Engagement of Na,K-ATPase ß3 subunit by a specific mAb suppresses T and B lymphocyte activation

Sawitree Chiampanichayakul1,2,3, Andreas Szekeres3, Panida Khunkaewla1, Seangduen Moonsom1, Vladimir Leksa3, Karel Drbal3, Gerhard J. Zlabinger5, Renate Hofer-Warbinek4, Hannes Stockinger3 and Watchara Kasinrerk1

1 Department of Clinical Immunology, Faculty of Associated Medical Sciences and 2 Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 3 Institute of Immunology–Vienna International Research Cooperation Center at NFI, and 4 Institute of Vascular Biology and Thrombosis Research, University of Vienna, Brunner Strasse 59, 1235 Vienna, Austria 5 Institute of Immunology, University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria

Correspondence to: W. Kasinrerk; E-mail: watchara{at}chiangmai.ac.th
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In order to identify new molecules involved in regulation of T cell proliferation, we generated various mAb by immunization of mice with the T cell line Molt4. We found one mAb (termed P-3E10) that down-regulated the in vitro T cell proliferation induced by CD3-specific OKT3 mAb. The P-3E10 mAb was also able to inhibit IFN-{gamma}, IL-2, IL-4 and IL-10 production of OKT3-activated T cells. The antigen recognized by P-3E10 mAb is broadly expressed on all hematopoietic as well as on all non-hematopoietic cell lines tested so far. Within peripheral blood leukocytes, the P-3E10 antigen was detected on lymphocytes, monocytes and granulocytes. Human umbilical vein endothelial cells (HUVEC) also scored positively. By evaluating the effect of P-3E10 mAb on these cell types we found that it also inhibited anti-IgM-induced B cell proliferation. However, it did not block growth factor-mediated proliferation of HUVEC, and spontaneous proliferation of SupT-1, Jurkat, Molt4 and U937 cell lines. Moreover, it did not influence phagocytosis of human blood monocytes and granulocytes. Biochemical analysis revealed that the P-3E10 antigen is a protein with a mol. wt of 45–50 kDa under non-reducing and 50–55 kDa under reducing conditions. By using a retroviral cloning system, the P-3E10 antigen was cloned. Sequence analysis revealed the P-3E10 antigen to be identical to the ß3 subunit of the Na,K-ATPase.

Keywords: lymphocyte inhibition, mAb, Na,K-ATPase ß3 subunit, retroviral cloning system


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Highly orchestrated cooperation of stimulatory and suppressive immune pathways is required in order to defeat pathogens without causing harm to self tissues. Under certain circumstances, such as autoimmunity or hypersensitivity, the immune system fails to reach this harmony and becomes rather a threat than a favor. In such cases, therapy is directed to suppress autoreactive responses and thus to restore the natural balance of the immune system. Characterization of the molecular mechanisms underlying negative immune regulation is supposed to provide targets for clinical interventions (14). The interaction between T cells and antigen-presenting cells (APC) seems to be a key event leading to activation of the cellular branch of immunity. Examples of molecules and their ligands involved in this interaction include CD4–MHC class II (5), CD8–MHC class I (6) and CD28/CTLA-4–CD80/CD86 (79). Therefore, the most suppressive therapeutic agents being developed target those molecules on the surface of both T cells and APC which are essential for regulation of T cell activation (10,11). For instance, immunosuppressive agents specific to the TCR complex molecules CD3, CD4 or CD8 as well as to the co-stimulatory molecules and their ligands CD28–CD80, CD152–CD86 or CD40–CD154 have been designed and used with success (1012). In addition, some molecules not directly involved in T cell activation also appear to be a reasonable target for immunosuppressive treatment, e.g. P-glycoprotein (13).

In an attempt to identify new molecules involved in regulation of T cell proliferation, we generated hybridomas from BALB/c mice immunized with the T cell line Molt4 and screened them for the ability to inhibit T cell activation. From among others, we selected one mAb (termed P-3E10) that down-regulated the T cell proliferation induced by immobilized CD3-specific mAb OKT3 as well as production of IFN-{gamma}, IL-2, IL-4 and IL-10 in vitro. To identify the molecule recognized by this mAb, we cloned the encoding cDNA using a retroviral expression cloning system (1416) and found that it is identical to the Na,K-ATPase ß3 subunit.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells, reagents and antibodies
All human hematopoietic and non-hematopoietic cell lines used in this study were maintained in RPMI 1640 medium supplemented with 10% FCS (Gibco, Grand Island, NY), 40 µg/ml gentamicin and 2.5 µg/ml amphotericin B in a humidified atmosphere of 5% CO2 at 37°C. Phoenix packaging cells, an ecotropic retroviral packaging cell line developed by Nolan et al. (14), were maintained in DMEM supplemented with 10% FCS.

Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden).

The CD99 mAb MT99/3 (IgG2a isotype), CD147 mAb M6-1E9 (IgG2a isotype) and the mAb KLH (IgG2a isotype) specific for the keyhole limpet hemocyanin molecule were generated by us [(17,18) and unpublished data]. The mAb MEM-188 (CD56; IgG2a isotype), MEM-M6/2 (CD147; IgG2a isotype) as well as the mAb against human {alpha}-fetoprotein AFP-01 (IgG1 isotype) were kindly provided by Dr V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic). Mouse IgG2a, UPC 10, was purchased from Sigma-Aldrich (St Louis, MO). The mAb 4G2 (IgG2a isotype) specific for E protein of dengue virus was kindly provided by Dr P. Malasit (Medical Biotechnology Unit, Mahidol University, Bangkok, Thailand). The IgG isotype mAb were purified by using a Protein A-coated Sepharose column (Zymed, San Francisco, CA) according to the methods described elsewhere (19).

Hybridoma production
mAb P-3E10 was generated by immunization of a female BALB/c mouse 3 times i.p. at 1-week intervals using 1 x 107 Molt4 cells. Then, the mouse was boosted i.v. using 1 x 106 cells. Splenocytes were collected and fused with P3-X63Ag8.653 myeloma cells by standard hybridoma fusion techniques using 50% polyethylene glycol and HAT medium selection. The IgG2a isotype of the mAb was determined using an isotyping ELISA kit (Sigma-Aldrich).

Proliferation assay for lymphocytes
Each culture was set up in a flat-bottom 96-well plate (Nunc, Roskilde, Denmark) in a final volume of 200 µl/well. Triplicate aliquots of 1 x 105 or 5 x 105 PBMC were activated using immobilized CD3 mAb OKT3 (20 ng/ml or 1 µg/ml; Ortho Pharmaceuticals, Raritan, NJ) or soluble goat anti-human IgM antibody (10 µg/ml; Hyland Diagnostics, Deerfield, MA) respectively in the presence or absence of various concentrations of tested mAb. The cultures were incubated for 3 days in a 5% CO2 incubator at 37°C and then 1 µCi/well of [3H]thymidine (Amersham Pharmacia Biotech, Freiburg, Germany) was added. The culture was incubated for an additional 18 h before harvesting. Incorporated radioactivity was counted in a liquid scintillation counter (MicroBeta; Wallac, Turku, Finland).

Proliferation assay for cell lines
For the cell line proliferation assay, triplicate aliquots of 1.5 x 104 cells were cultured with 0.5 µCi/well [3H]thymidine (Amersham Pharmacia) with or without 2.5 µg/ml P-3E10 mAb. The cultures were incubated for 3 and 5 h in a 5% CO2 incubator at 37°C. Then the culture was harvested and the incorporated radioactivity was counted in a liquid scintillation counter (Wallac).

Proliferation assay of human umbilical vein endothelial cells (HUVEC)
HUVEC were isolated by collagenase digestion. Briefly, human umbilical veins were flushed with Ringer’s lactate and then incubated with 0.5 mg/ml collagenase type II (Sigma-Aldrich) at 37°C for 30 min. Detached HUVEC were collected, washed, and then cultured in fibronectin-coated flasks (Nunc, Naperville, IL) using M199 medium that contained 20% supplemented calf serum (SCS; Hyclone, Logan, UT), 25 µg/ml EC growth supplement (Technoclone, Vienna, Austria), 5 U/ml heparin, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml fungizone. Confluent HUVEC were gently trypsinized, seeded onto 24-well plates (1.5 x 104 cells/well) and cultured in the presence of P-3E10 mAb or isotype-matched control mAb at a final concentration of 20 or 1 µg/ml in M199 medium supplemented with 20% SCS (Hyclone). As negative control, the cells were cultured in M199 supplemented with 1% SCS. After 3 days of cultivation, cells were fixed by methanol and stained by crystal violet. After intense washings, cells were solubilized in 0.5% Triton X-100 and the number of cells was determined by measuring the absorbance at 595 nm using an ELISA reader and a standard curve.

