science pharmaceutical expo biotech jobs
Originally published online as doi:10.1189/jlb.0503241 on November 21, 2003

Published online before print November 21, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0503241v1
75/2/307    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stamatos, N. M.
Right arrow Articles by Cross, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stamatos, N. M.
Right arrow Articles by Cross, A. S.
(Journal of Leukocyte Biology. 2004;75:307-313.)
© 2004 by Society for Leukocyte Biology

Desialylation of glycoconjugates on the surface of monocytes activates the extracellular signal-related kinases ERK 1/2 and results in enhanced production of specific cytokines

Nicholas M. Stamatos*,{dagger},1, Sabrina Curreli*, Davide Zella* and Alan S. Cross{dagger}

* Institute of Human Virology and
{dagger} Division of Infectious Diseases, Department of Medicine, University of Maryland Medical Center, University of Maryland, Baltimore

1 Correspondence: Institute of Human Virology, University of Maryland Medical System, 725 West Lombard St., Baltimore, MD 21201. E-mail: stamatos{at}umbi.umd.edu


arrow
ABSTRACT
 
Modulation of the sialic acid content of cell-surface glycoproteins and glycolipids influences the functional capacity of cells of the immune system. The role of sialidase(s) and the consequent desialylation of cell surface glycoconjugates in the activation of monocytes have not been established. In this study, we show that desialylation of glycoconjugates on the surface of purified monocytes using exogenous neuraminidase (NANase) activated extracellular signal-regulated kinase 1/2 (ERK 1/2), an intermediate in intracellular signaling pathways. Elevated levels of phosphorylated ERK 1/2 were detected in desialylated monocytes after 2 h of NANase treatment, and increased amounts persisted for at least 2 additional hours. Desialylation of cell surface glycoconjugates also led to increased production of interleukin (IL)-6, macrophage inflammatory protein (MIP)-1{alpha}, and MIP-1ß by NANase-treated monocytes that were maintained in culture. Neither increased levels of phosphorylated ERK 1/2 nor enhanced production of cytokines were detected when NANase was heat-inactivated before use, demonstrating the specificity of NANase action. Treatment of monocytes with gram-negative bacterial lipopolysaccharide (LPS) also led to enhanced production of IL-6, MIP-1{alpha}, and MIP-1ß. The amount of each of these cytokines that was produced was markedly increased when monocytes were desialylated with NANase before exposure to LPS. These results suggest that changes in the sialic acid content of surface glycoconjugates influence the activation of monocytes.

Key Words: sialidase • cellular activation • IL-6 • LPS


arrow
INTRODUCTION
 
Modulation of the sialic acid content of glycoproteins and glycolipids on the surface of diverse types of cells influences cellular activity. Removal of sialic acid from cell surface glycoconjugates affects the interaction of cells with other cells [1 2 3 4 5 ], with soluble regulatory molecules [6 7 8 ], and with numerous bacteria, protozoa, and viruses [9 10 11 12 13 14 15 16 17 ]. Desialylation of cell surface glycoconjugates has also been shown to have a role in cellular differentiation [18 19 20 ] and transformation [21 22 23 ]. Terminal sialyl residues are removed from glycoconjugates by neuraminidases (referred to as sialidases in mammalian cells), which comprise a family of enzymes that are widely distributed throughout nature [24 ]. The significance of sialidase(s) in the normal function of eukaryotic cells has been inferred from the heterogeneous clinical manifestations of individuals with genetic sialidase deficiencies [25 26 27 ].

The functional capacity of cells of the immune system is influenced by desialylation of cell surface glycoconjugates [1 2 3 4 5 6 , 16 , 17 , 28 29 30 ]. Treatment of purified T lymphocytes with exogenous, bacterial neuraminidase (NANase) promoted concanavalin A-induced cell proliferation [1 ] and the production of interleukin (IL)-4 following cell activation [6 , 29 ]. Desialylation of glycoconjugates on the surface of dendritic cells by NANase also resulted in enhanced T cell proliferation [4 , 5 ] and cytotoxic activity [2 ] in mixed cell assays. We and others [16 ,17 ] have shown that treatment of peripheral blood mononuclear cells (PBMCs) with NANase promoted infection with HIV-1. Desialylation of cell surface glycoconjugates that results from the increased endogenous sialidase activity of activated T cells and monocytes was associated with enhanced cytokine production by lymphocytes [6 ] and enhanced interaction of monocytes with hyaluronic acid, a component of the extracellular matrix [30 ]. The mechanism(s) by which desialylation of cell surface glycoconjugates affects the behavior of PBMCs is not understood.

