Published online before print October 2, 2007
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* Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea;
Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea;
Division of Biological Sciences and the Institute for Molecular Biology and Genetics, Chonbuk National University, Chonju, Korea;
Department of Molecular Biotechnology, Konkuk University, Seoul, Korea; and
|| Department of Biological Sciences and the Research Center for Women’s Diseases, Sookmyung Women’s University, Seoul, Korea
1 Correspondence: Department of Biological Sciences and the Research Center for Women’s Diseases, Sookmyung Women’s University, Chungpa-Dong, Yongsan-Gu, Seoul 140-742, Korea. E-mail: jslim{at}sookmyung.ac.kr
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Key Words: dendritic cells (DC) • siRNA
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It has been well established that optimal T cell activation requires two stimulatory signals. One of these signals is the TCR, which recognizes antigen in the form of peptide fragments bound to MHC class molecules on APC. The second signal is generally referred to as the costimulatory signal, which does not, by itself, induce any proliferation in resting T cells but significantly lowers the number of TCR complexes, which need to be triggered to induce the activation and acceleration of T cell responses [4 , 5 ]. A variety of costimulatory molecules has been described, which may alter the overall functional avidity of selection by affecting TCR-dependent signals or by inducing signals independently of TCR. CD5 and CD6 antigens are examples of similar costimulatory proteins, which are important during thymocyte selection [6 7 8 9 ]. In addition, CD6 is capable of providing the costimulatory signals needed to synergize with signals mediated through the TCR to enhance T cell proliferation [10 , 11 ].
Activated leukocyte cell adhesion molecule (ALCAM; CD166), also known as KG-CAM, neurolin, and BEN/DM-GRASP/SC1, is a novel member of the Ig superfamily, which is involved in homophilic adhesion and in binding to CD6 [12 , 13 ]. It is expressed on activated leukocytes [14 ], monocytes [15 ], hematopoietic progenitor cells [16 ], bone marrow stromal cells [17 ], and hematopoiesis-supporting osteoblastic cells [18 ]. ALCAM-mediated interactions are important during neural development [19 ], maturation of hematopoietic stem cells in blood-forming tissues [16 , 17 , 20 ], immune responses [21 ], and tumor progression [22 ]. Although ALCAM was shown to mediate homotypic and heterotypic cell-cell clustering through homophilic (ALCAM-ALCAM) and heterophilic (ALCAM-CD6) interaction [14 ], Hassan et al. [23 ] demonstrated recently that homophilic interaction was approximately 100 times weaker than heterophilic interaction. It was also observed that ALCAM/CD6 engagement made a positive contribution during specific immune responses. It has also been shown that a fraction of CD6 is physically associated with the TCR/CD3 complex and that CD6 and its ligand ALCAM colocalize with the TCR/CD3 complex at the central supramolecular activation clusters [24 ]. It is interesting that it has been shown in a more recently published work that ALCAM is another cell surface protein expressed by DC upon differentiation from monocytes and mediated adhesion to CD6, and ALCAM and CD6 interactions contribute to stabilization of the immunological synapse [25 ].
In this study, we examined N-myc downstream-regulated gene 2 (NDRG2) expression in monocyte-derived DC as well as primary DC from lymph nodes, thymus, and skin using anti-NDRG2 polyantibodies and mAb and found a DC-specific expression of NDRG2 protein. To assess whether NDRG2 expression affects APC function of DC, U937 and NDRG2-transfected U937 cells were evaluated for their surface expression of costimulatory or adhesion molecules. When U937 cells showing NDRG2 overexpression were stimulated with GM-CSF and IL-4, NDRG2 specifically inhibited the cytokine-induced reduction of ALCAM. Last, DC differentiated from NDRG2 small interfering RNA (siRNA)-transfected monocytes exhibited a decreased level of ALCAM expression and showed a decreased ability to stimulate allogeneic T cells. Thus, our data indicate that expression of NDRG2 during the differentiation of DC may contribute to their sustained ability to activate T lymphocytes by preserving the expression of costimulatory molecules.
