LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity

Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell surface glycoconjugates by one or both sialidase promotes cytokine production.

Abstract

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1^–/^– and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.

Introduction

Sialic acid is present on glycoproteins and glycolipids that are widely distributed throughout nature. This amino sugar imparts structural and functional diversity to glycoconjugates, given its terminal position and capacity for different substitutions and glycosidic linkages [1]. Modulation of the sialic acid content of glycoconjugates on the surface of diverse types of cells influences the interaction with ligands, microbes, and neighboring cells [2–8] and plays a role in cellular activation, differentiation, transformation, and migration [6, 9–17]. The extent of sialylation of these glycoconjugates has traditionally been attributed to the action of sialyltransferases, which are localized in the ER and Golgi apparatus. We and others [6, 9, 13, 16, 18–20] have demonstrated recently that sialidases, a family of enzymes that removes terminal sialyl residues from glycoconjugates, also contribute to the dynamic changes in carbohydrate complexity on the cell surface.

Four genetically distinct forms of mammalian sialidases (Neu1–4) have been characterized, each with a predominant cellular localization and substrate specificity [21–26]. It is widely accepted that Neu1 associates with other proteins to form a multienzyme complex in lysosomes [27], where it catabolizes glycoproteins and glycolipids. Yet, recent studies have shown that Neu1 is also expressed on the surface of diverse types of cells, where it regulates the activity of molecules involved in adhesion and intracellular signaling [6, 9–11, 20, 28]. Neu3 has been considered a plasma membrane-associated protein that preferentially desialylates gangliosides [13, 21], thereby modulating cellular activation, differentiation, and transformation [12, 29, 30]. Recently, it has been shown that Neu3 also associates with early endosomes [31]. The cytosolic sialidase (Neu2) can desialylate glycoproteins and gangliosides [24] and plays a role in differentiation and malignancy [32]. The function of Neu4 sialidase has not been established, but two different isoforms are present in lysosomes or mitochondria [25, 26].

Endogenous sialidase activity increases in cells of the immune system during cell activation [20, 33–35] or differentiation [28, 36]. Sialidase activity in these cells has been implicated in various activities to include cytokine production, transendothelial migration, retention of cells in bone marrow, antigen uptake, and interaction with the ECM [3, 15, 20, 28, 35, 37]. We have shown previously that expression of Neu1 and Neu3 is up-regulated in activated human lymphocytes [20] and monocyte-derived macrophages [28, 36]. One potential role of this endogenous sialidase activity was suggested by the enhanced LPS-induced production of IL-6, MIP-1α, and MIP-1β in human monocytes that were exposed to exogenous bacterial NANase [38]. Desialylation of glycoconjugates on the surface of monocytes may promote cytokine production by activating the ERK 1/2 and/or p38 MAPK signal transduction pathways [38, 39].

Monocytes and DCs have multiple capacities that include, among others, ligand–cell and/or cell–cell interactions, cell migration, and cytokine production. During maturation, DCs acquire the ability to recognize and process antigens and to present them to T lymphocytes. Whereas DCs are activated by various ligands and/or cytokines during inflammatory and infectious states, DCs themselves can also produce proinflammatory cytokines in response to microbial products, including bacterial cell wall LPS or viral dsRNA, through PRRs (e.g., TLR4). Cytokine production by mature DCs influences innate and adaptive immune responses, such as commitment of naïve T cells into Th1 or Th2 cells.

Although the maturation of monocytes into DCs is associated with a changing glycome [2, 40], much remains to be determined relating to how these changes influence the functional capacity of DCs. In this manuscript, we use the pharmacologic sialidase inhibitors, zanamivir and DANA, to show that Neu3, and possibly Neu1, enhances LPS-induced cytokine production in primary human monocyte-derived cells. A role for Neu3 in this process is supported further by our results using DCs from Neu1^–/^– mice. Sialidase-mediated change in the carbohydrate composition of specific cell surface glycoconjugates thus may play a pivotal role in development of the adaptive immune response.

MATERIALS AND METHODS

Purification of human monocytes and differentiation into DCs

PBMCs were isolated from leukocytes acquired commercially (leukapheresis product from SeraCare Life Sciences, Milford, MA, USA) and from local donors (guided by a protocol approved by the University of Maryland School of Medicine Institutional Review Board, Baltimore, MD, USA) by centrifugation over Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) gradients using standard procedures. Monocytes were purified from PBMCs by negative selection using StemSep® separation columns (Stem Cell Technologies, Vancouver, BC, Canada) as described previously [36]. Monocyte-derived immature and mature DCs were generated in culture by growth in RPMI-1640 medium containing 10% FBS and human rGM-CSF at 50 ng/ml and rIL-4 at 50 ng/ml (both from R&D Systems, Minneapolis, MN, USA), as described previously [2], with the exception that LPS from Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA) was used at 10 ng/ml, and mature DCs were harvested 24 h after the addition of LPS. Immature DCs were characterized phenotypically by the loss of cell surface CD14, the de novo expression of CD206 (mannose receptor), CD83, and CD1a, and the up-regulation of HLA-DR, CD40, CD80, and CD86; mature DCs expressed increased amounts of the latter six cell surface markers. Macrophages were generated from purified monocytes by growth in RPMI-1640 medium containing 10% heat-inactivated HS (Gemini Bioproducts, Calabasas, CA, USA) and human rM-CSF (R&D Systems), as described previously [36]. Where indicated, cells were grown in the presence of 1–2 mM sialidase inhibitors, zanamivir (GlaxoSmithKline, Research Triangle Park, NC, USA), or DANA (Calbiochem, La Jolla, CA, USA).

Preparation of murine monocytes and DCs

Experiments with mice were performed in accordance with guidelines approved by the University of Maryland School of Medicine and St. Jude Children′s Research Hospital Institutional Animal Care and Use Committees. Bone marrow cells were harvested from the femurs of 8- to 12-week FVB WT and Neu1^–/^– [41] mice, treated with ammonium chloride potassium lysis buffer (Gibco, Invitrogen, Carlsbad, CA, USA) and used to isolate monocytes and to generate DCs. Bone marrow monocytes were purified from total bone marrow cells by negative selection using an EasySep® mouse monocyte enrichment kit (Stem Cell Technologies), following the protocol suggested by the manufacturer. DCs were generated in culture by maintaining 3 × 10⁶ total bone marrow cells in six-well tissue-culture plates in RPMI-1640 medium containing 10% FCS, 2 mM GlutaMax, 1 mM pyruvate, and 1% MEM nonessential amino acids (all from Gibco, Invitrogen), as well as with 50 μM 2-ME (Sigma-Aldrich), murine rGM-CSF at 20 ng/ml, and murine rIL-4 at 20 ng/ml (R&D Systems). Nonadherent cells were harvested after 5 days in culture, and CD11c⁺ DCs were isolated using an EasySep® mouse CD11c-positive DC selection kit (Stem Cell Technologies). Purified DCs were placed in culture at 5 × 10⁵ cells/well in 12-well tissue-culture plates in fresh complete medium with or without 5 ng/ml LPS. Medium from cells was harvested after 12 h in culture and evaluated by ELISA for cytokine expression, and cells were collected for analysis of sialidase activity.