Determination of cytokine production
PBMC (1 x 105) in the presence or absence of various concentrations of tested mAb (in a total volume of 200 µl) were culture in a flat-bottom 96-well plate (Nunc) precoated with mAb OKT3 (1 µg/ml). After incubation at 37°C in a CO2 incubator for 24 or 72 h, the culture supernatants were harvested.

Cytokines were measured by sandwich ELISA using matched pairs of antibodies. Capture as well as detection antibodies to human IL-10 were obtained form R & D Systems (Minneapolis, MN). For the determination of IFN-{gamma} a mAb (clone 25718.111) from R & D Systems was used as capture antibody and a mAb (clone GZ4) from Roche Diagnostics (Mannheim, Germany) as detection antibody. For IL-2 and IL-4, ELISA kits from Euroclone (Wetherby, UK) were used. Standards consisted of human recombinant material were obtained from R & D Systems. Assays were set up in duplicates and performed according to the recommendations of the manufacturers.

Phagocytosis assay
Escherichia coli was grown in LB broth (Gibco) overnight at 37°C. Cells were washed twice and resuspended in PBS. The optical density of the bacterial suspension was measured at 600 nm and adjusted to 2.5. For phagocytosis assay, 100 µl of EDTA–blood was incubated with 25 µl of the bacterial suspension in the presence or absence of 10 µg/ml of P-3E10 mAb or isotype-matched control mAb at 37°C for 30 min. The sample was then smeared on a grass slide and stained with Wright’s stain. Phagocytic cells were counted by light microscopy.

Immunofluorescence analysis
mAb binding to cells was analyzed by indirect immunofluorescence using FITC-conjugated sheep F(ab')2 anti-mouse Ig antibodies (Immunotech/Coulter, Miami, FL). To block non-specific FcR-mediated binding of mAb, cells were pre-incubated for 30 min at 4°C with 10% human AB serum before staining. Membrane fluorescence was analyzed on a FACSCalibur (Becton Dickinson, Sunnyvale, CA) flow cytometer. Individual populations of blood cells were gated according to their forward and side scatter characteristics.

Labeling of cells and immunoprecipitation
For surface labeling, PBS-washed cells were biotinylated with Sulfo-NHS-LC-biotin (Pierce, Rockford, IL) (5 mM) for 1 h at 4°C. The reaction was quenched by washing once with 1 mM glycine in PBS and then twice with PBS. Cells (1 x 107) were solubilized in 1 ml lysis buffer (1% NP-40, 50 mM Tris–HCl, pH 8.2, 100 mM NaCl, 2 mM EDTA, 5 mM iodoactamide, 1 mM PMSF and 10 µg/ml aprotinin). Cell lysates were precleared with Protein A–Sepharose beads coated with non-specific mAb. Precleared lysates were then mixed with specific mAb-coated Protein A–Sepharose beads at 4°C for 24 h. After immunoprecipitation and SDS–PAGE, biotinylated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in PBS for 1 h at room temperature. The blocked membrane was incubated for 1 h at room temperature with avidin–peroxidase (Dako, Glostrup, Denmark) and the biotinylated proteins were visualized by the chemiluminescence detection system (Pierce).

Retroviral cloning of the P-3E10 molecule
The retroviral library construction was performed as described previously (15,16). In brief, a cDNA from human myeloid KG1a cells was cloned into the retroviral expression vector pBabeMN, kindly provided by G. Nolan (Stanford University).

For transfection of the library, Phoenix cells at 50% confluence were harvested by trypsinization, and 3 x 107 cells were added to a cocktail of 50 ml DMEM, 1% NuSerum (Genome Therapeutics, Waltham, MA), 200 µg/ml DEAE–dextran, 25 µM chloroquine diphosphate and 60 µg of the pBabeMN retroviral library. The cells were kept in suspension for 2 h at 37°C, washed once and cultivated in a 175 cm2 flask (Nunc) in DMEM containing 10% FCS at 37°C. At 24 h post-transfection the medium was renewed. After an additional 48 h of cultivation at 32°C, the virus-containing supernatant was collected, supplemented with 10 µg/ml hexadimethrene bromide (Sigma) and added to 1 x 106/ml BW5147 mouse thymoma cells in 10 ml RPMI 1640 medium containing 10% FCS.