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Generation of DC in vitro
Human PBMC of healthy donors (Red Cross Blood Center, Daejeon, Republic of Korea) were isolated by density centrifugation on a Histopaque 1077 (Sigma Chemical Co.). The Red Cross Blood Center Committee (Seoul, Republic of Korea) approved use of these cells. Monocytes were purified by negatively depleting T, B, and NK cells, erythrocytes, and granulocytes using mouse antibody-reactive immunomagnetic beads (Dynal, Oslo, Norway). In brief, anti-CD2-, -CD7-, -CD16-, -CD19-, -CD56-, and -CD235a-labeled PBMC were incubated with the immunomagnetic beads for 30 min at 4ºC with gentle rotation, and positive cells were removed using a Dynal magnet. Purified monocytes were found to be >90% positive for the CD14 marker. Immature DC were generated by culturing monocytes in human DC medium containing GM-CSF (400 ng/ml) and IL-4 (20 ng/ml) in a 12-well plate at a concentration of 5 x 105 cells per well.
Preparation of NDRG2 antibody
Mouse polyclonal antibodies and NDRG2 mAb were generated using recombinant protein consisting of amino acids 150–357 of NDRG2 (NM_201541). In brief, NDRG2 C terminus cDNA (corresponding to 150–357 aa) was cloned into the pET28a vector by PCR and restriction enzyme digestion methods. The cloned pET28a-NDRG2-C plasmid was transformed into BL21 DE3 to produce recombinant protein production. Recombinant NDRG2-C protein expression was induced by the addition of isopropylthiogalactoside to a final concentration of 1 mM at 37°C for 4 h. The recombinant NDRG2-C protein extraction and purification were performed according to the protocol for the QIAexpressionist system (Qiagen, Hilden, Germany). The eluted recombinant NDRG2-C protein was mixed with Freund’s adjuvant, and the protein/adjuvant complex was injected into BALB/c mice. During boosting, anti-NDRG2 antibody production was examined by ELISA. After the fifth boosting, antisera were collected for polyclonal antibody and transferred to microtubes for subsequent experiments. NDRG2 mAb was produced using BALB/c mice and a myeloma cell line, P3/NS/1-Ag4-1. Spleen cells of mice immunized with recombinant NDRG2-C protein were harvested, and roughly 50–60% of all cells were placed in culture after fusion. Cell culture supernatants from selected cell lines were tested by ELISA. Among the growing clones, which tested positive, the 19C-4 clone (isotype IgG1) was used for further study. mAb was produced in ascites from BALB/c mice injected with 19C-4 cells.
Immunohistochemistry
For immunohistochemistry, tissues were fixed routinely in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were cut from the paraffin blocks and mounted on 3-amino-propyltriethoxysilane-coated slides. After deparaffinization and antigen retrieval using a pressure cooker with 10 mM sodium citrate buffer (pH 6.0) at full power for 4 min, primary antibody raised against NDRG2 was diluted at 1/3200 with background-reducing diluent (Dako, Carpinteria, CA, USA). Immunostaining using antibodies against NDRG2, S-100 protein (Dako, 1/400 dilution), and CD1a (Dako, 1/50 dilution) was performed using the mouse EnVision kit (Dako). After preincubation with blocking serum for 15 min, the primary antibody was applied and incubated for 30 min in a humid chamber and then washed with PBS. Slides were incubated for 30 min with the EnVision peroxidase reagent (Dako) and were then incubated sequentially with 3,3-diaminobenzidine chromogen for 5 min. The slides were counterstained with Meyer’s hematoxylin and mounted. Careful rinses with several changes of PBS were performed between each stage of the procedure. Negative controls were used by excluding the primary antibody or by using preimmune IgG1.
