. Cells were collected after a 5-h incubation at 37 °C and lysed with 65 µl of lysis buffer (0.1 M Tris-HCl pH 7.8, 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA). To evaluate transfection efficiencies in selected experiments, cells were transfected with the plasmid pGL3 control (Promega, Madison, WI), which constitutively expresses luciferase from the SV40 promoter. Cells were also transfected with the plasmid pEGFP-N1 (CLONTECH) containing the cDNA for the green fluorescent protein (GFP) under control of the cytomegalovirus promoter. Efficiency was estimated from the number of cells presenting green fluorescence at 24 h after transfection.

Luciferase Activity-- Luciferase enzymatic activity was determined in cell lysates using a luminometer (Monolight 2010 Luminometer, Ann Arbor, MI). Briefly, 50 µl of cell lysate were mixed with 100 µl of buffer (30 mM triglycine, pH 7.8, 3 mM ATP, 15 mM MgSO₄, 10 mM dithiothreitol), and 100 µl of substrate (250 µM D-luciferin, pH 6.5). Light produced was measured during 20 s.

Cell Lysates-- Cells were lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM HEPES, 0.5% sodium deoxycholate, 1% Nonidet P-40, 10 mM 2-mercaptoethanol, pH = 7.5) containing 1 mM sodium vanadate, 1 mM p-nitrophenyl phosphate, 2 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin A, 25 µg/ml leupeptin, and 25 µg/ml 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 Blot-- Total cell lysates or MAP kinase immunoprecipitates were resolved on 12% SDS-PAGE. Proteins were then electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% bovine serum albumin, 5% nonfat dry milk (Carnation; Nestle Food Co., Glendde, CA) and 0.1% Tween 20 in PBS) overnight at room temperature. Membranes were subsequently probed with anti-pan ERK monoclonal antibody at 0.1 µg/ml in blocking buffer, for 1 h at room temperature. Membranes were washed with PBS six times for 5 min each and incubated with horseradish peroxidase-conjugated F(ab')₂ goat anti-mouse IgG (Cappel, Aurora, OH), for 1 h at room temperature. After washing six more times with PBS, antibody-reactive proteins were detected with a chemiluminescence substrate (Pierce) according to the manufacturer's instructions.

Anti-phosphotyrosine Monoclonal Antibodies-- A series of new monoclonal antibodies to anti-phosphotyrosine were obtained following standard techniques (42, 43). Briefly, BALB/c mice were immunized with phosphotyrosine-coupled KLH in Freund's adjuvant. Splenocytes from these animals were fused to SP2/O myeloma cells and hybridomas selected by ELISA. Positive hybridomas secreted antibodies binding to phosphotyrosine-coupled ovalbumin, but not to tyrosine-coupled ovalbumin. Several hybridomas were cloned and characterized. Clone AFT8, an IgG1 producer, was selected for anti-phosphotyrosine Western blots. Monoclonal antibody AFT8 was purified from ascitis fluid by affinity chromatography in a protein A-Sepharose column.

Immune Complex Kinase Assay-- MAP kinase was immunoprecipitated from THP-1 cell lysates (1.5 × 10⁷ cell equivalent) 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 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 [-³²P]ATP (1.11 TBq/mmol; 2 mCi/ml) (NEN Life Science Products) and 10 µg of myelin basic protein (MBP) (Sigma) and incubated at room temperature for 30 min. Reaction was stopped by adding 30 µl of 3× SDS sample buffer and boiling for 5 min. Samples were electrophoresed on 12% SDS-polyacrylamide gels. The phosphorylated substrate bands were analyzed by autoradiography. To evaluate the amount of protein immunoprecipitated, an aliquot of the sample was separated and Western blotted with anti-MAPK antibodies.