For the isolation of P-3E10-reactive cells, infected BW5147 cells (4 x 107) were washed with PBS containing 1% BSA and incubated with P-3E10 mAb for 30 min on ice. After another washing step the cells were incubated with goat anti-mouse IgG microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s instructions. After washing, cells were resuspended in 500 µl of MACS sorting buffer (0.5% BSA/2mM EDTA in PBS) and loaded onto MS+ separation columns (Miltenyi Biotec) for positive selection of P-3E10 transduced cells. The isolated fraction was cultured in RPMI 1640 medium supplemented with 10% FCS. After three rounds of sorting, >95% of the isolated cells stained positively with P-3E10 mAb. Then, single-cell clones were obtained by limiting dilution.

For recovery of the P-3E10 cDNA, total RNA was extracted from the single-cell clone using Tri-Reagent (Sigma). RT-PCR was performed with Stratascript (Stratagene, La Jolla, CA) and the Advantage-GC polymerase system (Clontech, Palo Alto, CA) using primers flanking the multiple cloning site of the retroviral vector pBabeMN. The PCR was run for 30 cycles (30 s at 94°C, 30 s at 58°C and 4 min at 68°C). The purified PCR product was subcloned back into pBabeMN and transformed into E. coli DH5{alpha}. The plasmid DNA was isolated using a Qiagen Miniprep column according to the manufacturer’s recommendation (Qiagen, Hilden, Germany). To confirm that the isolated plasmid encodes the P-3E10 antigen, we used it to infect BW5147 cells as described above, and analyzed the transductants for P-3E10 expression by indirect immunofluorescence and flow cytometry. The plasmid was sequenced at the VBC-Genomics sequencing facility (Bioscience Research, Vienna, Austria).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
mAb P-3E10 inhibits the OKT3-induced T cell proliferation
In an attempt to identify new molecules involved in the regulation of T cell proliferation, various mAb against leukocyte surface molecules were generated using the T cell line Molt4 as an immunizing agent. The mAb were examined for their ability to modulate T cell proliferation in mononuclear cell preparations isolated from peripheral blood. We found that one of the mAb, named P-3E10, inhibited the OKT3-induced T cell proliferation in vitro. As shown in Fig. 1, T cell proliferation was significantly inhibited by mAb P-3E10 (n = 16). In contrast, isotype-matched control mAb, KLH and UPC 10, had no effect on the response of T cells to immobilized OKT3.



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Fig. 1. P-3E10 mAb inhibits CD3-induced T cell proliferation. PBMC were activated with immobilized OKT3 mAb at 20 ng/ml (A) or 1 µg/ml (B) in the presence of the indicated concentrations of P-3E10 mAb, isotype-matched control mAb (KLH and UPC 10) or medium. The bars represent mean ± SD of 10 and six healthy donors for (A) and (B) respectively.

 
Inhibition of cytokine production of T cells by P-3E10 mAb
PBMC were activated with immobilized OKT3 mAb in the presence or absence of soluble P-3E10 mAb, and IFN-{gamma}, IL-2, IL-10 and IL-4 were measured in the culture supernatants by ELISA. In the presence of P-3E10 mAb, production of all cytokines tested was inhibited (Fig. 2). The isotype-matched control mAb, however, had no such effect.



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Fig. 2. P-3E10 mAb inhibits cytokine production. PBMC were activated with immobilized CD3 mAb OKT3 in the presence of indicated concentrations of soluble P-3E10 mAb, isotype-matched control mAb or medium alone. The culture supernatants were harvested, and IFN-{gamma}, IL-2, IL-10 and IL-4 were measured by ELISA.

 
Cellular distribution of the P-3E10 antigen
To characterize the molecule recognized by P-3E10 mAb, various cell types were stained. All peripheral blood leukocytes (n = 10) including lymphocytes, monocytes and granulocytes were positive with mAb P-3E10 (Fig. 3A). Then we examined expression on hematopoietic cell lines. As shown in Fig. 3(B), all cell lines tested, including B cell lines (Daudi, JY and RAJI), T cell lines (Molt4, Sup T1 and Jurkat) and myeloid cell lines (KG1a, HL-60 and THP-1), were strongly positive with P-3E10 mAb. We also analyzed several non-hematopoietic cells and cell lines, including HUVEC, 293 human embryonic renal epithelial, MCF-7 breast cancer, OVMZ ovarian cancer and TCL kidney cancer cell lines, and found that all were clearly stained by P-3E10 mAb (data not shown). Thus, our results indicate that the P-3E10 antigen is a broadly expressed plasma membrane molecule.