Western blot analysis
Cells were homogenized on ice in Pro-PREPTM protein extraction solution (iNtRon Biotechnology, Seongnam, Republic of Korea) containing protease inhibitor, 1 mM PMSF, 1 mM EDTA, 1 µM pepstatin A, 1 µM leupeptin, and 0.1 µM aprotinin. After 30 min on ice, the extracts were centrifuged (13,000 g) for 10 min at 4ºC, and the supernatants were recovered. The protein concentration was measured (Bio-Rad, Hercules, CA, USA), and 
30 µg of each extract was loaded for 12% SDS-PAGE gel and was then transferred from the gel onto a polyvinylidene difluoride membrane (Amersham Biosciences, Little Chalfont, UK). The nonspecific antibody-binding sites on the membrane were blocked by incubation of the membrane in PBS (pH 7.0) containing 0.1% Tween 20 and 4% nonfat dry milk for 1 h at room temperature. The membrane was washed in PBS containing 0.1% Tween 20 and incubated overnight at 4ºC with NDRG2 antibody. After washing for 1 h, the membrane was incubated with HRP-conjugated secondary antibody (Sigma Chemical Co.) for 1 h at room temperature. The resulting blot was visualized by ECL-Plus Western detection reagents (Amersham Biosciences).
Confocal laser scan microscopy (CLSM)
Monocyte-derived DC were grown on 12-mm diameter glass coverslips coated with poly-L-lysin in 12-well plates. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. After washing with PBS, blocking was completed with 1% BSA (Sigma Chemical Co.) in PBS for 30 min at room temperature. Cells were incubated with mouse anti-NDRG2 polyclonal antibody, followed by incubation with tetramethyl rhodamine isothiocyanate-conjugated secondary antibody and FITC-conjugated HLA-DR antibody. Cells were then treated with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) in PBS for 10 min and washed with 0.3% Triton X-100 in PBS. Coverslips, which held cells, were mounted onto cover glasses with Vectashield mounting medium, and the slides were kept in darkness prior to examination. Confocal images were obtained using a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Jena, Germany).
Construction and transfection of NDRG2 in U937 cells
The NDRG2 cDNA region containing the entire open reading frame was amplified by PCR with primers (5'-ATGGCGGAGCTGCAGG-3' and 3'-TCAACAGGAGACCTCCAT-5'). The PCR products were cloned into topoisomerase plasmid (Invitrogen, Carlsbad, CA, USA), and the nucleotide sequence was determined by automatic sequencing. After digestion with XhoI and BamHI, NDRG2 was subcloned into pcDNA3.1 (Invitrogen). For stable expression of NDRG2, U937 cells were transfected with pcDNA3.1-NDRG2 by electroporation with a Gene Pulser (Bio-Rad) at 960 µF and 300 V, resulting in U937-NDRG2. After stable transfection, U937 cells were maintained in culture medium containing 1 mg/ml Geneticin disulfate salt (G418, Sigma Chemical Co.). Two weeks after the transfection, single cells were plated in 96-well plates (0.5 cell/well) for growth. Expression of NDRG2 in cell clones was confirmed by Northern and Western blotting.
Transfection of siRNA in monocytes and DC
A mixture of four siRNAs (SMARTPool) specific for NDRG2 or ALCAM and control SMARTPool siRNA were purchased from Dharmacon (Lafayette, CO, USA). siRNAs were introduced into monocytes and DC using a monocyte or DC nucleofection kit (Amaxa, Cologne, Germany), according to the instructions of the manufacturer. In brief, 1 µg SMARTPool siRNA was mixed with 0.1 ml cell suspension, transferred to a 2-mm electroporation cuvette, and nucleofected with a NucleofectorTM apparatus (Amaxa), as described. After electroporation, cells were transferred immediately to 3 ml-supplemented medium or DC medium and were then cultured in six-well plates. Using pmaxGFP (Amaxa), transfection efficiency was evaluated in monocytes or DC after 24 h of transfection. Using cytometric analysis, GFP expression was detected in 70–75% of the monocytes and in 25–29% of DC (data not shown).