HA-MAPK Immune Complex Kinase Assay-- In some experiments, HA epitope-tagged MAP kinase was transfected into THP-1 cells. After overnight culture to allow for cell recovery, the THP-1 cells were stimulated in various forms. HA-MAPK was immunoprecipitated from THP-1 cell lysates (1.5 × 10⁷ cell equivalent) with 14 µg/ml of anti-HA monoclonal antibody 12CA5 (Boehringer Mannheim). The antibody was first incubated with cell lysates for 2 h and then 20 µl of protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) were added and the mixture incubated for another 4 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 subjected to a kinase assay just as described above.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Fc Receptor Stimulation Induces Interleukin 1 Production-- THP-1 monocytic cells were stimulated with insoluble immune complexes for various periods of time, and IL-1 produced and secreted in the supernatant was measured with a commercial ELISA kit. FcR cross-linking by immune complexes induced a rapid and strong production of IL-1 by these cells (Fig. 1). IL-1 production reached a maximum (around 400 pg/ml) at about 24 h of stimulation. Previous treatment of cells with the selective tyrosine kinase inhibitor herbimycin A (44) abolished cytokine production, indicating that FcR-mediated production of IL-1 requires protein-tyrosine kinase activity.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Fc receptor stimulation induces interleukin 1 production. 1 × 106 THP-1 cells in 0.5 ml of RPMI 1640 complete medium were incubated for various periods of time in the absence (medium) () or in the presence of 40 µl of IIC (). IL-1 production was determined by ELISA. Some cultures were treated with 10 µM herbimycin A (Herb. A) () for 60 min prior to IIC stimulation. Error bars represent standard deviations. When not seen, error bars are smaller than the symbols. Data are representative of three experiments with similar results.

Fc Receptor Stimulation Induces Tyrosine Phosphorylation-- Because treatment of cells with the selective tyrosine kinase inhibitor herbimycin A (44) abolished cytokine production, we decided to look more directly at the effect of this drug on the FcR response. Stimulation of THP-1 cells with insoluble immune complexes caused rapid phosphorylation on tyrosine of several proteins. Prominent phosphotyrosine bands are increased at 1 min of stimulation at around 30, 35, 40, 44, and 70 kDa (Fig. 2). Treatment of cells with herbimycin A prior to stimulation completely abolished tyrosine phosphorylation of these proteins. Moreover, several other bands that were tyrosine-phosphorylated in the resting state also showed a significant reduction in presence of herbimycin A (Fig. 2). This result is in agreement with previous data indicating that Fc receptors recruit tyrosine kinases for their signaling (3, 10). It also shows that herbimycin A is working well and it is blocking the tyrosine phosphorylation needed for FcR-mediated IL-1 production.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Fc receptor stimulation induces tyrosine phosphorylation. 1 × 107 THP-1 cells in 0.5 ml of RPMI 1640 complete medium were stimulated for 1 min in the absence (medium) or in the presence of 40 µl of insoluble immune complexes (IIC). Some cells were also treated with 10 µM herbimycin A (Herb. A) for 60 min prior to IIC stimulation. Cell lysates were prepared as described and proteins resolved by SDS-PAGE. Western blot for anti-phosphotyrosines was done with 5 µg/ml monoclonal antibody AFT8.