Circulating peripheral blood monocytes are activated by exposure to a variety of extracellular stimuli, enabling them to potentiate diverse immune activities. The surface of monocytes is comprised of sialylated glycoproteins [31 ] and glycolipids [32 ] that can be desialylated in vivo by interaction with neuraminidase-expressing microorganisms [15 ,33 ,34 ] or by endogenous cellular sialidases [30 ,35 36 37 ]. In this manuscript, we demonstrate that desialylation of glycoconjugates on the surface of human monocytes in vitro with NANase (i) stimulates phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), a critical step in cellular activation through the mitogen-activated protein kinase (MAPK) kinase (MEK)/ERK intracellular signaling pathway; (ii) results in the increased production of specific cytokines, namely interleukin (IL)-6, macrophage-inflammatory protein (MIP)-1{alpha}, and MIP-1ß; and (iii) enhances the cellular response to bacterial lipopolysaccharide (LPS).


arrow
MATERIALS AND METHODS
 
Isolation of PBMCs and purification of monocytes
PBMCs were isolated by leukophoresis of blood from individual HIV-1 and hepatitis B and C seronegative donors followed by centrifugation over Ficoll-Paque Plus (Amersham Pharmacia Biotech, 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 per the manufacturer’s suggested protocol. The purity of monocytes exceeded 98% as determined by flow cytometry after staining cells with fluorochrome-labeled antibodies (Ab) to CD3 and CD14, and viability was greater than 97% as determined by trypan blue dye exclusion.

Pretreatment of purified monocytes with NANase
Purified monocytes were suspended at 5 x 106 or 1 x 107 cells/ml in RPMI-1640 medium (Gibco, Grand Island, NY) containing 10% heat-inactivated (HI) fetal calf serum (Gemini Bioproducts, Calabasas, CA; referred to as complete medium in this manuscript) and were exposed to 100 mU/ml NANase (crystalline, type X, from Clostridium perfringens; Sigma Chemical Co., St. Louis, MO) at 37°C for 15 min–4 h, as noted in each experiment. Where indicated, monocytes were resuspended in phosphate-buffered saline (PBS), pH 7.4, containing 0.5% bovine serum albumin (BSA; Pentex bovine albumin fraction V, Miles Inc., Kankakee, IL) and were treated with NANase as noted above. After treatment with NANase under all conditions, the cells were washed three times with 10 ml PBS at 4°C. NANase was inhibited by being placed in boiling water for 10 min before addition to cells.

Culture conditions for purified monocytes
Mock- and NANase-treated monocytes were maintained at 4 x 105 or 2 x 106 cells/ml in complete medium at 37°C in a 5% humidified CO2 incubator. Monocytes (1x107 cells/condition) were resuspended in 5 ml (2x106 cells/ml) or 25 ml (4x105) complete medium and were cultured in conical 50 ml polypropylene tubes (Falcon, Becton Dickinson, Franklin Lakes, NJ) at 5 ml cell suspension per tube. In the experiment shown in Figure 3 , mock- and NANase-treated monocytes were prepared as described above and were grown in culture for 24 h in the presence of 0.1 ng/ml LPS (from Escherichia coli; Sigma Chemical Co.).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Desialylation of monocytes enhances production of cytokines in response to LPS. Monocytes were purified from the PBMCs of an individual human donor and were mock-treated or treated with NANase at 100 mU/ml in complete medium as described in Materials and Methods. Mock-treated and NANase-treated cells (1x107 total cells per condition at 2x106 cells/ml) were maintained in culture in 50 ml conical tubes in complete medium with and without 0.1 ng/ml LPS. The amount of each cytokine in a sample of culture medium was determined by ELISA 24 h after cells were mock- or NANase-treated. The data are expressed as nanograms/ml. (Note that data in Tables 1 and 2 are expressed in picograms/ml and total picograms, respectively.)

Analysis of cell surface sialic acid content
Mock- and NANase-treated monocytes (2x105) were suspended in 0.1 ml PBS, pH 7.4, containing 2% human serum (Gemini Bioproducts, Calabasas, CA) and were incubated with fluorescein isothiocyanate (FITC)-labeled lectin Arachis hypogaea (PNA; EY Laboratories, San Mateo, CA) at 10 µg/ml or were left unstained. PNA binds to the galactose moiety that is exposed on cell surface glycoconjugates after removal of terminal sialic acid. Monocytes were incubated with PNA for 45 min at 4°C before unbound lectin was removed by washing the cells at 4°C with 2 ml PBS, pH 7.4, containing 2% human serum. Cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry using a Becton Dickinson FACSCaliber (Mountain View, CA). Data were analyzed using FlowJo data analysis software.

Determination of ERK 1/2 phosphokinase activity
Mock- or NANase-treated monocytes were maintained in culture in complete medium in 50 ml polypropylene tubes. After collection at the indicated times by centrifugation, an equal number of cells from each condition was lysed for 15 min at 4°C in a solution containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. After a 5-min centrifugation at 21,000 g in an Eppendorf 5417R microfuge to remove cell debris, the supernatants were collected, and 25 µg protein from each cell lysate was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and probed with rabbit Ab to phosphorylated ERK 1/2 (New England Biolabs, Beverly, MA) or to total ERK 1/2 (Santa Cruz Biotechnology, Santa Cruz, CA) as described elsewhere [38 ]. The blots were incubated with a 1:1000 dilution of horseradish peroxidase-conjugated anti-rabbit Ab (Amersham, Arlington Heights, IL), developed using the enhanced chemiluminescence plus kit (Amersham), and were exposed to Kodak M35A-X-OMAT film. To reprobe the same membrane with different Ab, previously bound Ab were stripped in a solution containing 0.1 M glycine, pH 2.9.