RT-PCR and Northern blot analysis
Total RNA was extracted from the cultured cells using the acid guanidinium thiocyanate-phenol-chloroform extraction method [26
]. First-strand cDNA was synthesized from total RNA under RNase-free conditions. The reaction was performed with 10 µg total RNA using a ProSTARTM first-strand RT-PCR kit (Stratagene, La Jolla, CA, USA), according to the instructions of the manufacturer. PCR was performed in a GeneAmp PCR system 2700 (Perkin-Elmer/Centus, Norwalk, CT, USA) using the following first-strand cDNA and Taq polymerase (TaKaRa Shuzo, Kyoto, Japan) for each primer: 5'-GCCATGTACGTTGCTATCCAGGCTG-3' and 3'-AGCCGTGGCCATCTCTTGCTCGAAG-5' for β-actin, 5'-AATGGCCCTTGTTGCCCT-3' and 3'-TCCTTCCCCACACTCGTT-5' for NDRG2, and 5'-AGTTCCTGCCGTCTGCTCTT-3' and 3'-TTTTTCCTTTACCTGGGTCA-5' for ALCAM. PCR-amplified products were separated on 1.5% agarose gels containing 0.1 µg/ml ethidium bromide and visualized under UV light. Northern blot analysis was performed according to a method described previously [27
]. In brief, total RNA (3–5 µg) was separated in a 1% denaturing formaldehyde-agarose gel and transferred to a nylon membrane (Boehringer Mannheim, Mannheim, Germany). After UV fixation, a random primer DNA labeling kit (Roche, Mannheim, Germany) was used to hybridize the membrane at 68ºC in ExpressHybTM solution (Clontech, Palo Alto, CA, USA) with the cDNA probes labeled with [32p]dCTP (Perkin-Elmer/Centus). In the case of NDRG2, a cDNA probe was prepared from the RT-PCR product using a specific primer set, 5'-ATGGCGGAGCTGCAGG-3' and 3'-TCAACAGGAGACCTCCAT-5'. The probed membrane was then washed in 2x SSC and 0.1% SDS at room temperature and exposed to autoradiographic film using an intensifying screen for 2–3 days at –70ºC.
Proliferation assay of allogeneic PBL and measurement of IL-2 production
Monocyte-depleted PBL (1x105 cells), isolated from buffy coats of donor blood, were incubated with graded numbers of irradiated (3000 rad) DC transfected with control or NDRG2- or ALCAM-specific SMARTPool siRNA. Experiments were performed in each well of 96-well round-bottom plates. Cell proliferation was quantified by measuring the thymidine uptake of cells after they had been incubated with 1 µCi [methyl-3H]thymidine (Amersham Pharmacia, Piscataway, NJ, USA) during the last 18 h of the 4-day culture. Cells were harvested onto glass fibers, and radioactivity was measured using a scintillation counter. Results were presented as mean cpm of cultures performed in triplicate. For determining IL-2 production, DC (5x105) were incubated with allogeneic PBL (1x106), and IL-2 production on days 1–3 was measured from supernatants using an ELISA kit (Endogen).
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, NDRG2 expression increased gradually during differentiation (Fig. 1C)
. In addition, NDRG2 protein was not detected at all in leukemia, lymphoma, B-lymphocyte, or NK cell lines (Fig. 1D)
. Taken together, these results indicate that NDRG2 protein is expressed specifically during the differentiation of DC, as shown previously in NDRG2 mRNA expression in DC.
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Figure 1. NDRG2 protein is expressed specifically in human DC. (A–D) Cell lysate was prepared from human cell lines, MUTZ-3 DC, PBMC, primary monocytes, and monocyte-derived DC. Using anti-NDRG2 mAb, NDRG2 protein expression was determined by Western blot analysis. (E) Analysis of the cellular distribution of NDRG2 in monocyte-derived DC by CLSM. DC were stained with Hoechst 33342, FITC-conjugated HLA-DR mAb, and NDRG2 polyclonal antibody, respectively.