Measurement of sialidase and PPCA activities

Sialidase activity in cell lysates was determined using the artificial substrate 4-MU-NANA (Sigma-Aldrich), mixed bovine brain gangliosides (Calbiochem), or endogenous cellular sialylconjugates as described previously [36]. One unit of sialidase activity was defined as the amount of enzyme that released 1 nmole sialic acid/h at 37°C. PPCA activity in lysates from human monocytes and DCs was measured as described elsewhere [42]. The amount of activity measured in each sample was adjusted based on protein concentration to represent activity/mg protein.

Isolation of RNA and real-time RT-PCR

Total RNA was isolated from monocytes and monocyte-derived DCs and macrophages using an RNeasy mini kit (Qiagen, Valencia, CA, USA), following the protocol suggested by the manufacturer. The RNA preparation was treated with DNase I (Gibco, Invitrogen) at 37°C for 30 min to remove contaminating DNA. DNase was then removed by binding to Blue Sorb DNase affinity slurry (Clonogene, St. Petersburg, Russia). Semiquantitative real-time RT-PCR was performed using a QuantiTect SYBR Green RT-PCR kit (Qiagen) with an ABI sequence detection system (ABI Prism 5700) to detect gene expression of NEU1 (GenBank Accession NM_000434), NEU2 (GenBank Accession NM_005383), NEU3 (GenBank Accession AB008185), and NEU4 (GenBank Accession NM_080741) using RNAs, obtained as described above. Gene expression of 18S rRNA (GenBank Accession X03205) was also measured for use as an internal control. Total RNA (10 ng) was used to quantitate expression of each gene using primers and reaction conditions that were described previously [20, 36]. No NEU2 RNA was detected. The fold change in expression of NEU1, NEU3, and NEU4 RNAs in DCs was normalized to the fold increase of each relative to the amount of 18S RNA. All reactions were run in triplicate, and the accuracy of each reaction was monitored by analysis of melting curves and product size on gel electrophoresis.

Western blot analysis of cellular proteins

Monocytes and monocyte-derived cells were collected and solubilized as described previously [36], and protein concentration was determined using BCA (Sigma-Aldrich). Protein (5–25 μg) from each cell lysate was resolved by electrophoresis on a 4–20% SDS-polyacrylamide gel using Tris-glycine-SDS running buffer (gel and running buffer from Gibco, Invitrogen), electrotransferred by a semi-wet method to a Sequi-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA), and probed with polyclonal rabbit antibodies to Neu1 or Neu3 at 0.5 μg/ml. Polyclonal anti-Neu1 IgGs were purified from sera of rabbits (kindly provided by Alexei Pshezhetsky, University of Montreal, Canada), which were immunized with human rNeu1 sialidase and were characterized as described elsewhere [43]. Rabbit polyclonal anti-Neu3 IgGs were generated by immunizing rabbits with a recombinant protein spanning aa 243–342 of the human Neu3 sialidase and were affinity-purified using a recombinant protein fragment (Novus Biologicals, Littleton, CO, USA). The specificity of these antibodies was demonstrated by analyzing proteins from lysates of COS-7 cells that were transfected with pCMV-NEU1 or pcDNA3.1-NEU3 sialidase expression vectors (kind gifts from A. Pshezhetsky, University of Montreal, and from Taeko Miyagi, Miyagi Cancer Center and Research Institute, Miyagi, Japan). The respective blots were incubated with a 1:10,000 dilution of goat HRP-conjugated anti-rabbit IgGs (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), developed using an ECL substrate kit (Amersham Biosciences, Piscataway, NJ, USA) and exposed to Kodak X-ray film.

Identification of cell surface proteins by biotinylation

Proteins on the surface of 3 × 10⁷ monocytes and 1.5 × 10⁷ monocyte-derived immature and mature DCs were labeled with sulfo-NHS-SS biotin and separated from intracellular proteins by binding to immobilized Neutravidin using components of the Pinpoint cell surface protein isolation kit (Pierce, Rockford, IL, USA), as recommended by the manufacturer. Nonbiotinylated, intracellular proteins that did not bind to the Neutravidin column were collected in 0.3 ml lysis buffer provided in the kit, and the protein concentration was determined by the BCA reagent as above. Biotinylated, cell surface proteins were eluted from the Neutravidin column in 0.20 ml SDS-PAGE gel-loading buffer. Proteins from a portion of each sample (0.025 ml of the 0.20 ml biotinylated samples and 20 μg nonbiotinylated proteins from monocytes and immature and mature DCs in 0.070, 0.024, and 0.020 ml of the total 0.30 ml vol, respectively) were analyzed by immunoblot using rabbit polyclonal anti-Neu1 and anti-Neu3 IgGs (described above). Blots were also probed with 0.5 μg/ml rabbit polyclonal IgGs against PPCA [20] and 0.25 μg/ml rabbit anti-β-actin mAb (Cell Signaling Technology, Danvers, MA, USA).

Immunofluorescent staining of cell surface proteins and glycoconjugates and analysis by flow cytometry

Proteins on the surface of monocytes and monocyte-derived cells were detected by incubating cells at 1 × 10⁶ cells/ml PBS containing 2% heat-inactivated HS (Gemini Bioproducts) and anti-CD32 FcR antibodies (1.5 μg; Stem Cell Technologies) at 4°C for 15 min to minimize nonspecific binding of reagents. To verify cell purity and maturation status in each experiment, cells were stained at 4°C for 30 min with PE-, allophycocyanin-, or FITC-conjugated mAb to CD3, CD14, CD19, CD206, CD40, CD80, CD83, CD86, and CD1a and isotypic control IgGs (all mAb from BD PharMingen, San Diego, CA, USA), following the procedure recommended by the manufacturer. Where indicated, cells were stained with polyclonal rabbit anti-Neu1 IgGs at 100 μg/ml or anti-Neu3 IgGs at 50 μg/ml. Bound rabbit IgGs were detected using 8 μg/ml FITC-conjugated goat anti-rabbit F(ab′)₂ fragment (Alexa Fluor 488, Molecular Probes, Eugene, OR, USA) at 4°C for 30 min. Where indicated, cells were reacted with 2 μg/ml FITC-conjugated lectins ECA or PNA (both lectins from EY Laboratories, San Mateo, CA, USA). ECA binds to the exposed galactose on glycoconjugates bearing a Galβ1-4GlcNAc motif; PNA binds to Galβ1-3GalNAc. Following incubation with antibodies or lectins, cells were washed with 2 ml PBS containing 2% HS and fixed with 1% paraformaldehyde. Cells were analyzed using a FACSCaliber (Becton Dickinson, Mountain View, CA, USA), and data were analyzed using FlowJo data analysis software (Tree Star, Ashland, OR, USA).