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Fig. 3. Distribution of the antigen recognized by the mAb P-3E10 on peripheral blood leukocytes (A) and various hematopoietic cell lines (B). The indicated cells were stained with P-3E10 mAb (open) or AFP-01 control mAb (solid) by indirect immunofluorescence. Data are representative of 10 independent donors (A) and three independent experiments (B).

 
Effect of mAb P-3E10 on non-T cells
Because of the broad expression of the P-3E10 molecule, we tested the effect of the P-3E10 mAb on cells other than T cells. When we treated anti-IgM-induced B cells with 10 µg/ml of mAb P-3E10, similar to the results obtained with T cells, proliferation was inhibited by 65 ± 14% (mean ± SD; n = 3) (Fig. 4). However, in contrast to lymphocytes, the mAb did not block proliferation of HUVEC. Furthermore, it had also no effect on the growth of the hematopoietic cell lines Sup T-1, Jurkat, Molt4 and U937. Moreover, we analyzed the influence of P-3E10 on phagocytosis of myeloid cells, monocytes and granulocytes, but no effect was observed. The percentages of monocytes that had phagocytosed E. coli in the presence or absence of P-3E10 mAb were 72 ± 4.6 and 70 ± 6.2; those of granulocytes 56 ± 5.3 and 53 ± 3.8 (mean ± SD; n = 3), respectively.



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Fig. 4. P-3E10 mAb inhibits anti-IgM-induced B cell proliferation. PBMC were activated with 10 µg/ml of soluble anti-IgM antibody in the presence of 10 µg/ml P-3E10 mAb, isotype-matched control mAb (4G2) or medium alone. The bars represent mean ± SD of percent proliferation inhibition of three healthy donors.

 
Biochemical characterization of the surface molecule recognized by mAb P-3E10
To biochemically characterize the molecule bearing the P-3E10 antigen, we performed an immunoprecipitation experiment. As shown in Fig. 5, we were able to precipitate a 50- to 55-kDa protein under reducing conditions from lysates of surface-biotinylated Sup T1 cells using P-3E10 mAb. The protein shifted to 45–50 kDa under non-reducing conditions (Fig. 5) indicating that it contains intramolecular disulfide bonds. In comparison the isotype-matched control mAb MT99/3 (to the CD99 antigen) precipitated, as described previously (17), a protein band of 32 kDa both under reducing and non-reducing conditions.



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Fig. 5. Biochemical characterization of the cell surface molecule recognized by P-3E10 mAb. SDS–PAGE analysis of immuno precipitates obtained with either P-3E10 mAb or CD99 mAb MT99/3 from lysates of surface biotin-labeled Sup T1 cells. Electrophoresis was performed under reducing and non-reducing conditions. Molecular markers are shown on the left in kDa.

 
Molecular cloning of the cDNA coding for the antigen recognized by P-3E10 mAb
A KG1a cDNA library in the retroviral vector pBabeMN and the packaging cell line Phoenix were used to produce ecotropic viruses for transducing the target cell line BW5147. BW5147 transductants expressing the P-3E10 antigen were sorted by P-3E10 mAb using MACS and cloned to single-cell cultures by limiting dilution. One strongly positive cell clone was selected to isolate the P-3E10 cDNA. For this, RT-PCR was performed with the RNA extracted from the clone using primers flanking the multiple cloning site of the retroviral vector pBabeMN. The PCR product was digested with EcoRI to remove the plasmid sequences flanking the cDNA. Sequencing of the cDNA revealed a length of 1451 bp with an open reading frame of 840 bp coding for 279 amino acids (Fig. 6). Comparison of the sequence using the BLAST program at the NCBI (Bethesda, MD) resulted in 100% homology to the Na,K-ATPase ß3 subunit (20). This molecule is a type II transmembrane protein with a single transmembrane segment. The predicted extracellular domain contains six cysteine residues, which is in accord with our biochemical data in Fig. 5.



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Fig. 6. Nucleotide and deduced amino acid sequence of the cDNA encoding the P-3E10 antigen. Nucleotide positions are indicated on the left and amino acid positions on the right. The putative transmembrane-spanning domain is underlined. The six cysteines are boxed.