Fc Receptor Stimulation Induces Activation of the Nuclear Factor NF-B-- Because the IL-1 gene, as well as other early immediate genes (such as those for IL-8 and TNF), requires activation of the nuclear factor NF-B for transcriptional activation (34, 45), we evaluated NF-B activation in response to FcR cross-linking in monocytes. THP-1 cells were transiently transfected with the NF-B reporter plasmid, HIV-luc, and luciferase activity was measured in cell lysates. Stimulation of transfected THP-1 cells by immune complexes resulted in a strong activation of the nuclear factor NF-B, as indicated by an increase (around 4-fold) in luciferase activity (Fig. 3A). Pretreatment of THP-1 cells with herbimycin A also completely blocked NF-B activation (Fig. 3A). Specificity of this response was tested by transfecting cells with the plasmid E18pal-luc that activates luciferase transcription in response to activation of the nuclear factor Ets. Stimulation of THP-1 cells with insoluble immune complexes did not induce luciferase activity from this plasmid (Fig. 3B). Treatment of the cells with 15 µg/ml lipopolysaccharide induced luciferase activity from this plasmid (Fig. 3B), indicating that the response observed after immune complex stimulation is not due to general cell activation.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Fc receptor stimulation induces activation of the nuclear factor NF-B. A, 3 × 106 THP-1 cells transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in 4 ml of serum-free medium and left untreated () or stimulated by 40 µl of IIC (). Some cultures were treated with 10 µM herbimycin A () for 60 min before IIC stimulation. Five hours later, cells were collected and cell lysates prepared as described under "Experimental Procedures." Luciferase activity, representing NF-B activation, was determined in a luminometer. B, 3 × 106 THP-1 cells transiently transfected with the E18 pal-luc reporter plasmid, were placed in 4 ml of serum-free medium and left untreated () or stimulated by 40 µl of IIC () or by 15 µg/ml lipopolysaccharide (). Five hours later, luciferase activity was determined as described. Data are means ± S.E. of three different determinations.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Immune complex stimulation is mediated by Fc receptors. 3 × 106 THP-1 cells, transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in 4 ml of serum-free medium and left untreated () or stimulated by 40 µl of IIC (), by anti-ferritin IgG (), by F(ab')2 fragments of this anti-ferritin antibody (), by ferritin (), or by immune complexes formed with ferritin and the F(ab')2 fragments of this anti-ferritin antibody ( ). Five hours later, cells were collected and cell lysates prepared as described under "Experimental Procedures." Luciferase activity, representing NF-B activation, was determined in a luminometer. Data are means ± S.E. of three different determinations.

FcR-dependent NF-B Activation Is Independent of Ras-- There is evidence that cross-linking of several immunoreceptors in leukocytes activates various elements of the Ras consensus signaling pathway (26-32). To explore the possibility that FcR signaling leading to nuclear factor activation in monocytic cells also involved elements of the Ras pathway, THP-1 cells were co-transfected with the NF-B-driven reporter plasmid and an expression plasmid directing the synthesis of a mutant form of Ras or Raf. Transfected THP-1 cells were then stimulated with insoluble immune complexes and NF-B activation evaluated by measuring luciferase activity in cell lysates. Expression of either wild-type Ras (not shown) or the dominant negative mutant Ras N17 did not affect FcR-mediated NF-B activation (Fig. 5A). That the mutant Ras proteins were functional in these cells was confirmed by co-transfection of THP-1 cells with the corresponding Ras construct and the mitogen-responsive reporter system Gal-Elk/5XGal-luc, which detects Ras pathway activation (40, 41). Cells were serum-starved for 48 h and then stimulated with 10% serum in the medium. Five hours after serum stimulation, a 3-fold increase in Ras activity was detected by measuring the luciferase activity (Fig. 5B). Co-expression of the dominant negative mutant Ras N17 blocked serum-induced activation of the reporter system to basal levels (Fig. 5B). Moreover, the presence of the activated oncogenic Ras L61 enhanced the Ras signaling initiated by serum (Fig. 5B). These results also confirmed that, in these cells, the mutant forms of Ras were affecting Ras signaling activity as expected.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   FcR-dependent NF-B activation is independent of Ras. A, 3 × 106 THP-1 cells transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in serum-free medium and left untreated () or stimulated with insoluble immune complexes (). Cells were co-transfected with the vector pZIP or with the dominant negative mutant form of Ras N17 cloned into pZIP. Luciferase activity, representing NF-B activation, was determined 5 h later. Data are means ± S.E. of eight different determinations. B, THP-1 cells were co-transfected with the Ras-reporter system Gal-Elk/5XGal-luc, and pZIP, Ras N17, or the activated oncogenic form of Ras L61. After transfection cells were serum-starved for 48 h and then left untreated () or stimulated with 10% serum (). Luciferase activity, indicating Ras pathway activation, was determined 5 h later. Data are means ± S.E. of three different determinations.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   FcR-dependent NF-B activation is independent of Raf. A, 3 × 106 THP-1 cells transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in serum-free medium and left untreated () or stimulated by insoluble immune complexes (). Cells were co-transfected with the vector pCGN or with the dominant negative mutant form of Raf 23-284 cloned into pCGN. Luciferase activity, representing NF-B activation, was determined 5 h later. Data are means ± S.E. of five different determinations. B, THP-1 cells co-transfected with the Ras reporter system Gal-Elk/5XGal-luc, and pCGN, or Raf 23-284, were serum-starved for 48 h and then left untreated () or stimulated with 10% serum (). Luciferase activity, indicating Ras pathway activation, was determined 5 h later. Data are means ± S.E. of three different determinations.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   The monocytic cell line U937 also presents FcR-dependent NF-B activation independent of Ras. 3 × 106 U937 cells transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in serum-free medium and left untreated () or stimulated with insoluble immune complexes (). Cells were co-transfected with the vector pZIP or with the dominant negative mutant form of Ras N17 cloned into pZIP, and with the vector pCGN or with the dominant negative mutant form of Raf 23-284 cloned into pCGN. Luciferase activity was determined 5 h later. Data are means ± S.E. of three different determinations.

