Extracellular Signal-Regulated Kinase, Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase, and p38^mapk Are Involved in IL-10-Mediated Selective Repression of TNF-α-Induced Activation and Maturation of Human Peripheral Blood Monocyte-Derived Dendritic Cells

Dendritic cells (DCs)² are unique, professional, major APCs capable of stimulating resting T cells in the primary immune response, and are more potent APCs than monocytes/macrophages or B cells (1). DCs are also critically involved in the autoimmune diseases, graft rejection, and HIV infection, and the generation of T cell-dependent Abs (1, 2, 3, 4). They capture and process Ag in nonlymphoid tissues, then migrate to T cell-dependent areas of secondary lymphoid organs through afferent lymph or the blood stream to prime native T cells and initiate the immune response (5). During this process, DCs lose Ag-capturing/processing ability as they differentiate into mature, fully stimulatory APCs (6).

The characterization of DCs is difficult because they represent only a small subpopulation that includes interdigitating reticulum cells in lymphoid organs, blood DCs, Langerhans cells in the epidermis of the skin, and dermal DCs (1). Recently, an in vitro culture system that enables progenitors in peripheral blood, bone marrow, and cord blood to differentiate into DCs has been established, and it has revealed the basic mechanisms underlying the properties of DCs (7, 8, 9, 10). Previous studies have shown that TNF-α promotes activation and maturation of DCs, whereas IL-10 suppresses several DC properties in vitro (8, 9, 11, 12, 13, 14, 15, 16). However, little is known about the effect of dual stimulations of TNF-α and IL-10 on morphological, phenotypical, and functional states of DCs.

TNF-α secreted by activated T cells, monocytes/macrophages, and DCs is a pleiotropic cytokine that has growth modulatory, cytotoxic, and inflammatory activities (14, 17). The effects of TNF-α are mediated by two distinct cell surface receptors of ∼55 kDa (TNF-R1, CD120a) and 75 kDa (TNF-R2, CD120b) (18), and TNF-R1 is involved in TNF-α-induced phenotypical and functional changes in DCs (10). TNF-R1 receptor engagement by TNF-α initiates complex signaling events, including protein tyrosine kinase (PTK)-dependent cascades and a ceramide-mediated pathway leading to the activation of two transcription factors, NF-κB and activator protein 1, which regulate the expressions of numerous immune/inflammatory response genes (18).

IL-10, which is a cytokine produced by activated T cells, B cells, keratinocytes, monocytes/macrophages, and DCs, suppresses cytokine synthesis by activated T cells, NK cells, monocytes/macrophages, and DCs (15, 16). IL-10 can also block the ability of monocytes/macrophages and DCs to act as APCs via down-regulation of MHC products and costimulatory molecules (15, 16). The biological functions of IL-10 are mediated through the cell surface receptor IL-10R, which is a member of the IFN receptor-like subgroup of the cytokine receptor family (19, 20). Stimulation with IL-10 results in tyrosine phosphorylation and activation of the Janus kinase (JAK) family, including JAK1, Tyk2, and their effectors, and the STAT, STAT1α and STAT3 (21). Recent studies have shown that IL-10 induces activation of phosphatidylinositol 3-kinase (PI-3 kinase) and p70 S6 kinase (22). However, these intracellular events do not appear to be responsible for the suppressive effects of IL-10 on immune/inflammatory responses, and the signal transduction events causing these effects remain unclear (21, 22).

Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses (23, 24, 25, 26). The various members of the MAPK families differ in their substrate specificity and are activated by distinct upstream regulators and extracellular stimuli (23, 24, 25, 26). Currently, the MAPK family is comprised of three subfamilies, namely: 1) the extracellular signal-related kinase (ERK) subfamily, including p44^mapk/erk1 (ERK1) and p42^mapk/erk2 (ERK2); 2) the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) subfamily, including the p46 SAPK/JNK and p54 SAPK/JNK isoforms and their variants; and 3) the p38^mapk subfamily (23, 24, 25, 26). Previous studies have shown that ligation of TNF-α with TNF-R1 initiates activation of ERK2, SAPK/JNK, and p38^mapk in several cells and cell lines (23, 24, 25, 26). On the other hand, stimulation with IL-10 does not activate the p21^ras/Raf/ERK2 pathway in human monocytes and murine cell lines (20, 27). However, much less is known about the intracellular signaling that are responsible for mediating the TNF-α and IL-10-regulated DC properties.