Quantitation of cellular sialic acid content

Monocytes (5×10⁶) and mature DCs (2×10⁶) that were grown in culture in the absence and presence of sialidase inhibitors were resuspended in 0.10 ml PBS/0.5% BSA (Pentex bovine albumin fraction V, Miles Inc., Kankakee, IL, USA) containing 500 μU/ml bacterial NANase (crystalline, type X, from Clostridium perfringens; Sigma-Aldrich) and incubated at 37°C for 3 h. After treatment with NANase, cells were collected by centrifugation, and 0.02 ml each supernatant was analyzed for sialic acid content using a Dionex DX600 chromatography system (Dionex Corp., Sunnyvale, CA, USA) equipped with an electrochemical detector (ED50, Dionex Corp.), as described previously [36].

Determination of cytokine expression

Cytokine levels were determined in the medium of cells 12 h after exposure to LPS. ELISA was used to measure IL-12p40, TNF-α, and IL-6 (R&D Systems).

Characterization of ganglioside composition of DCs

Monocytes were differentiated in culture into immature and mature DCs in the absence or presence of 1 mM zanamivir or DANA and were pulse-labeled for 2 h with 0.1 μCi/ml [3H]-sphingosine (specific activity range: 15–30 Ci/mmol; 555–1110 GBq/mmol; Perkin-Elmer, Waltham, MA, USA) after 72 h in culture. After the 2-h pulse, the medium was removed, and cells were maintained in the original conditioned medium for an additional 48 h to allow metabolic radiolabeling of all sphingolipids. Thereafter, a portion of cells was treated with LPS for an additional 24 h to generate mature DCs. All cells were harvested 72 h after the addition of radioactive label. Neutral lipids and gangliosides from cells were extracted with chloroform/methanol 2:1 (v/v) and subjected to two-phase partitioning in chloroform/methanol 2:1 (v/v) and 20% water. [3H]-Sphingolipids of aqueous and organic phases were separated by HPTLC using chloroform/methanol/CaCl₂, 60/40/9, and chloroform/methanol/water, 110/40/6, respectively; identified by comparison with radiolabeled standards; and quantified by digital radiochromatoimaging using a Beta-Imager 2000 instrument. Data are shown as dpm/mg total protein.

RESULTS

Differentiation of human monocytes into DCs results in increased expression of Neu1 and Neu3 sialidases

We have shown previously that differentiation of human monocytes into macrophages is associated with increased expression of Neu1 and Neu3 sialidases [36]. To determine whether monocyte-derived DCs also up-regulate expression of these sialidases, sialidase activity was analyzed in lysates of human monocytes and monocyte-derived immature and mature DCs from five donors using exogenous substrates: 4-MU-NANA, which specifically detects Neu1 in monocytes and macrophages [36], or mixed bovine gangliosides, which are hydrolyzed by Neu2, Neu3, or Neu4. With 4-MU-NANA as substrate, the 8.2 ± 1.3 units of sialidase activity in monocytes increased to 173.3 ± 23.4 units in immature DCs and remained elevated at 163.5 ± 11.9 units in mature DCs (Fig. 1A, left panel). When exogenous mixed bovine gangliosides were added as substrate to a lysate of monocytes, 23.8 ± 3.7 units of sialidase activity were detected (Fig. 1A, middle panel). Differentiation of monocytes into immature and mature DCs resulted in an increase in this activity to 41.5 ± 4.5 and 45.2 ± 5.2 units, respectively (Fig. 1A, middle panel). The amount of sialidase activity in DCs detected with 4-MU-NANA was approximately twofold greater than what was measured in macrophages generated from the same monocytes. In contrast, the amounts of sialidase activity in DCs and macrophages detected with exogenous gangliosides were comparable.

Figure 1. Differentiation of monocytes into DCs is associated with increased activity and mRNA expression of Neu1 and Neu3.

Monocytes were purified from the peripheral blood of human donors and were differentiated into immature (imDC) and mature (mDC) DCs by growth at 37°C in RPMI-1640 medium with 10% FCS and human rGM-CSF and rIL-4, as described in Materials and Methods. (A) Sialidase activity. Sialidase activity from a lysate of 5 × 10⁶ cells was determined immediately after isolation of monocytes and from the same number of immature and mature DCs. Sialidase activity was measured with 4-MU-NANA or mixed bovine gangliosides [(+) Gangliosides] added as substrate or in the absence of exogenous substrate [(+) Endogenous Sialylconjugates], as described in Materials and Methods. Sialidase activity is reported in units that reflect the release of 1 nmole sialic acid/h at 37°C/mg protein. (B) Expression of mRNA. Total RNA was isolated from monocytes and monocyte-derived immature and mature DCs after 6 days in culture. To obtain mature DCs, LPS had been added to immature cells after 5 days in culture, and all cells were harvested after an additional 24 h in culture. RNA (10 ng) was used with primers that were specific for NEU1–4 in SYBR Green semi-quantitative real-time RT-PCR, as described in Materials and Methods. The fold change in NEU1, NEU3, and NEU4 RNAs in monocyte-derived cells was normalized to the amount of change in expression of 18S RNA in each type of cell. The amount of RNAs encoding these sialidases in monocytes was assigned the value of 1. Sialidase activity and RNA quantitation in macrophages generated from monocytes from the same donors are shown for comparison. The results represent the mean ± sem from five independent experiments using cells from five different donors. Statistical significance compared with monocytes (*P<0.002; **P<0.04; ***P<0.05) is shown by asterisks.

The observed increase in sialidase activity of monocyte-derived DCs that was detected with both substrates described above was supported by measuring sialidase activity using endogenous sialylconjugates. Monocytes expressed 3.7 ± 1.5 units of sialidase activity in the absence of exogenous substrates (Fig. 1A, right panel). Sialidase activity increased to 13.4 ± 2.4 and 12.1 ± 2.7 in immature and mature DCs, respectively, levels similar to the activity that was detected in macrophages in the absence of exogenous substrate (Fig. 1A, right panel). These results using endogenous sialylconjugates, which are the natural substrates for sialidases, confirm the up-regulation of sialidase activity during differentiation of monocytes that was detected with 4-MU-NANA and mixed bovine gangliosides.