FcR-mediated NF-B Activation Requires MEK Activation-- Although Ras and Raf did not seem to be involved in activation of the nuclear factor NF-B, many reports have indicated that MAPK is activated after cross-linking of FcR. MEK is a cytoplasmic serine-threonine kinase that directly activates MAPK by phosphorylating its TEY domain on threonine and tyrosine residues (47). To determine if MEK was participating in FcR signaling leading to nuclear factor activation, we used the selective MEK inhibitor PD98059, in THP-1 cells transiently transfected with the NF-B reporter plasmid. Treatment of cells with PD98059 for 60 min before stimulation with insoluble immune complexes resulted in complete inhibition of FcR-mediated NF-B activation (Fig. 8). This result clearly showed the participation of MEK in the signal transduction pathway from Fc receptors leading to activation of this nuclear factor. Also, PD98059 affected the production of IL-1 by these cells after IIC stimulation. However, a concentration of PD98059 that completely blocked NF-B activation (Fig. 8) inhibited IL-1 production only about 40% (Fig. 9). Increasing concentrations of the MEK inhibitor did not further block IL-1 production.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   FcR-mediated NF-B activation requires MEK activation. THP-1 cells transiently transfected with the NF-B reporter plasmid, HIV-luc, were placed in serum-free medium and left untreated () or stimulated by insoluble immune complexes (), or treated with 30 µM PD98059 () for 60 min before IIC stimulation. Five hours later, cells were collected and cell lysates prepared as described under "Experimental Procedures." Luciferase activity, representing NF-B activation, was determined in a luminometer. Data are means ± S.E. of three determinations.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   MEK activity is needed for FcR-mediated IL-1 production. 1 × 106 THP-1 cells in 0.5 ml of RPMI 1640 complete medium were incubated for various periods of time in the absence (medium) () or in the presence of 40 µl of IIC (). IL-1 production was determined by ELISA. Some cultures were treated with 30 µM PD98059 () for 60 min prior to IIC stimulation. Error bars represent standard deviations. When not seen, error bars are smaller than the symbols. Data are representative of three experiments with similar results.