Here, we show that TNF-α and IL-10 exhibit the opposite effects on morphological, phenotypical, and functional states of human peripheral blood monocyte-derived DCs, and dual stimulations of these cells with these cytokines antagonized their respective effects on several DC properties. Furthermore, stimulation of DCs with TNF-α induced tyrosine phosphorylation and activation of ERK2, p38^mapk, and SAPK/JNK, whereas IL-10 failed to induce this activation. Dual stimulations with TNF-α and IL-10 suppressed TNF-α-induced modulation of these MAPKs. Our results suggest that the repression of MAPK cascade may be crucially involved in IL-10-mediated negative regulation of TNF-α-induced changes of human peripheral blood monocyte-derived DC properties.

The medium used was RPMI 1640 supplemented with 2 mM l-glutamine, 50 μg/ml streptomycin, 50 U/ml penicillin, and 10% heat-inactivated FCS. Granulocyte-macrophage CSF (GM-CSF) was kindly provided by Kirin Brewery (Tokyo, Japan). IL-4, IL-10, TNF-α, and RANTES were purchased from PeproTech (London, U.K.). FITC-labeled dextran (FITC-DX) and lucifer yellow (LY) were purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine mAbs (clone RC20) were purchased from Transduction Laboratories (Lexington, KY). ERK2, p38^mapk and SAPK/JNK immunoblotting kits and their kinase assay kits were purchased from New England Biolabs (Beverly, MA). A mAb to IL-10 was purchased from PharMingen (San Diego, CA).

DCs were generated from PBMC, as described previously (8, 9, 10), with some modification (28). Briefly, PBMC were obtained from 30 ml of leukocyte-enriched buffy coat from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), and the light density fraction from the 42.5–50% interface was recovered. The cells were resuspended in culture medium and allowed to adhere to 6-well plates (Costar, Cambridge, MA). After 2 h at 37°C, nonadherent cells were removed, and adherent cells (∼90% CD14⁺ cells) were cultured in 3 ml of medium supplemented with GM-CSF (50 ng/ml) and IL-4 (250 ng/ml). After 7 days of culture, DCs were harvested, washed, and subsequently cultured in the presence or absence of TNF-α (50 ng/ml), IL-10 (50 ng/ml), or a mixture of TNF-α and IL-10 for an additional 4 days. The resulting cells were used for subsequent experiments. These cell populations have the typical dendritic morphology and phenotype (>85% CD1a⁺ cells; >95% CD11c⁺ cells; <1% CD14⁻ cells; >75% CD86⁺ cells;> 85% HLA-DR⁺ cells). The cell morphology was monitored by light microscopy.

T cells were prepared using a T cell-enrichment immunocolumn (Biotex Laboratories, Edmonton, Canada) from leukocyte-enriched buffy coat as, described above. T cell preparations were typically >90% pure, as indicated by anti-CD3 mAb (Becton Dickinson, Mountain View, CA) staining.

For surface marker analysis, DCs were cultured with one of the following mAbs conjugated to phycoerythin (PE) for direct fluorescence: CD83 (Coulter Immunology, Hialeah, FL), CD86 (PharMingen), and HLA-DR (Becton Dickinson). Cells were also stained with the corresponding PE-conjugated isotype-matched control mAb (Becton Dickinson). Thereafter, the cells were washed twice and suspended in PBS containing 0.2 μg/ml propidium iodide (Sigma, St. Louis, MO) to exclude dead cells. Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer (Becton Dickinson) and CELLQuest Software.