 
To confirm that the isolated Na,K-ATPase ß3 subunit cDNA encodes the P-3E10 antigen, the cDNA was re-ligated via EcoRI into the pBabeMN vector and transduced into BW5147 cells, which were subsequently tested for binding of the P-3E10 mAb. The transduced BW cells were specifically stained by P-3E10 mAb, demonstrating that the P-3E10 antigen is indeed a determinant on the Na,K-ATPase ß3 subunit.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In our study, we prepared a set of mAb reactive with the Molt4 T cell line. The generated mAb were screened for the ability to modulate CD3-induced T cell proliferation. P-3E10 mAb was found to be of interest, as it significantly inhibited T cell proliferation as well as IFN-{gamma}, IL-2, IL-4 and IL-10 production in vitro. P-3E10 mAb also inhibited anti-IgM-induced B cell proliferation. However, it had no effect on HUVEC and hematopoietic cell line proliferation, as well as phagocytosis of monocytes and granulocytes. By using a retroviral cloning system we proved that the P-3E10 antigen is a determinant on the human Na,K-ATPase ß3 subunit (20).

The Na,K-ATPase is a membrane-associated enzyme responsible for the active transport of Na+ and K+ in most animal cells (2123). By using the energy from the hydrolysis of one molecule of ATP, it transports three Na+ out in exchange for two K+ that are taken in. By coupling the hydrolysis of ATP to the movement of Na+ and K+ ions across the plasma membrane, the enzyme produces the electrochemical gradient that is the primary energy source for the active transport of nutrients, the action potential of excitable tissues and the regulation of cell volume (2123). In all tissues, Na,K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its {alpha} and ß subunits. At present, as many as four different {alpha} polypeptides ({alpha}1, {alpha}2, {alpha}3 and {alpha}4) and three distinct ß isoforms (ß1, ß2 and ß3) have been identified in mammalian cells (23). The {alpha} subunits are multispanning membrane proteins with a molecular mass of ~100 kDa that are responsible for the catalytic and transport properties of the enzyme. The ß polypeptides cross the membrane once, depending on the degree of glycosylation in different tissue, and their molecular mass ranges from 40 to 60 kDa. The ß subunit is essential for the normal activity of the enzyme, and it appears to be involved in the occlusion of K+, and the modulation of K+ and Na+ affinity of the enzyme (22,23). In addition, in vertebrate cells, the ß subunit may act as a chaperone, stabilizing the correct folding of the {alpha} subunit to facilitate its transport to the plasma membrane (22,23).

In immune cells, induction of Na,K-ATPase-mediated K+ fluxes in mitogen-activated lymphocytes has been reported (2428). Increase in mRNA encoding the {alpha} and ß subunits of Na,K-ATPase in phytohemagglutinin-activated lymphocytes was also demonstrated (26,29). Several mechanisms have been proposed to explain the increase in the Na,K-ATPase activity in lymphocyte activation (26,28,30). Prasad et al. have argued that the increase in intracellular calcium may lead to a subsequent protein kinase-induced enhancement of Na,K-ATPase enzymatic activity (28). Na,K pump activation, accompanying human lymphocyte blast transformation, also plays a critical role in the expression of IL-2 receptor and initiates IL-2 expression (24,31,32). Inactivation of Na,K-ATPase by specific inhibitors causes inhibition of both mitogen- and antigen-induced lymphocyte activation (3133).

In the present study we demonstrate that engagement of the Na,K-ATPase ß3 subunit by a mAb down-regulates both T and B lymphocyte proliferation as well as production of IFN-{gamma}, IL-2, IL-4 and IL10 of T cells. This is, to the best of our knowledge, the first study to demonstrate that a specific mAb to the Na,K-ATPase ß chain is able to block lymphocyte activation. Although, the precise mechanism of this inhibition is unknown, engagement of Na,K-ATPase by P-3E10 mAb may block Na,K-ATPase activation and subsequently suppress downstream events, which in turn lead to the inhibition of lymphocyte activation. Further investigation of the mechanisms of the Na,K-ATPase ß3 chain in inhibition of lymphocyte activation may lead to a better understanding of immune regulation, which may provide new avenues for clinical intervention.


    Acknowledgements
 
We would like to thank Dr Garry Nolan for providing the retroviral vector pBabeMN-lacZ and the Phoenix packaging cell line, and Dr Václav Horejsí for providing mAb. This work was supported by the Thailand Research Fund, the Royal Golden Jubilee PhD program of Thailand, the National Center for Genetic Engineering and Biotechnology (BIOTEC) of the National Sciences and Technology Development Agency, and the Competence Center for Biomolecular Therapeutics in Austria.


    Abbreviations
 
APC—antigen-presenting cell

PBMC—peripheral blood mononuclear cell

HUVEC—human umbilical vein endothelial cell

SCS—supplemented calf serum


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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