Determination of cytokine levels
Enzyme-linked immunosorbent assay (ELISA) were used to determine cytokine levels in the medium of cultured monocytes using kits for IL-6, tumor necrosis factor {alpha} (TNF-{alpha}), and IL-10 (R&D Systems, Minneapolis, MN) and IL-1ß, interferon-{gamma} (IFN-{gamma}), MIP-1{alpha}, MIP-1ß, and regulated on activation, normal T expressed and secreted (RANTES) chemokine (Endogen, Woburn, MA).


arrow
RESULTS
 
Most of the potential sialylation sites of accessible glycoconjugates on the surface of freshly isolated monocytes are sialylated
To determine the relative degree of sialylation of glycoconjugates on the surface of freshly isolated human monocytes, purified monocytes were mock- and NANase-treated and were evaluated by flow cytometry for the relative amount of lectin PNA that specifically bound to the exposed penultimate galactose residue of desialylated cell surface glycoconjugates [28 ,35 ,39 ]. FITC-labeled PNA bound to the surface of freshly isolated, mock-treated monocytes, yielding a mean channel fluorescence (MCF) of 57 (Fig. 1 , shaded area), which was approximately tenfold greater than the fluorescence of unstained, mock-treated cells (Fig. 1 , thin line). After monocytes were treated with 100 mU/ml NANase, there was a greater than 20-fold increase in the amount of PNA that bound to the cell surface (MCF, 1286; Fig. 1 , bold line). Thus, at least 95% of potential sialylation sites on surface glycoconjugates that were accessible to exogenous NANase were sialylated in freshly isolated monocytes.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Treatment of purified monocytes with NANase removes cell surface sialic acid. Monocytes were purified from the peripheral blood of a human donor and were mock-treated and treated with NANase at 100 mU/ml at 37°C for 1 h. Mock-treated (shaded area) and NANase-treated (solid bold line) cells were stained with FITC-labeled PNA, which binds to the penultimate galactose residue of glycoconjugates after removal of terminal sialic acid. Alternatively, mock-treated cells were left unstained (thin solid line). Cells were fixed in 1% paraformaldehyde and were analyzed for the amount of PNA bound to the cell surface by flow cytometry as described in Materials and Methods. Relative amounts of desialylation were determined by comparing the MCF of FITC-labeled cells under each condition. These data are from the monocytes of an individual donor and are representative of data using cells from three donors.

Desialylation of monocytes activates ERK 1/2
The MAPKs ERK 1/2 are intermediates in the MEK/ERK intracellular signaling pathway, which are phosphorylated in cells that have been activated by a variety of extracellular stimuli (reviewed in refs. [40 ,41 ]). To determine whether desialylation of cell surface sialoconjugates with exogenous NANase activated monocytes, as evidenced by signaling through the ERK 1/2 pathway, freshly isolated monocytes were treated with NANase and analyzed for an increase in phosphorylation of ERK 1/2. Monocytes that were mock-treated supported a low level of ERK 1/2 phosphorylation (Fig. 2A and 2B ). This level of baseline activity increased minimally in mock-treated monocytes that were maintained in culture over a 4-h period (Fig. 2A) . In contrast, NANase-treated monocytes expressed increased amounts of phosphorylated ERK 1/2 when analyzed 2 h after desialylation (Fig. 2A and 2B) . This difference in amount of phosphorylated ERK 1/2 in NANase-treated cells did not result from increased synthesis of ERK 1/2, as there was no difference in the amount of total ERK 1/2 in mock- and NANase-treated cells (Fig. 2B) . Enhanced phosphorylation of ERK 1/2 in NANase-treated cells continued to be detected when analyzed after 4 h of treatment but at a level that was lower than what occurred after 2 h. Increased phosphorylation of ERK 1/2 was not observed in monocytes that were treated with heat-inactivated (HI) NANase (Fig. 2B) , dismissing the possibility that the observed cellular response was caused by endotoxin or by antigenic stimulation of monocytes by exposure to exogenous NANase.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 2. Desialylation of glycoconjugates on the surface of monocytes stimulates phosphorylation of MAPKs ERK 1/2. Monocytes were purified from the peripheral blood of an individual human donor and were mock-treated or treated with 100 mU/ml NANase (with and without heat inactivation) at 37°C for 15 min–4 h. Cells were collected at the indicated time, and 25 µg protein from each cell lysate was separated by SDS-PAGE and analyzed for the amount of total ERK 1/2 and the portion of total ERK 1/2 that was phosphorylated (p-ERK 1/2), as indicated in Materials and Methods. The relative intensity of total and phosphorylated ERK 1/2 on Western blots was quantified by densitometry using Molecular Analyst software (Bio-Rad, Hercules, CA). (A) Histogram showing the relative amount of phosphorylated ERK 1/2 in mock- and NANase-treated cells at the indicated times in comparison with the amount of phosphorylated ERK in mock-treated cells at time 0 (relative value set to one; data for time 0 not shown in figure). (B) Autoradiograph of phosphorylated and total ERK in samples as described: Lane 1, Negative control of mock-treated monocytes before culture; lane 2, mock-treated monocytes after 2 h in culture; lane 3, cells treated with HI NANase (inactivated in boiling water for 10 min) after 2 h in culture; lane 4, NANase-treated monocytes after 2 h in culture. The numbers on the right side of the radiograph represent protein molecular weight markers for 48.5 and 35.3 kDa. The results are representative of data from three independent experiments using cells from three different donors.