The methods used to determine the endocytotic activity of in vitro-generated DCs have previously been described (10). Briefly, FITC-DX or LY was added to a final concentration of 1 mg/ml to the cells, and the cells were cultured for 60 min at 37°C. After incubation, cells were washed four times with ice-cold PBS and analyzed by flow cytometry, as described above.

The in vitro migration of cells was assessed in a Transwell cell culture chamber (Costar) as described previously (28). In brief, polycarbonate filters with 8.0-μm pore size were precoated with 5 μg of gelatin in a volume of 50 μl on the lower surface and dried overnight at room temperature. The coated filters were washed in PBS and then dried immediately before use. DCs (10⁶/100 μl) were added to the upper compartment of the chamber. RANTES diluted in serum-free culture medium (1–100 ng/ml) was loaded in the lower compartment. After a 2-h incubation, the filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope at a magnification of ×400, and each assay was performed in triplicate. The data were expressed as number (no.) of migrated cells/field.

Responding T cells (10⁵) from an unrelated individual (allogeneic MLR) were cultured in 96-well flat-bottom microplates (Costar) with different numbers (10²-5 × 10⁴) of irradiated (15 Gy from a ¹³⁷Cs source) stimulator cells. Thymidine incorporation was measured on day 5 by an 18-h pulse with 0.5 μCi/well of [³H]thymidine (1 μCi/well; sp. act., 5 Ci/mmol) (Amersham Life Science, Buckinghamshire, U.K.).

Monocyte-derived DCs (10⁶) were starved in serum-free medium for 24 h at 37°C and subsequently kept for 4 h on ice to reduce the basal level of tyrosine phosphorylation of intracellular proteins. The cells were either unstimulated or incubated with TNF-α (50 ng/ml), IL-10 (50 ng/ml), or a mixture of TNF-α and IL-10 in the presence or absence of anti-IL-10 mAb (10 μg/ml) for 5 min at 37°C. The cells were washed twice in cold PBS and resuspended in 100 μl of lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate). The nuclei and the insoluble cell debris were removed by centrifugation at 4°C for 10 min at 14,000 g. The postnuclear extracts were collected and used as total cell lysates. Total cell lysates or the immunoprecipitates were suspended in 2× SDS sample buffer (313 mM Tris-HCl (pH 6.8), 10% SDS, 2-ME, 50% glycerol, and 0.01% bromphenol blue) and heated at 95°C for 3 min. The protein samples were fractionated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The nonspecific Ab binding sites on the membrane were blocked with 1% BSA, 0.1% Tween 20 in saline (10 mM Tris-HCl (pH 7.4), 100 mM NaCl) for 20 min at 37°C. The membrane was then incubated for 16 h at 4°C with HRP-conjugated anti-phosphotyrosine mAb and washed for 15 min with 0.5% Tween 20 in saline. Immunoblotting of ERK2, SAPK/JNK, or p38^mapk were performed according to the instruction manual. Briefly, the membranes were incubated for 16 h at 4°C with mAbs to anti-tyrosine phosphorylated ERK2, SAPK/JNK, or p38^mapk, washed for 15 min, and incubated with HRP-conjugated secondary Abs for 1 h at room temperature. Blots were visualized by enhanced chemiluminescence (ECL; New England Biolabs). To ensure similar amounts of MAPKs in each sample, the same membrane was stripped off, reprobed with mAbs to ERK2, SAPK/JNK, or p38^mapk, and developed with HRP-conjugated secondary Abs by ECL.