To evaluate whether sialidase activity of monocyte-derived DCs was attributable mostly to Neu1 and Neu3, as was shown in macrophages [36], the relative amounts of RNAs encoding Neu1–4 in monocytes and in immature and mature DCs were determined by real-time RT-PCR. RNAs encoding Neu1, Neu3, and Neu4 were detected in monocytes, whereas no RNA encoding Neu2 was detected in monocytes or monocyte-derived DCs (data not shown). At all times analyzed in monocytes, the absolute amount of Neu1 RNA exceeded that of Neu3 and Neu4 (the crossover comparative thresholds during PCR for Neu1, Neu3, and Neu4 RNAs were 27.5±0.1, 29.9±0.3, and 28.7±0.2, respectively). As monocytes differentiated into immature DCs, the amount of RNAs encoding Neu1 and Neu3 increased 25.9 ± 1.1- and 2.4 ± 0.2-fold, respectively, normalized to the change in amount of 18S rRNA (Fig. 1B, left and middle panels). In contrast, during differentiation, the amount of Neu4-specific RNA declined 14.5-fold to the limit of detection (Fig. 1B, right panel). The expression of Neu1-specific RNA remained up-regulated in mature cells (11.0±0.9-fold increase) but at a lower level than in immature DCs. The relative amount of RNA encoding Neu3 changed minimally after further differentiation of immature DCs into mature DCs. Thus, as was shown previously for Neu1 in monocyte-derived macrophages [36], the increase in RNA encoding Neu1 reflected the increase in activity detected with 4-MU-NANA in immature DCs. Similarly, the increase in Neu3 RNA, coupled with the absence or marginal detection of RNA encoding Neu2 and Neu4, suggests that Neu3 is solely responsible for the increased sialidase activity detected with mixed gangliosides.

Given the increase in sialidase activity measured with 4-MU-NANA and mixed bovine gangliosides and in the amount of RNA encoding Neu1 and Neu3 in monocyte-derived DCs, it was determined whether there was a corresponding increase in the total amount of Neu1 and Neu3 proteins. Monocyte and DC proteins were separated by SDS-PAGE and analyzed on an immunoblot using rabbit polyclonal anti-Neu1 and -Neu3 IgGs. The specificity of each antibody for Neu1 or Neu3 is shown on an immunoblot, where only the 42- to 44-kDa Neu1 doublet or 48-kDa Neu3 was detected by the respective antibody in lysates prepared from COS cells that were transfected with the Neu1 or Neu3 gene (Fig. 2A and B). No band was detected with either antibody in lysates from mock-transfected COS cells probed on the immunoblot. The anti-Neu1 IgGs recognized the 42-kDa Neu1 sialidase in monocytes, with a more intense band in immature and mature DCs (Fig. 2A). The anti-Neu3 IgGs recognized in immature and mature DCs a protein with MW of 48 kDa, the expected MW of Neu3 (Fig. 2B). Additional, higher MW bands at 50 and 52 kDa, possibly representing post-translationally modified Neu3, were also present in these cells. In monocytes, a faint 50-kDa band of Neu3 was seen following a longer exposure of the immunoblot (data not shown). The anti-Neu3 IgGs also detected, in lysates from monocytes and derived cells, higher MW proteins that remain to be identified (Fig. 2B). In summary, there was a correlation between the increase in sialidase activity that was detected with 4-MU-NANA and the increased amounts of Neu1 RNA and protein in monocyte-derived DCs. Similarly, the increase in sialidase activity detected with mixed bovine gangliosides reflected the up-regulation of Neu3 gene expression and the presence of the 48- to 50-kDa form of Neu3 during monocyte differentiation into DCs.

Figure 2. The amount of Neu1 and Neu3 proteins increases during monocyte differentiation.

Monocytes (Mono) and monocyte-derived immature DCs, mature DCs, and macrophages (Mac) were collected, and total cellular protein from a lysate of each was separated by electrophoresis on 4–20% SDS-polyacrylamide gels, transferred to PVDF membranes, and analyzed for the amount of Neu1 (A) and Neu3 (B) protein using specific antibodies, as described in Materials and Methods. The same amount of total cellular protein from monocytes and monocyte-derived cells was analyzed in each lane with the respective antibodies (5 μg for samples probed with anti-Neu1 IgGs and 20 μg for samples probed with anti-Neu3 IgGs). An equal amount of protein (2 μg) from COS cells that were transfected with a vector expressing Neu1 (COS-Neu1), Neu3 (COS-Neu3), or empty vector (COS-control) was also analyzed on the immunoblot using anti-Neu1 IgGs (A) and anti-Neu3 IgGs (B). The tick marks on the left side of the radiograph represent protein MW markers. These results from one donor are representative of data from five independent experiments using cells from four different donors.

Cellular localization of Neu1 and Neu3 during differentiation of human monocytes

Although Neu1 and Neu3 have been described in various cells as lysosome- and plasma membrane-associated sialidases, respectively, each has been found in intracellular and cell surface locations [20, 22, 28, 31, 44, 45]. To determine if Neu1 and Neu3 are present on the outer cell surface of primary human monocytes and monocyte-derived DCs, surface proteins of intact cells were biotinylated, separated from intracellular proteins by binding to immobilized Neutravidin, and analyzed by immunoblot using anti-Neu1 and anti-Neu3 IgGs. When the immunoblot was probed with an antibody against β-actin, a protein known to be located soley intracellularly, a band was detected only in the nonbiotinylated fraction of monocytes and DCs, confirming that intracellular proteins were not biotinylated (data not shown). The 42- to 44-kDa Neu1 was detected in the fraction of biotinylated, cell surface proteins from immature DCs and increased in intensity as cells matured further (Fig. 3A, Biotinylated). This form of Neu1 was also present in the fraction of nonbiotinylated, intracellular proteins from monocytes and monocyte-derived cells (Fig. 3A, Non-Biotinylated). There was also a prominent band that migrated at 68 kDa in the biotinylated material from monocytes and DCs (Fig. 3A, Biotinylated). Neu1 exists in lysosomes in a multienzyme complex that contains β-galactosidase and PPCA, a protein that protects and activates Neu1 [46]. Detection of this 68-kDa protein from lysates of monocytes and DCs on an immunoblot using anti-PPCA IgGs (shown for DCs, Fig. 3A) suggests that Neu1 is tightly bound to the 20- or 32-kDa subunit of PPCA on the surface of these cells, as we showed previously in lymphocytes [20]. PPCA is present in monocytes (181 units of enzyme activity were detected in cell lysates), and the specific activity increased 2.5-fold (456 units detected) during differentiation of monocytes to DCs.

Figure 3. A portion of Neu1 and Neu3 is present on the surface of monocytes and DCs.