Desialylation of monocytes leads to increased secretion of cytokines
NANase-treated monocytes that were maintained in culture in complete medium were also evaluated for the production of cytokines. NANase-treated monocytes produced greater amounts of IL-6 within 24 h after NANase treatment than did mock-treated cells from five donors that were evaluated (Table 1 ). Increased amounts of the chemokines MIP-1{alpha} and MIP-1ß were also detected in the medium of NANase-treated monocytes in four of five donors (Table 1) . In contrast, the level of RANTES in medium from cells from each donor was not affected by NANase treatment. Analysis for the cytokines TNF-{alpha}, IL-1ß, IFN-{gamma}, and IL-10 revealed undetectable levels of each in the medium of mock- and NANase-treated cells from all five donors (data not shown). The specificity of NANase treatment was again supported by the finding that the increased levels of IL-6, MIP-1{alpha}, and MIP-1ß were not detected in the medium of cells that were treated with HI NANase (Table 1 , Donors A, C, and E). Similar results were obtained when cells for treatment with NANase were resuspended in PBS, pH 7.4, containing 0.5% BSA, rather than in complete medium (Donors B and E). This confirmed that the effect of NANase was a result of desialylation of cell surface glycoconjugates and not a result of desialylation of exogenous components in the medium. Thus, desialylation of freshly isolated monocytes by exogenous NANase leads to increased production of cytokines in monocytes.


View this table:
[in this window]
[in a new window]
  		Table 1. Desialylation of Monocytes Using Exogenous Neuraminidase Stimulates Cytokine Production

Enhanced production of cytokines by NANase-treated cells occurs independently of cell concentration
To determine whether the enhanced cytokine production of NANase-treated cells resulted from increased homotypic adhesion of desialylated cells, freshly isolated monocytes were treated with 100 mU/ml NANase and maintained in culture at more dilute cell concentrations than were used in the experiments in Table 1 to reduce the amount of cell-to-cell contact. Monocytes from two donors (1x107 cells from each), which were treated with NANase at 1 x 107 cells/ml in complete medium and subsequently maintained in culture at 2 x 106 cells/ml, released 8099 and 9796 picograms of IL-6 into the culture medium (Table 2 ). In contrast to the presentation of data in Table 1 , where cytokine levels are expressed in pg/ml, the data in Table 2 show the total amount of IL-6 that was released into the culture medium. Similar total amounts of IL-6 were released into the medium when the same number of cells from each donor (1x107 cells) was treated with NANase at 5 x 106 cells/ml (twofold dilution) and subsequently maintained in culture at 4 x 105 cells/ml (fivefold dilution; Table 2 ). Thus, the amount of IL-6 that was produced in NANase-treated cells did not appear to depend on the cell concentration during NANase treatment or subsequent cell culture, suggesting that the NANase effect may be a result of its effect on individual cells as opposed to resulting from cell-to-cell adhesion.


View this table:
[in this window]
[in a new window]
  		Table 2. The Amount of IL-6 Produced by NANase-Treated Monocytes is Independent of the Concentration of Cells in Culture

Desialylation of monocytes enhances production of cytokines in response to LPS
Monocytes respond to microbial products such as gram-negative bacterial LPS by initiating production of a wide variety of inflammatory mediators. To determine whether the effect of LPS on monocytes was affected by the state of sialylation of cell surface glycoconjugates, the production of IL-6, MIP-1{alpha}, and MIP-1ß in monocytes was determined after monocytes were first treated with NANase and then exposed to LPS. A low concentration of LPS (0.1 ng/ml) was used to elicit a suboptimal cytokine response. In the absence of exposure to LPS or treatment with NANase, no or minimal cytokine production was observed (Fig. 3 and Table 1 ). In contrast, desialylation of cell surface glycoconjugates by treatment with NANase or exposure of cells to 0.1 ng/ml LPS resulted in production of detectable amounts of IL-6 (NANase, 1.9 ng/ml; LPS, 6.2 ng/ml), MIP-1{alpha} (NANase, 0.2 ng/ml; LPS, 0.2 ng/ml), and MIP-1ß (NANase, 1.4 ng/ml; LPS, 0.7 ng/ml). However, when monocytes were pretreated with NANase and subsequently exposed to LPS, a significant increase in inflammatory mediator production occurred with production of 26.9 ng/ml IL-6, 5.0 ng/ml MIP-1{alpha}, and 4.6 ng/ml MIP-1ß. Thus, desialylation of cell surface glycoconjugates promoted a state in monocytes that increased their responsiveness to an exogenous mediator such as LPS.