FcR Stimulation by Insoluble Immune Complexes Results in MAPK Activation-- Our results, described above, indicated that FcR signaling in monocytic cells did not involve Ras and Raf, but clearly activated MEK and the nuclear factor NF-B. To determine if MAPK was connecting MEK and NF-B, we decided to look directly at MAPK activation by immune complex kinase assays. Stimulation of THP-1 cells with IIC resulted in a clear and strong stimulation of MAPK activity (Fig. 10). The kinetics of FcR-mediated activation of MAPK showed maximal kinase activity by 1 min of IIC stimulation. This activity had returned to basal levels around 3 min (Fig. 10). This result was in agreement with previous reports that FcR cross-linking results in MAPK activation. Moreover, treatment of THP-1 cells with PD98059 for 1 h before IIC stimulation demonstrated that FcR-mediated MAPK activation is completely blocked when MEK activation is inhibited (Fig. 11). These data suggested that, in FcR signaling, MEK activation is an upstream event of MAP kinase activation, which then leads to nuclear factor NF-B activation.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Kinetics of MAPK activation after Fc receptor cross-linking with IIC. 1.5 × 107 THP-1 cells in 7.5 ml of serum-free RPMI 1640 medium were stimulated with 60 µl of IIC for various periods of time. MAPK activity was measured by immune complex kinase assays from cell lysates. A, MAPK was immunoprecipitated from equal number of cells with anti-pan ERK monoclonal antibody, and its kinase activity determined by myelin basic protein (MBP) phosphorylation. Following separation of the substrate (MBP) by SDS-PAGE, the gels were dried and autoradiographed. B, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination. Data are representative of three separate experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 11.   FcR-mediated NF-B activation requires MEK activation. THP-1 cells were treated with 30 µM of the selective MEK inhibitor PD98059 for 60 min before IIC stimulation. Cells were stimulated as in Fig. 10, during 1 min and then cell lysates prepared and MAPK activity determined by immune complex kinase assay. A, MAPK was immunoprecipitated from equal number of cells with anti-pan ERK monoclonal antibody, and its kinase activity determined by MBP phosphorylation. B, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination. Data are representative of three separate experiments.

FcR-dependent MAPK Activation Is Independent of Ras-- Data presented above indicated that MAPK is clearly activated by insoluble immune complexes and that the Ras and Raf dominant negative constructs did not inhibit NF-B activation. It was then important to determine directly if these dominant negative mutants did not blocked FcR-dependent MAPK activation. It is not easy to see the effect of these mutants on MAPK directly because the efficiency of transfection of monocytic cells is rather low, approximately 5%, as estimated by transfections with a plasmid that expresses the GFP (data not shown). Therefore, the effect of the dominant negative constructs is only on those cells that were successfully transfected.

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Ras N17 does not inhibit MAPK activation after Fc receptor cross-linking. 1.5 × 107 THP-1 cells, co-transfected with HA-MAPK and the dominant negative mutant Ras N17 or the empty vector pZIP, were stimulated with 60 µl of IIC (A and B) or with serum (C and D) for 1 min at 37 °C. MAPK activity was measured by immune complex kinase assays from cell lysates. A, HA-MAPK was immunoprecipitated from equal number of IIC-stimulated cells with anti-HA monoclonal antibody, 12CA5, and its kinase activity determined by MBP phosphorylation. Following separation of the substrate (MBP) by SDS-PAGE, the gels were dried and autoradiographed. B, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination from A. C, HA-MAPK was immunoprecipitated from equal number of serum-stimulated cells with anti-HA monoclonal antibody, 12CA5, and its kinase activity determined by MBP phosphorylation. D, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination from C. Data are representative of two separate experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 13.   Raf 23-284 does not inhibit MAPK activation after Fc receptor cross-linking. 1.5 × 107 THP-1 cells, co-transfected with HA-MAPK and the dominant negative mutant Raf 23-284 or the empty vector pCGN, were stimulated with 60 µl of IIC (A and B) or with serum (C and D) for 1 min at 37 °C. MAPK activity was measured by immune complex kinase assays from cell lysates. A, HA-MAPK was immunoprecipitated from equal number of IIC-stimulated cells with anti-HA monoclonal antibody, 12CA5, and its kinase activity determined by MBP phosphorylation. Following separation of the substrate (MBP) by SDS-PAGE, the gels were dried and autoradiographed. B, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination from A. C, HA-MAPK was immunoprecipitated from equal number of serum-stimulated cells with anti-HA monoclonal antibody, 12CA5, and its kinase activity determined by MBP phosphorylation. D, Western blot of MAPK using anti-pan ERK monoclonal antibody showing the same amount of protein immunoprecipitated in each determination from C. Data are representative of two separate experiments.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Membrane receptors for the Fc portion of immunoglobulin G class antibodies (FcR) are expressed on almost every type of hematopoietic cells. Cross-linking of these receptors by aggregated IgG, in the form of antigen-antibody complexes, triggers a very wide array of responses important for host defense and for modulation of the immune response (3). There is a great deal of interest in understanding the signaling mechanisms that lead to the various cell responses. One of the most important functions activated by immune complexes on myelomonocytic and NK cells is the production of inflammatory cytokines such as IL-1, IL-8, and TNF. This means that FcR cross-linking induces transcription of the genes encoding these response (2, 18). To have initiation of transcription of these genes, activation of diverse nuclear factors has to take place. Very little is known about the signal transduction pathway from FcR to nuclear factors in the cell nucleus.