Proteins on the surface of monocytes and monocyte-derived immature and mature DCs were labeled with sulfo-NHS-SS biotin, separated from intracellular proteins by binding to immobilized Neutravidin, and analyzed by immunoblot using anti-Neu1 (A) and anti-Neu3 (B) IgGs. Protein from the biotinylated fraction of mature DC was also analyzed with anti-PPCA IgGs (A). Proteins from an equal portion of each biotinylated sample (0.03 ml from the 0.20-ml total volume) and from 20 μg nonbiotinylated proteins (0.070, 0.024, and 0.020 ml from a total volume of 0.30 ml from monocytes and immature and mature DCs, respectively) were committed to the respective lanes. An aliquot of monocytes and monocyte-derived immature and mature DCs was removed prior to biotinylation, and cells were stained with anti-Neu1 (C) or anti-Neu3 (D) IgGs and analyzed by flow cytometry. Thin, dotted lines: preimmune IgGs; thick lines: anti-Neu1 or anti-Neu3 IgGs.

A small amount of Neu3 was detected in the biotinylated and nonbiotinylated fractions of monocytes, as seen on longer exposure of the immunoblot (data not shown). Neu3 was also detected on the surface of immature and mature DCs (Fig. 3B, Biotinylated), but a large portion of Neu3 was not biotinylated, suggesting an additional, major intracellular localization in these cells. This finding in DCs is in contrast to the predominant cell surface localization of Neu3 that has been reported previously [22, 45]. It is of note that the additional, high MW proteins (>70 kDa), which were detected by the anti-Neu3 IgGs on an immunoblot of whole cell lysates, were seen only in the nonbiotinylated, intracellular fraction (Fig. 3B, Non-Biotinylated).

Analysis by flow cytometry of intact monocytes stained with each antibody indicated that Neu1 and Neu3 are present on the cell surface (Fig. 3C and D). Neu1 was detected on the surface of monocytes, and the amount remained relatively constant during differentiation (Fig. 3C), consistent with the results of the biotinylation experiment. Neu3 was also detected on the surface of monocytes, but in contrast to the findings with Neu1, the amount of Neu3 detected on the cell surface decreased during differentiation into DCs (Fig. 3D). The quantitatively different results of cell surface localization of Neu3 shown in Fig. 3B and D may reflect disparate techniques: immunoblot analysis of denatured proteins versus antibody detection of native Neu3 on the surface of intact cells. It is also conceivable that epitopes of cell surface Neu3 are masked during differentiation of monocytes, possibly by associating with caveolin [47] and other proteins in lipid rafts [47, 48]. It is clear from these data that at the very least, a portion of Neu1 and Neu3 is present on the surface of monocytes during various stages of maturation.

Differentiation of monocytes into DCs in the presence of pharmacologic sialidase inhibitors results in an increase in cell surface sialic acid content

The roles of Neu1 and Neu3 sialidases and the identity of their physiologic substrates during monocyte differentiation into DCs are unknown. To determine whether Neu1 and Neu3 modulate the total amount of cell surface sialic acid during differentiation of monocytes, monocyte-derived DCs were grown in the presence of competitive, pharmacologic sialidase inhibitors (DANA or zanamivir), and changes in sialic acid content were analyzed by lectin staining and HPLC (Dionex Corp.). DANA effectively inhibits all four human sialidases, with IC₅₀ from 43 to 168 μM [49, 50], whereas zanamivir, an inhibitor designed specifically to inhibit influenza virus NANase [51], effectively inhibits mammalian sialidases Neu2 (IC₅₀ of 16 μM), Neu3 (IC₅₀ of 7 μM), and Neu4 (IC₅₀ of 487–690 μM) but not Neu1 (IC₅₀ of 2713 μM) [49]. As there is no detectable Neu2 in monocytes or monocyte-derived cells, and expression of Neu4 is down-regulated to the limit of detection in DCs, Neu3 is the predominant zanamivir-inhibitable sialidase activity in DCs. It has been strongly proposed that DANA and zanamivir act only at the cell surface [42] (GlaxoSmith-Kline, unpublished results). Differentiation of monocytes in the presence of 1 mM DANA or 2 mM zanamivir had a minimal effect on cell viability or apoptosis, as measured by staining with Annexin V and 7-amino-actinomycin D, and led to no differences in expression of cell surface markers specific for immature or mature DCs (data not shown). These results demonstrate that inhibition of cell surface sialidase activity does not affect cell viability nor differentiation of monocytes into DCs.

Monocytes and mature DCs, grown in the presence of DANA or zanamivir, were stained with tagged lectins PNA and ECA and analyzed by flow cytometry. In the absence of terminal sialic acid, PNA binds to the penultimate galactose on glycoconjugates bearing a Galβ1-3GalNAc motif; ECA binds to Galβ1-4GlcNAc. When grown in the absence of inhibitors, but after exposure to LPS, mature DCs unexpectedly stained less intensely with PNA or ECA than did monocytes, indicating more cell surface sialic acid on the mature cells (Fig. 4 and Table 1). The MFI of staining with PNA declined from 793 in monocytes to 341 in mature DCs; the MFI of staining with ECA declined from 95 in monocytes to 51 in mature cells (shaded regions of histograms, Fig. 4; Table 1). The binding of both lectins was reduced further in DCs that were grown in the presence of 1 mM zanamivir (i.e., indicating more cell surface sialic acid), with even less binding of PNA or ECA when zanamivir was used at 2 mM (Fig. 4 and Table 1). Similarly, growth of cells in the presence of DANA reduced the staining of cells by both lectins. These data suggest that mature DCs, in spite of up-regulated sialidase activity, contain more cell surface sialic acid than monocytes. The additional increase in cell surface sialic acid when inhibitors are present suggests that Neu1 and/or Neu3 selectively desialylate specific glycoconjugates on the cell surface during monocyte differentiation into DCs.

Figure 4. Changes in binding of PNA and ECA to the surface of monocyte-derived mature DCs grown in the absence or presence of sialidase inhibitors.

Monocytes were differentiated in culture into mature DCs in the absence or presence of 1 or 2 mM zanamivir or 1 mM DANA and were stained with FITC-conjugated lectins PNA (A) or ECA (B) and evaluated by flow cytometry, as described in Materials and Methods. Thin, dotted line: unstained cells grown without inhibitor; shaded area: cells grown without inhibitors; heavy, solid line: cells grown in the presence of 1 mM zanamivir or DANA; dashed line: cells grown in the presence of 2 mM zanamivir. The histograms for unstained cells, grown in the presence of each inhibitor, overlapped the histogram of unstained, control cells and are not shown. Data shown are representative of data from four experiments using cells from four different donors.

Table 1.