arrow
DISCUSSION
 
We have shown in this report that desialylation of glycoconjugates on the surface of monocytes leads to increased phosphorylation of ERK 1/2 and to enhanced production of specific cytokines. The effects of desialylating cell surface glycoconjugates were demonstrated by maintaining mock- and NANase-treated monocytes in growth conditions that minimized the baseline level of cellular activation. The results of treating monocytes with NANase were specific for desialylation of cell surface glycoconjugates and not simply a result of NANase-induced antigenic stimulation, as enhanced cellular activation did not occur when NANase was heat inactivated before exposure to cells. In addition, similar results were obtained when monocytes were resuspended in PBS, rather than in serum-containing medium, before treatment with NANase, confirming that the NANase effect on monocytes was not a result of desialylation of a component(s) in the medium.

The MEK/ERK MAPK pathway is one of several intracellular signaling pathways (e.g., p38, c-Jun) that is stimulated by cell surface contact with a wide range of stimuli (reviewed in refs. [40 41 42 ]). In this study, we determined the effect of NANase treatment of monocytes on the MEK-ERK intracellular signaling pathway, but it is possible that other signaling pathways were similarly activated. We observed a complex profile of cytokine expression in desialylated monocytes (increased IL-6, MIP-1{alpha}, MIP-1ß; unaffected TNF-{alpha}, IL-1, IFN-{gamma}, IL-10, RANTES). The enhanced production of IL-6 by NANase-treated monocytes from every donor in our experiments may be the result of activated nuclear factor-{kappa}B [43 ] and may reflect the vigorous expression of IL-6 in comparison with other cytokines that occurs in inflammatory states [44 ,45 ]. The qualitative and quantitative differences in cytokine production from all donors in our experiments may partly be explained by the well-recognized genetic polymorphisms that control the production of specific cytokines in different individuals in response to inflammatory stimuli [46 47 48 ] and/or by differences in the baseline state of activation of the purified monocytes from independent donors in separate experiments. Our finding of enhanced cytokine production in desialylated monocytes is consistent with reports from other laboratories that demonstrate increased production of specific cytokines in lymphocytes that were desialylated with exogenous microbial NANases [6 ,29 ].

There are several possible mechanisms to explain how desialylation of glycoconjugates on the surface of monocytes is involved in intracellular signaling. Modulation of the sialylation status of cell surface receptors and regulatory molecules may modify the ability of these surface-recognition molecules to bind extracellular ligands such as gram-negative bacterial LPS, cytokines, growth factors, and hormones. Alternatively, desialylation of regulatory molecules on the surface of monocytes may lead to receptor activation independently of binding to exogenous ligands by inducing conformational changes in the involved molecules and/or receptor dimerization. In this study, we found that desialylation of glycoconjugates on the surface of monocytes, including possibly the Toll-like receptor 4 complex that recognizes LPS and through which LPS transmits its intracellular signal [49 ], enhanced the cellular response to LPS as determined by cytokine production. This finding suggests that desialylation of monocytes affects their functional activity by potentiating the effect of soluble mediators, such as LPS. It is also possible that desialylation of cell surface glycoconjugates increases cell-to-cell interactions as we demonstrated previously in polymorphonuclear leukocytes [28 ,50 ] and as others showed in PBMCs [1 ,4 ].

Several pathways can be envisioned by which desialylation of cell surface glycoconjugates occurs on monocytes in vivo during their normal immune function. Monocytes express increased endogenous sialidase activity and have hyposialylated cell surface glycoconjugates when they are activated [30 ]. Monocytes also express increased endogenous sialidase as they differentiate into macrophages or immature dendritic cells (manuscript in preparation). Thus, glycoconjugates on the surface of monocytes can be desialylated by endogenous sialidase(s) after immune activation or by the sialidase(s) on neighboring monocytes, lymphocytes, neutrophils, or endothelial cells, all of which express sialidase activity [3 ,6 ,30 ,35 36 37 ] that can be induced by inflammatory stimuli. Alternatively, interaction with NANase-expressing microorganims such as influenza virus or trypanosomes [15 ,34 ] can potentially lead to desialylation of cell surface glycoconjugates on monocytes.

Activation of monocytes is clearly a multistep process that may include desialylation of cell surface glycoconjugates. It should be noted that sialidases likely work in a microenvironment with sialyltransferases, enzymes that add sialyl residues to glycoconjugates and restore the basal state of cell surface sialylation [51 ]. Thus, control of cell function may depend on a delicate balance of activity of these enzymes.


arrow
ACKNOWLEDGEMENTS
 
This work was supported in part by National Institutes of Health Grants KO8 HL72176-01 to N. M. S. and AI 42818-01 to A. S. C. N. M. S. is grateful to Peter John Gomatos for discussion throughout this work and critique of the manuscript. We thank James Mitchell for assistance during the early stages of this work.