It has been observed that the 5' regulatory sequences of the cytokine genes induced by FcR cross-linking (IL-1, IL-8, TNF), all contain sites for the nuclear factor NF-B (34-36). We, therefore reasoned that NF-B activation would be an ideal way for monitoring the FcR signaling pathway leading to gene induction. We decided to investigate directly if FcR cross-linking on THP-1 monocytic cells also resulted in production of IL-1 and activation of the nuclear factor NF-B. Insoluble immune complexes indeed caused NF-B activation, as indicated by luciferase production from the NF-B-specific reporter plasmid (Fig. 3). This response is clearly mediated by Fc receptors because F(ab')₂ fragments of antibodies or antibody complexes made with them, were unable to stimulate the NF-B-specific reporter plasmid (Fig. 4). So, FcR stimulation in monocytic cells initiates a signal transduction pathway that activates nuclear factors and induces IL-1 gene expression.

FcR, and also the antigen receptors on T lymphocytes and B lymphocytes, present a common feature that is important for signaling by all these immunoreceptors (5). They all contain a conserved motif, known as ITAM for immunoreceptor tyrosine-based activation motif (7, 9), which contains phosphorylation sites important for signal transduction. Polyvalent ligands induce receptor cross-linking and activation of Src family (48, 49) and Syk/ZAP-70 family related kinases (50-52), which associate with the phosphorylated ITAM in the cytoplasmic tail of the receptor. After FcR aggregation, these activated kinases catalyze the phosphorylation of cellular substrates on tyrosine residues (Fig. 2) (10). However, the nature of these substrates and other molecules involved in the signal transduction pathway is not clearly identified.

Activation of tyrosine kinases leading finally to nuclear factor activation resembles the signal transduction pathway defined for receptor tyrosine kinases that induces mitogenic signals in response to growth factors such as epidermal growth factor and platelet-derived growth factor. This signaling pathway is also known as the Ras pathway (20, 37). Receptor tyrosine kinase activity induces transient formation of Ras-GTP and activation of Raf kinase at the membrane, followed by sequential activation of MEK and MAPK (22). MAPK is then responsible for activation of several transcription factors (25). Therefore, it has been thought that the classical Ras signal transduction pathway (37) may be involved in FcR signaling leading to activation of nuclear factors and cytokine production.

Moreover, recent reports indicate that MAPK is activated after FcR cross-linking in various cell types (26-32), supporting the idea that the Ras pathway is used by FcR to induce gene transcription. However, no direct evidence of Ras involvement has been provided in these reports. A recent publication indicates that an increase in Ras-GTP was observed after cross-linking of FcRIIIA on NK cells (33), further supporting the idea for FcR using the Ras signaling pathway. Because MAPK activation does not necessarily mean that the Ras pathway is being utilized, and because diverse signaling pathways may be used in different cell types, a direct evaluation for the involvement of the various elements of the Ras pathway in different cell types becomes important.

In this report, we investigated the participation of the Ras signaling pathway in the activation of cytokine genes upon FcR cross-linking on monocytes, probing for the different elements involved in this signaling cascade.