Kinase activities of ERK2, SAPK/JNK, or p38^mapk were determined according to the instruction manual. In brief, monocyte-derived DCs (4 × 10⁶) were either unstimulated or incubated with TNF-α (50 ng/ml), IL-10 (50 ng/ml), or a mixture of TNF-α and IL-10 for 5 min at 37°C. The cells were subsequently lysed with 100 μl of lysis buffer (1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin), and the lysates were subjected to immunoprecipitation with anti-tyrosine phosphorylated ERK2 mAb (for ERK kinase activity), c-Jun fusion protein (for SAPK/JNK kinase activity), or anti-tyrosine phosphorylated p38^mapk mAb (for p38^mapk kinase activity), followed by protein G-Sepharose 4 fast flow (Pharmacia). The complexes were washed three times with lysis buffer and twice with kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM β-glycerol phosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl₂) before resuspending in 20 μl of kinase buffer containing 100 μM ATP, 1 μg of Elk-1 (for ERK kinase activity), or activating transcription factor (ATF)-2 (for p38^mapk kinase activity). The mixtures were incubated at 30°C for 30 min. Reactions were terminated with the addition of 20 μl of 2× SDS sample buffer. Sample were heated at 95°C for 3 min, separated by 12% SDS-PAGE, and transferred onto PVDF membranes. The membranes were probed with mAbs to phospho-Elk-1 (for ERK kinase activity), -c-Jun (for SAPK/JNK kinase activity) or -ATF-2 (for p38^mapk kinase activity), and developed with HRP-conjugated secondary Abs by ECL.

The physiological states of DCs are believed to be strictly regulated by extracellular stimuli, including cytokines, adhesion/costimulatory molecules, and bacterial products. The activation and maturation states of DCs are correlated with morphological, phenotypical, and functional changes including up-regulation and down-regulation of cell surface expression levels of MHC products and adhesion/costimulatory molecules, migratory capacity, Ag uptake, and processing capacity, and T cell stimulatory capacity.

Previous studies have shown that TNF-α promotes activation and maturation states of DCs, whereas IL-10 suppresses several DC properties (8, 9, 11, 12, 13, 14, 15, 16). However, little is known about the effect of dual stimulations of TNF-α and IL-10 on DC properties and the intracellular signaling responsible for mediating these states following these cytokine exposures. In an attempt to clarify the effect of dual stimulations of TNF-α and IL-10 on DC properties, GM-CSF plus IL-4-generated human peripheral blood monocyte-derived immature DCs were cultured with or without TNF-α (50 ng/ml), IL-10 (50 ng/ml), or a mixture of TNF-α and IL-10 for 4 days, and morphological, phenotypical, and functional analyses of the resulting cells were performed. It has been previously shown that the expression levels of CD86 and HLA-DR increase during activation and maturation of DCs (8, 11, 12, 13, 14), and CD83 has been identified as a selective marker of mature DCs (9). Fig. 1 shows that treatment of monocyte-derived DCs with TNF-α resulted in increased expression of CD86 and HLA-DR, and CD83 was induced on the cell surface, whereas cell surface expression levels of CD86 and HLA-DR were inhibited by IL-10 when compared with unstimulated cells. On the other hand, the combination of TNF-α and IL-10 antagonized their respective effects on cell surface expression levels of CD86 and HLA-DR. Interestingly, IL-10 did not affect TNF-α-induced cell surface expression of CD83.

Saunders et al. (29) have previously reported that the development of murine DCs from thymic precursors is correlated with cluster formations. Therefore, we examined the relationship between maturation of monocyte-derived DCs and their cluster formation (Fig. 2). Light microscopy revealed that treatment of the cells with TNF-α results in the formation of homotypic aggregates, whereas IL-10 failed to induce these events, indicating that maturation of monocyte-derived DCs is also associated with their cluster formation. We also found that IL-10 had little or no effect on TNF-α-induced aggregation of these cells. These results indicate that IL-10 selectively abrogates TNF-α-induced changes of properties of monocyte-derived DCs in terms of morphologic and phenotypic features.