Received May 25, 2003; revised September 24, 2003; accepted October 23, 2003.


arrow
REFERENCES
 
    1
  1. Hunig, T. (1983) The role of accessory cells in polyclonal T cell activation II.Induction of interleukin 2 responsiveness requires cell-cell contact Eur. J. Immunol. 13,596-601[Medline]
    2. 2
  2. Boog, C. J. P., Neefjes, J. J., Boes, J., Ploegh, H. L., Melief, C. J. M. (1989) Specific immune responses restored by alteration in carbohydrate chains of surface molecules on antigen-presenting cells Eur. J. Immunol. 19,537-542[Medline]
    3. 3
  3. Taira, S., Nariuchi, H. (1988) Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia J. Immunol. 141,440-446[Abstract]
    4. 4
  4. Fanales-Belasio, E., Zambruno, G., Cavani, A., Girolomoni, G. (1997) Antibodies against sialophorin (CD43) enhance the capacity of dendritic cells to cluster and activate T lymphocytes J. Immunol. 159,2203-2211[Abstract/Free Full Text]
    5. 5
  5. Oh, S., Belz, G. T., Eichelberger, M. C. (2001) Viral neuraminidase treatment of dendritic cells enhances antigen-specific CD8(+) T cell proliferation, but does not account for the CD4(+) T cell independence of the CD8(+) T cell response during influenza virus infection Virology 286,403-411[CrossRef][Medline]
    6. 6
  6. Chen, X-P., Enioutina, E. Y., Daynes, R. A. (1997) The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase J. Immunol. 158,3070-3080[Abstract]
    7. 7
  7. Hayes, G. R., Lockwood, D. H. (1986) The role of cell surface sialic acid in insulin receptor function and insulin action J. Biol. Chem. 261,2791-2798[Abstract/Free Full Text]
    8. 8
  8. Schooley, J. C. (1985) Neuraminidase increases DNA synthesis of spleen cells induced by native and asialylated erythropoietin Exp. Hematol. 13,994-998[Medline]
    9. 9
  9. Liu, C. K., Wei, G., Atwood, W. J. (1998) Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal {alpha}(2-6)-linked sialic acids J. Virol. 72,4643-4649[Abstract/Free Full Text]
    10. 10
  10. Maganti, S., Pierce, M. M., Hoffmaster, A., Rodgers, F. G. (1998) The role of sialic acid in opsonin-dependent and opsonin-independent adhesion of Listeria monocytogenes to murine peritoneal macrophages Infect. Immun. 66,620-626[Abstract/Free Full Text]
    11. 11
  11. Matsui, S., Okuno, T., Shiraki, K. (1994) Functional roles of terminal glycomoieties in varicella-zoster virus infection Virology 198,50-58[CrossRef][Medline]
    12. 12
  12. Ming, M., Chuenkova, M., Ortega-Barria, E., Pereira, M. E. (1993) Mediation of Trypanosoma cruzi invasion by sialic acid on the host cell and trans-sialidase on the trypanosome Mol. Biochem. Parasitol. 59,243-252[CrossRef][Medline]
    13. 13
  13. Ohuchi, M., Feldmann, A., Ohuchi, R., Klenk, H. D. (1995) Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity Virology 212,77-83[CrossRef][Medline]
    14. 14
  14. Palese, P., Tobita, K., Ueda, M., Compans, R. W. (1974) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase Virology 61,397-410[CrossRef][Medline]
    15. 15
  15. Schenkman, S., Jiang, M. S., Hart, G. W., Nussenzweig, V. (1991) A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells Cell 65,1117-1125[CrossRef][Medline]
    16. 16
  16. Stamatos, N. M., Gomatos, P. J., Cox, J., Fowler, A., Dow, N., Wohlhieter, J. A., Cross, A. S. (1997) Desialylation of peripheral blood mononuclear cells promotes growth of HIV-1 Virology 228,123-131[CrossRef][Medline]
    17. 17
  17. Sun, J., Barbeau, B., Sato, S., Tremblay, M. J. (2001) Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process Virology 284,26-36[CrossRef][Medline]
    18. 18
  18. Hasegawa, T., Yamaguchi, K., Wada, T., Takeda, A., Itoyama, Y., Miyagi, T. (2000) Molecular cloning of mouse ganglioside sialidase and its increased expression in Neuro2a cell differentiation J. Biol. Chem. 275,8007-8015[Abstract/Free Full Text]
    19. 19
  19. Kopitz, J., Muhl, C., Ehemann, V., Lehmann, C., Cantz, M. (1997) Effects of cell surface ganglioside sialidase inhibition on growth control and differentiation of human neuroblastoma cells Eur. J. Cell Biol. 73,1-9[Medline]
    20. 20