The involvement of Ras signal pathway elements in the FcR signal transduction pathway was investigated by measuring NF-B activation in the presence of specific inhibitors. Expression of either wild-type Ras or the dominant negative mutant Ras N17, in THP-1 cells did not have any effect on FcR-mediated NF-B activation. Similarly, expression of the dominant negative mutant form of Raf-1, Raf 23-284, did not prevent NF-B activation by insoluble immune complexes. These mutants have been shown to efficiently block signal transduction via the classical Ras pathway (38, 39) (see also Figs. 5B and 6B). These results were obtained in the monocytic cell line THP-1. In order to confirm that this was a more general behavior of monocytes, the experiments were repeated in a different monocytic cell line. Data on U937 cells (Fig. 7) also showed that dominant negative mutant forms of Ras and Raf did not have any effect on activation of NF-B by immune complexes. Thus, in monocytic cells, Ras and Raf are not directly involved in the FcR-mediated signal transduction pathway that leads to activation of NF-B. Although Ras and Raf did not seem to be involved in activation of the nuclear factor NF-B, many reports have indicated that MAPK is activated after cross-linking of FcR, so downstream elements in the Ras pathway may still be used for FcR signaling.

MEK is a cytoplasmic serine-threonine kinase that directly activates MAPK (47) and it is found downstream of Raf in the Ras signaling cascade activated by receptor tyrosine kinases (20). The selective MEK inhibitor, PD98059, completely blocked NF-B activation, indicating that MEK participated in FcR-mediated signal transduction. Immune complex stimulation of THP-1 cells also resulted in activation of MAPK, as indicated by in vitro kinase assays using MBP as substrate for the kinase (Fig. 10). In addition, PD98059 was able to block MAPK activation back to basal levels (Fig. 11). This established a link between FcR and the pathway MEK, MAPK, and NF-B. Moreover, evaluating directly MAPK activity only in transfected cells by using the HA-MAPK, it was found that the dominant negative mutants of Ras and Raf did not affect FcR-dependent activation of this kinase (Figs. 12 and 13). These data further supported the idea that FcR cross-linking activates a MAPK pathway without using the proteins Ras and Raf.

Treatment of THP-1 cells with a concentration of PD98059 that completely blocked NF-B activation resulted only in partial inhibition of IL-1 production. To initiate transcription of the IL-1 gene, more than one nuclear factor is required. The promoter region of this and many other genes contains multiple and different sites for nuclear factor binding (36, 53, 54). NF-B is one of the nuclear factors identified to bind at the 5' regulatory region of the IL-1 gene (34-36). Thus, blockage of MEK and MAPK activity by the inhibitor PD98059 resulted in failure to activate NF-B, and therefore reduced FcR-mediated IL-1 production in monocytes. Full transcriptional activation of the IL-1 gene needs NF-B and cooperation from other transcription factors (36, 53, 54). This cooperation effect has been demonstrated in the THP-1 cells for the IL-8 gene (41).

Stimulation of monocytic cells was done with immune complexes. These interact and stimulate all types of Fc receptors. However, it is known that the various types of Fc receptors activate different cellular responses (1, 3, 55), so it would be very interesting to know what type of receptor is responsible for the MAPK and nuclear factor activations that are connected to the induction of cytokine production by monocytes. We are now investigating this by stimulating cells with the monoclonal antibodies IV.3 and 3G8, which are specific for FcRII and FcRIII, respectively. Our preliminary results indicate that both receptors FcRII and FcRIII, expressed on monocytic cells, are capable of inducing NF-B activation, although they seem to do it at lower levels compared with immune complex stimulation.