Immature DCs capture and process Ags via their high endocytic capacity, and they lose their endocytic/processing activities of Ags and mature into potent immunostimulatory APCs during differentiation (6). Previous studies have shown that the endocytic capacity of DCs was suppressed by TNF-α during their maturation process (8, 10), whereas IL-10 enhanced this function (16). To assess the effect of dual stimulations of TNF-α and IL-10 on the endocytic capacity of DCs, mannose receptor-mediated endocytosis of FITC-DX and macropinocytosis of LY via a cytoskeleton-dependent type of fluid-phase endocytosis were monitored. As shown in Fig. 3 A, TNF-α inhibited the uptake of FITC-DX and LY and their accumulation into DCs, whereas IL-10 increased the endocytosis of these molecules in these cells compared with those of unstimulated cells. On the other hand, comparable results were observed using the combination of TNF-α and IL-10 when compared with unstimulated immature DCs.

Trafficking of DCs from local nonlymphoid areas to lymphoid tissue is believed to be a crucial event in the process of presentation of antigenic materials to naive or memory T cells (5, 6). Recent studies have shown that TNF-α induced down-regulation of chemotactic migratory capacity in response to several chemokines (30). On the other hand, Sozzani et al. (31) have previously reported that IL-10 enhanced the chemotactic migratory capacity of human monocytes. To assess the relationship between activation and maturation states of DCs and their migratory property, the effect of dual stimulations of TNF-α and IL-10 on the ability of monocyte-derived DCs to migrate in response to RANTES was examined using a Transwell cell culture chamber. Fig. 3 B shows that monocyte-derived DCs following stimulation with TNF-α exhibited lower chemotactic migratory capacity, whereas IL-10 enhanced the capacity of these cells to migrate as compared with that of unstimulated cells. On the other hand, treatment of immature monocyte-derived DCs with the combination of TNF-α and IL-10 exhibited similar migratory capacity to that of unstimulated cells.

We further evaluated the allogeneic T-cell stimulatory capacity of monocyte-derived DCs following dual stimulations with TNF-α and IL-10 (Fig. 3 C). DCs stimulated with TNF-α possessed a high allostimulatory capacity as compared with untreated DCs whereas IL-10 suppressed the ability of DCs to stimulate allogeneic T-cell proliferation. On the other hand, dual stimulations of the cells with TNF-α and IL-10 inhibited their respective regulatory effects. These stimulatory effect correlated with cell surface expression levels of CD86 and HLA-DR. These results indicate that IL-10 represses TNF-α-induced functional changes of monocyte-derived DCs.

Engagement of TNF-α or IL-10 by their respective receptors increases the tyrosine phosphorylation of targeted intracellular proteins in various cell types, and these intracellular events appear to be crucial for some functions of these cells (18, 20, 21, 22, 23, 24, 25, 26). However, much less is known about signaling events associated with protein tyrosine phosphorylation cascades in DCs. Therefore, we examined the potential involvement of TNF-α or IL-10-induced tyrosine phosphorylation events in functionality (Fig. 4 A). Under the starved condition, several intracellular proteins were weakly tyrosine phosphorylated in unstimulated DCs whereas elevated tyrosine phosphorylation appeared in various proteins in DCs stimulated with TNF-α. Stimulation of cells with IL-10 caused tyrosine phosphorylation of several different intracellular proteins. On the other hand, dual stimulations with TNF-α and IL-10 initiated distinct tyrosine phosphorylation events, and the degree of tyrosine phosphorylation of several target proteins were significantly lower than these events following TNF-α stimulation. To address direct involvement of IL-10 in down-regulation of TNF-α-induced tyrosine phosphorylation of intracellular proteins, the cells were unstimulated or stimulated with TNF-α and IL-10 in the presence of a mAb to IL-10. Treatment of the cells with IL-10 and anti-IL-10 mAb suppressed IL-10-induced protein tyrosine phosphorylation events, whereas anti-IL-10 mAb inhibited IL-10-mediated repression of TNF-α-induced tyrosine phosphorylation of intracellular proteins. These results indicate that dual stimulations with TNF-α and IL-10 antagonize their respective several tyrosine phosphorylation events in monocyte-derived DCs.