  20. Wu, G., Ledeen, R. W. (1991) Stimulation of neurite outgrowth in neuroblastoma cells by neuraminidase: putative role of GM1 ganglioside in differentiation J. Neurochem. 56,95-104[CrossRef][Medline]
    21. 21
  21. Altevogt, P., Fogel, M., Cheingsong-Popov, R., Dennis, J., Robinson, P., Schirrmacher, V. (1983) Different patterns of lectin binding and cell surface sialylation detected on related high- and low-metastatic tumor lines Cancer Res. 43,5138-5144[Abstract/Free Full Text]
    22. 22
  22. Kakugawa, Y., Wada, T., Yamaguchi, K., Yamanami, H., Ouchi, K., Sato, I., Miyagi, T. (2002) Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression Proc. Natl. Acad. Sci. USA 99,10718-10723[Abstract/Free Full Text]
    23. 23
  23. Schengrund, C. L., Lausch, R. N., Rosenberg, A. (1973) Sialidase activity in transformed cells J. Biol. Chem. 248,4424-4428[Abstract/Free Full Text]
    24. 24
  24. Roggentin, P., Schauer, R., Hoyer, L. L., Vimr, E. R. (1993) The sialidase superfamily and its spread by horizontal gene transfer Mol. Microbiol. 9,915-921[Medline]
    25. 25
  25. d’Azzo, A., Andria, G., Strisciuglio, P., Galijaard, H. (2001) Galactosialidosis Scriver, C. R. Beaudet, A. L. Sly, W. S. Valle, D. eds. Metabolic and Molecular Bases of Inherited Disease ,3811-3826 McGraw-Hill New York, NY.
    26. 26
  26. Thomas, G. H., Beaudet, A. L. (2001) Disorders of glycoprotein degradation and structure: {alpha}-mannosidosis, ß-mannosidosis, fucosidosis, sialidosis, aspartylglucosaminuria and carbohydrate-deficient glycoprotein syndrome Scriver, C. R. Beaudet, A. L. Sly, W. S. Valle, D. eds. Metabolic and Molecular Bases of Inherited Disease ,3507-3534 McGraw-Hill New York, NY.
    27. 27
  27. Pshezhetsky, A. V., Ashmarina, M. (2001) Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology Prog. Nucleic Acid Res. Mol. Biol. 69,81-114[Medline]
    28. 28
  28. Cross, A. S., Sakarya, S., Rifat, S., Held, T. K., Drysdale, B-E., Grange, P. A., Cassels, F. J., Wang, L-X., Stamatos, N., Farese, A., Casey, D., Powell, J., Bhattacharjee, A. K., Kleinberg, M., Goldblum, S. E. (2003) Recruitment of murine neutrophils in vivo through endogenous sialidase activity J. Biol. Chem. 278,4112-4120[Abstract/Free Full Text]
    29. 29
  29. Oh, S., Eichelberger, M. C. (2000) Polarization of allogeneic T-cell responses by influenza virus-infected dendritic cells J. Virol. 74,7738-7744[Abstract/Free Full Text]
    30. 30
  30. Katoh, S., Miyagi, T., Taniguchi, H., Matsubara, Y, Kadota, J, Tominaga, A., Kincade, P. W., Matsukura, S., Kohno, S. (1999) Cutting edge: an inducible sialidase regulates the hyaluronic acid binding ability of CD44-bearing human monocytes J. Immunol. 162,5058-5061[Abstract/Free Full Text]
    31. 31
  31. Nong, Y. H., Remold-O’Donnell, E., LeBien, T. W., Remold, H. G. (1989) A monoclonal antibody to sialophorin (CD43) induces homotypic adhesion and activation of human monocytes J. Exp. Med. 170,259-267[Abstract/Free Full Text]
    32. 32
  32. Yohe, H. C., Wallace, P. K., Berenson, C. S., Ye, S., Reinhold, B. B., Reinhold, V. N. (2001) The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures Glycobiology 11,831-841[Abstract/Free Full Text]
    33. 33
  33. Gubareva, L. V., Kaiser, L., Hayden, F. G. (2000) Influenza virus neuraminidase inhibitors Lancet 355,827-835[CrossRef][Medline]
    34. 34
  34. McCullers, J. A., Bartmess, K. C. (2003) Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae J. Infect. Dis. 187,1000-1009[CrossRef][Medline]
    35. 35
  35. Galvan, M., Murali-Krishna, K., Ming, L. L., Baum, L., Ahmed, R. (1998) Alterations in cell surface carbohydrates on T cells from virally infected mice can distinguish effector/memory CD8+ T cells from naïve cells J. Immunol. 161,641-648[Abstract/Free Full Text]
    36. 36
  36. Landolfi, N. F., Leone, J., Womack, J. E., Cook, R. G. (1985) Activation of T lymphocytes results in an increase in H-2-encoded neuraminidase Immunogenetics 22,159-167[CrossRef][Medline]
    37. 37
  37. Lukong, K. E., Seyrantepe, V., Landry, K., Trudel, S., Ahmad, A., Gahl, W. A., Lefrancois, S., Morales, C. R., Pshezhetsky, A. V. (2001) Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail J. Biol. Chem. 276,46172-46181[Abstract/Free Full Text]
    38. 38