Increased Sialic Acid Content of Monocyte-Derived DCs Grown in the Presence of Inhibitors

	MFIa on flow cytometry when stained with:		Sialic acid contentb
	PNA	ECA	nmoles/5 × 106 cells	pmoles/μg protein
Monocytes	793	95	2.0 ± 0.1	31.7 ± 1.5
DCs - without inhibitor	341	51	8.9 ± 1.4	17.2 ± 2.7
DCs - zanamivir (1 mM)	259	29	20.3 ± 0.2	39.5 ± 0.4
DCs - zanamivir (2 mM)	160	12	24.0 ± 1.9	46.7 ± 3.7
DCs - DANA (1 mM)	276	41	17.9 ± 0.2	34.8 ± 0.5

^aMFI determined by FloJo software (Tree Star).

^bSialic acid content was analyzed using a Dionex DX600 chromatography system, as described in Materials and Methods. Results represent the mean ± sem from three independent experiments using cells from three different donors. Monocytes and mature DCs (5 × 10⁶) contained 63 and 514 μg protein, respectively.

The increase in sialic acid content of mature DCs and the role of sialidases in modulating this increase, which was suggested by changes in ECA and PNA binding, were corroborated directly by HPLC (Dionex Corp.) measurement of total sialic acid content of monocytes and monocyte-derived mature DCs, grown in the presence of DANA and zanamivir. On a per-cell basis, mature DCs contain more sialic acid than monocytes (8.9 compared with 2.0 nmoles/5×10⁶ cells; Table 1). However, given the greater amount of protein in DCs (514 μg protein/5×10⁶ cells) than in monocytes (63 μg protein/5×10⁶ cells), the amount of sialic, on the basis of protein content, declines from 31.6 to 17.3 pmoles sialic acid/μg protein during monocyte differentiation to DCs (Table 1). This relative cellular hyposialylation that occurs under normal growth conditions did not occur in mature DCs grown in the presence of zanamivir or DANA: mature DCs contained 39.5 and 34.8 pmoles sialic acid/μg protein, respectively, more than twice the amount of sialic acid in DCs in which sialidase was not inhibited (Table 1). These data correlate with changes in lectin-binding and demonstrate that Neu1 and/or Neu3 are active in removing sialic acid from cell surface glycoconjugates during monocyte differentiation into mature DCs. This occurs in spite of the increase in total cell surface sialic acid on a per-cell basis (Fig. 4 and Table 1).

GM3 ganglioside as a substrate for sialidase activity in monocyte-derived cells

Ganglioside GM3 is a physiologic substrate for Neu3 sialidase in various types of cells [21, 22]. GM3 synthase (ST3GalV) opposes the action of Neu3 by catalyzing addition of sialic acid to lactosylceramide to yield GM3 (Fig. 5A). To determine whether GM3 is desialylated by sialidases during monocyte differentiation, monocytes were differentiated into immature and mature DCs in the presence of sialidase inhibitors. Lipids were pulsed-labeled for 2 h with [³H]-sphingosine, and after a 48-h incubation, cells were treated with LPS or remained untreated. After an additional 24-h incubation, cells were disrupted, and gangliosides and neutral lipids were separated by HPTLC, and each was quantified. GM3 is the major ganglioside in monocytes (data not shown). The amount of GM3 increases as monocytes differentiate into DCs, and this increased amount is coincident with enhanced expression of ST3GalV synthase transcripts measured by RT-PCR (data not shown). The total amount of [³H]-GM3 increases even further in mature DCs grown in the presence of zanamivir and DANA, increasing from a baseline of 23,932 dpm/mg protein to 40,647 and 29,915, respectively (Fig. 5B and C). There was a corresponding decline in the amount of lactosylceramide in the cells grown under conditions of sialidase inhibition (Fig. 5C). Immature DCs grown in the presence of zanamivir showed similar changes in expression of GM3 and lactosylceramide. That the change in amount of GM3 in cells grown with zanamivir and DANA was not a result of enhanced expression of ST3GalV was confirmed by finding equivalent amounts of mRNA encoding ST3GalV in cells grown under all conditions (data not shown). Thus, Neu3 desialylates GM3 during maturation of monocytes to DCs, as shown by the increased amounts of GM3 when inhibitors are present.

Figure 5. Effect of cellular sialidase activity on amount of GM3 ganglioside as monocytes differentiate into DCs.

Monocytes were differentiated in culture into immature and mature DCs in the absence or presence of 1 mM zanamivir (imDC + Z, mDC + Z) or DANA (mDC + D) and were pulse-labeled for 2 h with 0.1 μCi/ml [3H]-sphingosine, as described in Materials and Methods. Schematic describing the synthesis and desialylation of GM3 by GM3 synthase and sialidase, respectively, is shown (A). Gangliosides and neutral lipids from cells were extracted, separated by HPTLC, and identified on radiograph by comparison with radiolabeled standards, as described in Materials and Methods (B, shown only for gangliosides). The amounts of labeled GM3 and lactocylceramide (LacCer) were quantified (C) by digital radiochromatoimaging using a Beta-Imager 2000 instrument. Data represent dpm/mg total protein. These results from one donor are representative of data from two independent experiments using cells from two different donors.

Inhibition of cellular sialidase(s) reduces LPS-induced cytokine production in mature DCs

Desialylation of glycoconjugates on the surface of human monocytes leads to enhanced production of cytokines in response to LPS [38, 39]. To determine whether inhibition of sialidase activity in maturing DCs and the consequent hypersialylation of surface glycoconjugate(s) reduces cytokine production after cells are exposed to LPS, monocytes were differentiated into DCs in the presence of DANA (expected to inhibit cell surface Neu1 and Neu3) or zanamivir (specifically inhibits cell surface Neu3). After 5 days in culture, monocyte-derived immature DCs expressed undetectable levels (<50 pg/ml) of IL-12p40, TNF-α, or IL-6 (data not shown). When cytokine production was evaluated at 12 h after exposure to LPS, the level of IL-12p40, TNF-α, and IL-6 rose markedly to 18.0, 7.0, and 11.7 ng/ml, respectively (Fig. 6). Production of each cytokine was reduced when cells were grown in the presence of the inhibitors (Fig. 6). The amount of IL-12p40, TNF-α, and IL-6 in the medium of cells exposed to 1 mM zanamivir was reduced to 7.2, 4.4, and 8.4 ng/ml, respectively. Growth of cells in the presence of DANA resulted in a more pronounced reduction in levels of IL-12p40, TNF-α, and IL-6 to 4.8, 2.3, and 3.3, respectively. These data demonstrate that sialidase activity supports LPS-induced cytokine production in mature DCs and strongly suggest that Neu3 and possibly Neu1 mediate this effect.