MAPK is a serine/threonine protein kinase whose activities are up-regulated through tyrosine and threonine residue phosphorylation by its upstream regulators (23, 24, 25, 26). Cross-linking of TNF-R1 with TNF-α initiates activation of various members of the MAPK families including ERK2, SAPK/JNK, and p38^mapk (23, 24, 25, 26), whereas stimulation with IL-10 does not activate the p21^ras/Raf/ERK pathway in several cells and cell lines (20, 27). To clarify the potential involvement of ERK2, SAPK/JNK, and p38^mapk in the changes of DC properties, cells were unstimulated or stimulated with TNF-α, IL-10, or the combination of these stimuli, and the level of MAPKs phosphorylations were assessed by immunoblotting with respective anti-tyrosine phosphorylated MAPKs mAbs (Fig. 4, B-D). Stimulation of the cells with TNF-α increased the amounts of tyrosine phosphorylated ERK2, SAPK/JNK, and p38^mapk, whereas IL-10 failed to induce tyrosine phosphorylation of these MAPKs. On the other hand, dual stimulations with TNF-α and IL-10 resulted in significantly reduced tyrosine phosphorylation of these MAPKs when compared with TNF-α stimulation. The total amounts of these MAPKs were unchanged following stimulation.

Enzymatically activated ERK2, SAPK/JNK, or p38^mapk phosphorylate their transcription factors substrates (ElK-1, c-Jun, or ATF-2, respectively) in response to a variety of cellular stimuli, and these events result in regulation of numerous immune/inflammatory response gene expressions (18, 23, 24, 25, 26). Therefore, we examined whether any kinase activities were associated with changes in the amount the tyrosine phosphorylation forms of these MAPKs (Fig. 4, B-D). Indeed, the kinase assays clearly show that TNF-α triggered activation of ERK2, SAPK/JNK, and p38^mapk, whereas IL-10 failed to activate them. Conversely, dual stimulations using TNF-α and IL-10 resulted in reduction of TNF-α-induced kinase activities of MAPKs. These results were concomitant with the amount of the tyrosine phosphorylation form of these MAPKs. These results indicate that ERK2, SAPK/JNK, and p38^mapk are targets for IL-10-mediated repression of TNF-α-induced activation of PTK-dependent signaling events in monocyte-derived DCs.

Activation and maturation states are believed be strictly regulated by various extracellular stimuli, including cytokines, adhesion/costimulatory molecules, and bacterial products, and these events are accompanied by the changes of their morphological, phenotypical, and functional properties (8, 9, 11, 12, 13, 14, 15, 16). However, little is known about the regulatory mechanism of DC properties in their activation and maturation states and the molecular mechanism responsible for regulation of these states of DCs. Here, we show that TNF-α-induced morphological, phenotypical, and functional changes are selectively modulated by IL-10 in monocyte-derived DCs. Furthermore, we show that IL-10 represses TNF-α-induced signaling events involving ERK2, SAPK/JNK, and p38^mapk in these cells.

We demonstrated that IL-10 inhibited TNF-α-induced up-regulation of the cell surface expressions of CD86 and HLA-DR, but not CD83 (Fig. 1), whereas IL-10 failed to abolish TNF-α-induced maturation-associated homotypic aggregation of monocyte-derived DCs (Fig. 2). On the other hand, dual stimulations of TNF-α and IL-10 antagonized their respective effects on the capacities for endocytosis, chemotactic migration, and allogeneic T cell stimulation (Fig. 3). These results suggest that IL-10 may selectively modulate TNF-α-induced changes of the properties of monocyte-derived DCs. TNF-α produced by activated monocytes/macrophages plays a crucial role in the promotion of DC development and their properties (14, 17), and IL-10 derived from monocytes, as well as activated Th cells, negatively regulate several DC properties (15, 16). Furthermore, it has been shown that DCs possess the ability to produce TNF-α and IL-10 (14, 15, 16). Thus, our results imply that TNF-α and IL-10 reciprocally control activation and maturation states of DCs in vivo.