How MEK is activated without Raf participation is not clear, but there is evidence for diverse ways to activate this kinase (56). MEK is phosphorylated and activated by an upstream kinase, which in the case of many serum growth factor receptors is the proto-oncogene Raf. Direct action of Raf over MEK is clearly established (57, 58). Another MEK kinase, also called MEKK, has been described upstream of MEK in the signaling pathway from a different type of receptors (56). MEKK is the mammalian counterpart of the yeast protein kinases Byr2 (from Schizosaccharomyces pombe) and Ste11 (from Saccharomyces cerevisiae), which in turn activate the protein kinases Byr1 and Ste7, respectively. Byr1 and Ste7 have considerably sequence homology to MEK and function in the pheromone-induced signaling pathway that leads to mating (56). Some pheromone receptors have a seven-membrane-spanning serpentine structure coupled to G proteins. Similarly, in mammalian cells, some serpentine receptors coupled to heterotrimeric G_i2 proteins can stimulate DNA synthesis via MAPK activation (59). For example, G_i2-coupled acetylcholine muscarinic M2 receptors have been reported to activate MEK and MAPK independently of Raf. The kinase responsible for this effect is MEKK (59). Moreover, in mouse (NIH3T3) and rat (Rat1a) fibroblasts, MEK and MAPK are activated in response to epidermal growth factor (recognized by a receptor tyrosine kinase) and also in response to thrombin (recognized by a serpentine G protein-coupled receptor). Raf is activated by epidermal growth factor but not thrombin (60). It seems, then, that MEKK is a conserved kinase for the regulation of G protein-coupled signal pathways in yeast and vertebrates and Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system (59, 61). Whether G protein subunits activate MEKK directly or through an unknown intermediary molecule remains to be determined (61).

FcR are not associated with G proteins. The kinase responsible for MEK activation after immune complexes stimulation remains unknown. Because G proteins may activate MEKK indirectly, it may be possible that this kinase is also used by FcR to activate MEK. Another possibility is that FcR may activate MEK via a different and yet undescribed kinase that has MEK kinase properties. It will be interesting to determine the involvement of MEKK in FcR signaling to the nucleus to activate gene transcription.

FcR signaling initiates with tyrosine phosphorylation (10). Herbimycin A, a selective inhibitor of tyrosine kinases, completely blocked both IL-1 production and activation of the NF-B-driven reporter plasmid in transient transfection assays, confirming that FcR aggregation triggers activation of tyrosine kinases (see Fig. 2) as an early event in the signal transduction pathway from Fc receptors to gene activation and production of cytokines in monocytes. Syk kinase (72 kDa) has been implicated in FcR signaling in several cell types. Syk belongs to the ZAP-70 kinase family. These enzymes are not myristoylated and therefore are exclusively cytoplasmic. Syk is present in all hematopoietic cells, whereas ZAP-70 is expressed in T cells and NK cells (62). In mast cells (RBL-2H3), MAPK activation has been clearly shown to by dependent of Syk, probably through the GTP/GDP exchange factor Vav (63). The link between FcR and MAPK may also be through Shc, which is phosphorylated by Syk and then binds to Grb2. This adaptor protein is known to associate with Sos to activate Ras upstream of MAPK (64), although in mast cells the FcR for IgE seems to connect Syk to MAPK via Ras. In monocytes, we did not find evidence for Ras or Raf involvement in MEK and MAPK activation after FcR cross-linking. Syk is the most likely tyrosine kinase involved in FcR signal transduction in THP-1 monocytic cells. In this report we did not look directly at Syk, but it will be interesting to confirm that Syk activation is required for MEK and MAPK activation in THP-1 cells. The mechanism that Syk may use to activate this downstream kinases bypassing Ras and Raf is unknown, but as discussed above, it may be through activation of MEKK. As of this report, there are no studies directly looking for a functional interaction between Syk and MEKK in any cell type.

Taken together, data presented in this work strongly suggest that the monocyte signaling pathway from Fc receptors leading to expression of different genes important to leukocyte biology (Fig. 14), initiates with tyrosine kinases and requires MAPK activation, but in contrast to other tyrosine kinase receptors, FcR-mediated MAPK activation does not involve Ras and Raf.

View larger version (19K): [in this window] [in a new window]   Fig. 14.   Model for FcR-mediated signal transduction in monocytic THP-1 cells. FcR are aggregated by immune complexes formed by IgG bound to polyvalent antigens. Tyrosine kinases, such as Syk, are activated and bound to the tyrosine-phosphorylated receptor. Direct substrates for these tyrosine kinases are not clearly defined. The signaling pathway leads to activation of MEK, which in turn phosphorylates and activates MAPK. These enzymes are then responsible for activation of the nuclear factor NF-B. There is not involvement of Ras and Raf proteins upstream of MEK.