Figure 6. Modulation of LPS-induced cytokine production in mature DCs grown in the presence of sialidase inhibitors.

Human monocytes were differentiated into immature DCs in the presence of 1 mM zanamivir or DANA and exposed to 10 ng/ml LPS after 5 days in culture, and an aliquot of the medium was collected after 12 h for analysis of IL-12p40, TNF-α, and IL-6 by ELISA. No cytokines were detected (<50 pg/ml) in the medium of immature DCs prior to the addition of LPS (data not shown). Data represent the mean ± sem from three separate wells of cells obtained from each of five different donors. Statistical significance compared with cells grown without inhibitors (*P<0.002; **P<0.05) is shown by asterisks.

Sialidase activity in DCs from Neu1^–/^– mice modulates LPS-induced cytokine production

As human and murine Neu1 and Neu3 sialidases share extensive amino acid homology (Neu1: 86%; Neu3: 68%), we generated DCs from WT and Neu1^–/^– mice to determine the influence of Neu1 sialidase on LPS-induced cytokine production. As seen with human monocyte-derived DCs (Fig. 1), murine DCs expressed greater amounts of sialidase activity than total bone marrow cells and bone marrow monocytes from which they were derived (Fig. 7A). With 4-MU-NANA as a substrate, the 0.4 units of sialidase activity in a lysate of total bone marrow cells and 2.5 units in bone marrow monocytes increased to 9.9 units in murine DCs (Fig. 7A). This activity was inhibited >95% in the presence of 1 mM DANA. As expected, DCs from Neu1^–/^– mice expressed marginal activity (0.4 units) with 4-MU-NANA as substrate (Fig. 7A).

Figure 7. Neu1 deficiency does not impair LPS-induced cytokine production in murine DCs.

Bone marrow cells were isolated from WT and Neu1^–/^– mice and were used to generate DCs, as described in Materials and Methods. (A) Sialidase activity. Sialidase activity from a lysate of 5 × 10⁶ total bone marrow (BM) cells and the same number of bone marrow monocytes and from 2 × 10⁶ DCs from WT mice was measured with 4-MU-NANA as substrate, as described in Materials and Methods. Sialidase activity from 2 × 10⁶ DCs from Neu1^–/^– mice and from WT mice in the presence of 1 mM DANA was similarly measured. Sialidase activity is reported in units that reflect the release of 1 nmole sialic acid/h at 37°C/mg protein. (B) Cytokine production. Murine bone marrow-derived DCs were generated in culture in the absence or presence of 1 mM DANA and exposed to 5 ng/ml LPS after 5 days in culture, and an aliquot of the medium was collected after 12 h for analysis of IL-12p40, TNF-α, and IL-6 by ELISA. No cytokines were detected (<50 pg/ml) in the medium of cells prior to the addition of LPS (data not shown). Data represent the mean ± sem from three separate wells of cells from one experiment generated from pooled bone marrow cells of two mice and are representative of results from three separate experiments. Statistical significance compared with WT cells grown without inhibitor (WT- DCs) is shown by asterisks; *P>0.50; **P<0.01.

To determine whether cytokine production in DCs from Neu1^–/^– mice differed from that of WT mice, DCs were generated from both sets of mice in the absence and presence of 1 mM DANA and challenged with LPS. As seen with human monocyte-derived DCs, murine DCs expressed undetectable levels (<50 pg/ml) of IL-12p40, TNF-α, and IL-6 prior to exposure to LPS (data not shown). When cytokine production was evaluated at 12 h after exposure to LPS, the level of IL-6 and TNF-α produced by DCs rose to 6.0 and 0.85 ng/ml, respectively (Fig. 7B). There was an equivalent increase in the level of IL-6 (6.3 ng/ml) and TNF-α (0.80 ng/ml) produced by DCs from Neu1^–/^– mice (Fig. 7B). In contrast to results with human DCs, the level of IL-12p40 remained undetectable in DCs from both sets of mice after treatment with LPS (data not shown). As demonstrated in human DCs (Fig. 6), growth of WT DCs in the presence of 1 mM DANA resulted in a reduction in levels of IL-6 and TNF-α to 4.2 and 0.45 ng/ml, respectively. That this reduction was not related to inhibition of Neu1 sialidase activity was demonstrated by an equivalent diminution in production of IL-6 and TNF-α to 4.1 and 0.47 ng/ml, respectively, in DCs from Neu1^–/^– mice (Fig. 7B). These results indicate that sialidase activity is involved in cytokine production in murine DCs, as in humans, and that a sialidase other than Neu1 (likely Neu3) mediates this process.

DISCUSSION

DCs perform many critical functions in the immune system. During inflammatory and infectious states, they recognize and phagocytize pathogens (e.g., bacteria, viruses) and process individual antigens for presentation to other cells of the immune system. During this response, they also produce proinflammatory cytokines that regulate innate and adaptive immune responses. Changes in the carbohydrate composition of key cell surface molecules by glycan-modifying enzymes, such as sialyltransferases and the four genetically distinct human sialidases, are important for some of these functions. Our data in this manuscript show that LPS-induced cytokine production in DCs is inhibited by specific pharmacologic inhibitors of sialidase activity, suggesting that sialidase-mediated desialylation of cell surface glycoconjugates plays a key role in this process. We demonstrate that Neu1 and Neu3 are up-regulated at the RNA, protein, and enzymatic levels during differentiation of primary human monocytes into DCs, similar to our previous finding in monocyte-derived macrophages [36]. As Neu2 is undetectable, and expression of Neu4 is down-regulated during differentiation of monocytes into DCs, sialidase modulation of cytokine production likely results from Neu1 and/or Neu3 activity. Involvement of Neu3 is strongly suggested by inhibition of the LPS-induced cytokine production by zanamivir, which inhibits Neu3 but not Neu1. DANA, which inhibits Neu1 and Neu3, also inhibits LPS-induced cytokine production to a further extent, suggesting that Neu1 activity also has a role.

Our results in DCs from Neu1^–/^– mice support a role for a sialidase other than Neu1 (likely Neu3) in LPS-induced cytokine production. We show in this report that DCs from Neu1^–/^– mice produce equivalent amounts of IL-6 and TNF-α, as do DCs from WT mice and that cytokine production in DCs from both sets of mice is similarly reduced by growth of cells with DANA. Our data differ from results reported recently by another laboratory, using PPCA^–/^– mice that have a secondary, partial impairment of Neu1, showing that Neu1 regulates the LPS-induced cytokine response of bone marrow macrophages [9, 52]. One explanation for this difference is that inactivation or reduced expression of PPCA may itself affect cytokine production. The Neu1^–/^– mouse used in our study is a unique tool, as it is a complete knockout of Neu1 activity with preserved PPCA activity [41]. Indeed, we have found that LPS-induced cytokine production in DCs from PPCA^–/^– mice (absent PPCA and inactive Neu1) and from mice in which an inactive PPCA is present (inactive PPCA and active Neu1) is reduced to a similar extent as in cells grown with DANA (data not shown).