Receptor engagement with TNF-α or IL-10 initiates intracellular events in various cell types (10, 18, 23, 24, 25, 26). To the best of our knowledge, we are the first to have detected PTK-dependent intracellular signaling events in DCs (Fig. 4,A). We observed that treatment of monocyte-derived DCs with the PTK inhibitors, genestein or herbimycin A, suppressed TNF-α- or IL-10-induced changes in several DC properties (data not shown). Furthermore, we demonstrated that stimulation of monocyte-derived DCs with either TNF-α or IL-10 induced distinct tyrosine phosphorylation of intracellular proteins in these cells (Fig. 4 A). These results suggest that PTK-dependent cascades may be involved in their respective effects on the activation and maturation states of monocyte-derived DCs.

Previous studies have shown that TNF-α-induced tyrosine phosphorylation of ERK2, SAPK/JNK, and p38^mapk resulted in their enzymatic activation leading to structural and functional changes in various cell type (23, 24, 25, 26). We demonstrated that stimulation of monocyte-derived DCs with TNF-α induced tyrosine phosphorylation and activation of ERK2, SAPK/JNK, and p38^mapk, whereas IL-10 failed to induce these events (Fig. 4, B-D). These results imply that ERK2, SAPK/JNK, and p38^mapk may be involved in TNF-α-induced changes of DC properties, whereas these MAPKs may not be responsible for IL-10-induced regulation of these cells.

Previous studies have shown that IL-10 causes activation of JAK1, Tyk2, PI-3 kinase, and p70 S6 kinase (21, 22). However, these intracellular events may not be involved in the anti-inflammatory properties of IL-10 (21, 22). Although the signaling events for IL-10-induced inhibitory effects are unknown, PTK-dependent cascades probably play a role. Indeed, we showed that dual stimulations with TNF-α and IL-10 abolished TNF-α-induced tyrosine phosphorylation-dependent kinase activities of ERK2, SAPK/JNK, and p38^mapk (Fig. 4, B-D). Recent studies have shown that the blockage in the p21^ras/Raf/ERK2 and SAPK/JNK pathways leads to anergic states in T cells and monocytes (27, 32, 33). These results led us to hypothesize that the IL-10-mediated signaling may repress TNF-α-induced activation of ERK2, SAPK/JNK, and p38^mapk. Thus, the blockage in the MAPKs cascades may lead to the suppressive effect of IL-10 on TNF-α-induced changes of monocyte-derived DCs. Conversely, the potential effect of TNF-α on IL-10-mediated signaling remains unclear. We showed that TNF-α inhibited IL-10-induced phenotypic and functional changes (Figs. 1 and 3). Previous studies have shown that TNF-α also activates a family of JAK/STAT, PI-3 kinase, and p70 S6 kinase in several cells (34, 35, 36), suggesting that these molecules may not be involved in the suppressive effect of TNF-α on IL-10-mediated changes of the properties of monocyte-derived DCs. Further study is needed to determine the molecular mechanism underlying these phenomena.

The molecular mechanism by which IL-10 blocks PTK-mediated activation of ERK2, SAPK/JNK, and p38^mapk cascades remain unclear. We (37) and others (38, 39, 40) have previously suggested that a family of protein tyrosine phosphatases may exist to antagonize a large number of kinases, and these phosphatases may be involved in dominant negative signaling in certain cells. Furthermore, a series of recent studies have shown that the phosphatases specifically dephosphorylate several members of the MAPK families (41). Although the precise relationship between IL-10-mediated intracellular events and their respective phosphatases remain unknown, our data suggests that the downstream section of the IL-10-mediated signaling cascade may negatively regulate PTK-dependent cascades involving MAPKs.

In summary, our results suggest that PTK-dependent cascades may be involved in TNF-α- or IL-10-mediated regulation of monocyte-derived DCs. Furthermore, the blockage in the MAPKs cascades may contribute to suppressive effects of IL-10 on TNF-α-induced changes of DC properties. Further characterization of the molecular events of DCs may elucidate the regulation of DC properties by extracellular stimuli.

We thank Miss H. Takahashi for her excellent assistance.