  38. Romerio, F., Riva, A., Zella, D. (2000) Interferon-{alpha}2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism Br. J. Cancer 83,532-538[CrossRef][Medline]
    39. 39
  39. Novogrodsky, A., Lotan, R., Ravid, A., Sharon, N. (1975) Peanut agglutinin, a new mitogen that binds to galactosyl sites exposed after neuraminidase treatment J. Immunol. 115,1243-1248[Abstract/Free Full Text]
    40. 40
  40. Chang, L., Karin, M. (2001) Mammalian MAP kinase signalling cascades Nature 410,37-40[CrossRef][Medline]
    41. 41
  41. Johnson, G. L., Lapadat, R. (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases Science 298,1911-1912[Abstract/Free Full Text]
    42. 42
  42. Seger, R., Krebs, E. G. (1995) The MAPK signaling cascade FASEB J 9,726-735[Abstract]
    43. 43
  43. Tuyt, L. M., Dokter, W. H., Birkenkamp, K., Koopmans, S. B., Lummen, C., Kruijer, W., Vellenga, E. (1999) Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-{kappa}B-dependent IL-6 expression in human monocytes J. Immunol. 162,4893-4902[Abstract/Free Full Text]
    44. 44
  44. Abraham, E., Anzueto, A., Gutierrez, G., Tessler, S., San Pedro, G., Wunderink, R., Dal Nogare, A., Nasraway, S., Berman, S., Cooney, R., Levy, H., Baughman, R., Rumbak, M., Light, R. B., Poole, L., Allred, R., Constant, J., Pennington, J., Porter, S. (1998) Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock.NORASEPT II Study Group Lancet 351,929-933[Medline]
    45. 45
  45. Gallagher, J., Fisher, C., Sherman, B., Munger, M., Meyers, B., Ellison, T., Fischkoff, S., Barchuk, W. T., Teoh, L., Velagapudi, R. (2001) A multicenter, open-label, prospective, randomized, dose-ranging pharmacokinetic study of the anti-TNF-{alpha} antibody afelimomab in patients with sepsis syndrome Intensive Care Med. 27,1169-1178[CrossRef][Medline]
    46. 46
  46. Westendorp, R. G., Langermans, J. A., Huizinga, T. W., Elouali, A. H., Verweij, C. L., Boomsma, D. I., Vandenbroucke, J. P. (1997) Genetic influence on cytokine production and fatal meningococcal disease Lancet 349,170-173[CrossRef][Medline]
    47. 47
  47. Read, R. C., Cannings, C., Naylor, S. C., Timms, J. M., Maheswaran, R., Borrow, R., Kaczmarski, E. B., Duff, G. W. (2003) Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease Ann. Intern. Med. 138,534-541[Abstract/Free Full Text]
    48. 48
  48. Kotb, M., Norrby-Teglund, A., McGeer, A., El-Sherbini, H., Dorak, M. T., Khurshid, A., Green, K., Peeples, J., Wade, J., Thomson, G., Schwartz, B., Low, D. E. (2002) An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections Nat. Med. 8,1398-1404[CrossRef][Medline]
    49. 49
  49. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene Science 282,2085-2088[Abstract/Free Full Text]
    50. 50
  50. Cross, A. S., Wright, D. G. (1991) Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells J. Clin. Invest. 88,2067-2076
    51. 51
  51. Broquet, P., Baubichon-Cortay, H., George, P., Louisot, P. (1991) Glycoprotein sialyltransferases in eukaryotic cells Int. J. Biochem. 23,385-389[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Curreli, Z. Arany, R. Gerardy-Schahn, D. Mann, and N. M. Stamatos
Polysialylated Neuropilin-2 Is Expressed on the Surface of Human Dendritic Cells and Modulates Dendritic Cell-T Lymphocyte Interactions
J. Biol. Chem., October 19, 2007; 282(42): 30346 - 30356.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Duca, C. Blanchevoye, B. Cantarelli, C. Ghoneim, S. Dedieu, F. Delacoux, W. Hornebeck, A. Hinek, L. Martiny, and L. Debelle
The Elastin Receptor Complex Transduces Signals through the Catalytic Activity of Its Neu-1 Subunit
J. Biol. Chem., April 27, 2007; 282(17): 12484 - 12491.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
X. Nan, I. Carubelli, and N. M. Stamatos
Sialidase expression in activated human T lymphocytes influences production of IFN-{gamma}
J. Leukoc. Biol., January 1, 2007; 81(1): 284 - 296.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0503241v1
75/2/307    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stamatos, N. M.
Right arrow Articles by Cross, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stamatos, N. M.
Right arrow Articles by Cross, A. S.