Several studies have reported that Neu1 sialidase activity modulates cytokine production in the THP-1 monocyte/macrophage cell line [9, 28]. Involvement of Neu1 was suggested based on experiments using anti-Neu1 IgGs, although an effect of these antibodies on Neu1 sialidase activity was not shown, and oseltamivir phosphate, another inhibitor of influenza virus NANase, whose effectiveness in inhibiting Neu1–4 remains to be shown [50]. It is possible that Neu1 and Neu3 affect cytokine production in human DCs, possibly acting through different mechanisms.

Although the increase in Neu1 expression in human DCs is greater than that of Neu3, the relative roles and importance of these sialidases in monocyte and DC function remain to be determined. Potential functions and specific substrates of cell surface Neu1 and Neu3 in other cell types have been suggested [6, 10, 11, 19, 20, 28, 37, 44], and the presence of Neu1 and Neu3 on the surface of monocytes and DCs suggests that both enzymes have the potential to modulate key immune cell functions. Our results show that during differentiaton of monocytes to immature or mature DCs, the amount of total cell sialic acid increases, but the amount/mg protein decreases. Thus, it would appear that the activity of Neu1 or Neu3 results in selective desialylation of specific cell surface glycoconjugates.

The physiologic substrates for Neu1 and Neu3 in primary human monocytes and maturing DCs and the mechanisms by which desialylation of these substrates leads to cytokine production are not known. It is possible that the action of sialidases during monocyte differentiation primes cells for responsiveness to LPS. It has been reported that LPS treatment of established macrophage and DC lines causes a rapid increase in sialidase activity [9], but the relation between this LPS-inducible sialidase activity in cell lines and the prolonged sialidase activity in primary human DCs grown in IL-4 and GM-CSF remains to be determined. It is of note that we were unable to enhance the LPS responsiveness of immature DCs by artificially desialylating cells with exogenous NANases from C. perfringens (cleaves α2-3, α2-6, and α2-8 linkages) and Streptococcus pneumoniae (cleaves α2-3 linkages) prior to exposure to LPS (data not shown). Although these treatments led to extensive desialylation, as measured by a significant increase in PNA binding, there was no difference in the amount of cytokines detected in the medium of NANase-treated cells in culture for 24 h compared with untreated cells. Thus, it is possible that desialylation of specific glycoconjugates by endogenous sialidase(s) during monocyte differentiation pre-empted the global desialylation of immature DCs in preparation of cells for exposure to LPS. It is also possible that desialylation of intracellular glycoconjugates is important for cytokine response, and these moieties would not have been cleaved by the exogenously-added NANases.

We show in this report that inhibition of sialidase activity with DANA or zanamivir prevents the relative hyposialylation of surface glycoconjugates that occurs normally during monocyte differentiation. It is possible that this hypersialylated state causes nonspecific (e.g., change in net cellular charge) or highly selective (e.g., sialylation of components of TLR4 complex) events that interfere with LPS-induced signaling. It should be noted that cytokine production was reduced greatest in cells grown in the presence of DANA, yet zanamivir caused the greatest increase in sialic acid content. These data suggest that desialylation of key regulatory molecules on the cell surface, rather than changes in global cell surface sialic acid, is important in affecting the LPS responsiveness of cells.

Desialylation of several potential substrates could affect the LPS responsiveness of DCs. Components of the TLR4 complex (TLR4, myeloid differentiation protein 2, and CD14) have multiple N-linked glycosylation sites [53], each of which can potentially be sialylated. It is possible that desialylation of these proteins promotes receptor dimerization, complex formation, and mobilization into lipid rafts, with subsequent intracellular signal transduction. Ganglioside GM3, which we have shown is desialylated by Neu3 in maturing DCs, may also affect LPS-induced cytokine production in DCs. GM3 has been shown to have an important role in modulating activity of the insulin and EGFRs [54, 55] and in cytokine production in PBMCs [56, 57]. GM3 is a known substrate of Neu3. We cannot exclude the potential of Neu1 to desialylate GM3. Although gangliosides are not a preferred substrate for Neu1, Neu1 activity in activated murine lymphocytes led to desialylation of gangliosides and increased production of IL-4 [56].

There is “cross-talk” between signaling intermediates of the TLR4 complex and other cell surface molecules, such as immunoregulatory siglecs [58, 59]. When siglecs are bound to sialic acid-containing glycans of glycoproteins and glycolipids, they transmit an inhibitory, intracellular signal. The relative hyposialylation (reduced sialic acid/mg protein) of cell surface glycoconjugates in DCs compared with monocytes may reduce siglec binding to sialic acid and remove inhibition of downstream events in the TLR4 signaling pathway. This concept is supported by a report that ligand engagement by siglec-7 and siglec-9 negatively regulates signaling of the TCR in Jurkat T cells [60]. The siglec signaling system may comprise only one of multiple pathways through which modulation of cell surface sialic acid content affects TLR4 signal transduction. Removal of sialic acid from the cell surface likely abrogates siglec engagement with sialic acid yet typically exposes a penultimate galactose residue that can be recognized by galectins, another class of immunoregulatory lectins.

We have shown in this report that sialidase activity in human monocyte-derived DCs enhances the LPS-induced production of three cytokines. It remains to be determined whether the production of other cytokines is similarly affected and whether this occurs in DCs that are generated from monocytes by factors other than GM-CSF and IL-4. It is likely that sialidases mediate additional functions of DCs. Desialylation of cell surface glycoconjugates in vivo may increase their chemotactic response to sites of inflammation, as was shown in PMNs [15]. Increased sialidase activity in DCs may be able to enhance the immunogenicity of processed antigens after removal of sialic acid masks of concealed epitopes. Finally, desialylation of glycoconjugates on the surface of DCs likely enhances the interactions with lymphocytes that are critical for cell-mediated immunity [2]. The results of this manuscript build on an expanding awareness that sialic acid modification of cell surface glycoproteins and glycolipids is an extremely important means to control cellular activity.

AUTHORSHIP

N.M.S. conceived the study and designed and supervised the research; N.M.S., I.C., D.v.V., E.J.B., N.P., and L-X.W. performed experiments; N.M.S., I.C., D.v.V., E.J.B., N.P., C.F., B.V., A.d'A., A.S.C., L-X.W., and P.J.G. analyzed the data; E.J.B., N.P., C.F., B.V., and A.S.C. contributed analytical techniques; and N.M.S. and P.J.G. wrote the paper.