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NDRG2 is expressed specifically in primary DC in human lymph node, thymus, and skin
To investigate the expression pattern of NDRG2 in human tissues, we performed immunohistochemistry against NDRG2 using normal human thymus, reactive lymph nodes, and skin. In addition, several pathologic lesions were studied for the expression of this molecule. In a reactive lymph node, NDRG2 was expressed characteristically in interdigitating DC (IDC), and lymphocytes and follicular DC were found to be negative for NDRG2 expression (Fig. 2A
). In the thymus, the expression of NDRG2 was noted in DC and thymic epithelial cells, including Hassall’s corpuscles (Fig. 2B)
. Its expression was also demonstrated in the epidermal Langerhans cells and dermal DC in the skin (dendrocytes; Fig. 3A
, upper). Expression of NDRG2 in pathologic lesions such as dermatopathic lymphadenitis was confirmed additionally but not in tuberculosis (Fig. 3A
, lower). When we prepared 3 µm serial sections to confirm the expression of NDRG2, IDC of the lymph node showed coexpression of NDRG2 and S-100 protein (Fig. 3B
, top). Epidermal Langerhans cells and LCH showed coexpression of NDRG2 and CD1a in staining of sequential sections (Fig. 3B
, middle and bottom). In LCH, most of the tumor cells were strongly reactive for our NDRG2 antibody (Fig. 3B
, bottom). When the immunostaining was performed for DC-SIGN, it was distributed mainly in the sinusoidal DC of reactive lymph nodes (data not shown). These results confirm that NDRG2 protein is expressed specifically in IDC of lymph nodes and Langerhans cells.
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Figure 2. Immunohistochemistry for NDRG2 expression in the human lymph node and thymus. (A) In lymph nodes, NDRG2 was expressed specifically in IDC, and all lymphocytes and follicular DC were found to be nonreactive. Upper left and right (100x original magnification), lower left (200x original magnification), lower right (400x original magnification). Arrows indicate typical positive staining in DC. (B) In the normal thymus, the expression of NDRG2 was noted in DC and thymic epithelial cells including Hassall’s corpuscles. M, Medullar; C, Cortex. Upper left (200x original magnification), upper right, lower left, and lower right (400x original magnification). Arrows indicate typical positive staining in DC.
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Figure 3. Expression of NDRG2 in the skin DC and lymphadenitis (A) and comparison of NDRG2 expression with other DC marker expression by staining of sequential sections (B). (A) In the skin, NDRG2 expression was demonstrated in the epidermal Langerhans cells (upper left, 200x original magnification) and dermal DC (upper right, 200x original magnification). Arrows indicate typical positive staining in Langerhans cells and dermal DC. The actively proliferating IDC in dermatopathic lymphadenitis (lower right, 100x original magnification) were also positive, and epithelioid histiocytes of tuberculosis were negative (lower left, 200x original magnification). (B) In parallel with NDRG2 expression, paracortex of lymph node in 3 µm serial sections was stained with anti-S-100 antibody (top, 400x original magnification). Expression of NDRG2 and CD1a in serial sections was compared in epidermal Langerhans cells (middle, 400x original magnification) and Langerhans cell histiocytosis (LCH; bottom, 400x original magnification).
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Figure 4. NDRG2 inhibits the GM-CSF/IL-4-induced ALCAM reduction in U937 cells. (A) U937 cells were transfected with pcDNA3.1 or pcDNA3.1-NDRG2, and NDRG2 expression was detected by Northern blotting. (B) Several clones overexpressing NDRG2 were selected by G418 (1 mg/ml), and NDRG2 protein expression in the clones and DC was detected by immunoblotting with anti-NDRG2 mouse mAb. (C) U937-mock and three U937-NDRG2 clones were cultured with or without GM-CSF (400 ng/ml) and IL-4 (20 ng/ml) for 4 days, and ALCAM expression was analyzed by flow cytometry. (D) U937-mock and U937-NDRG2 (clone 61) were cultured in the presence of GM-CSF and IL-4 for the times indicated and stained with PE-conjugated ALCAM mAb (BD Biosciences) for flow cytometric analysis. (E) ALCAM mRNA expression was detected 2 days after culture in the absence or presence of GM-CSF and IL-4 by RT-PCR. M, Mock; N, NDRG2.
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Figure 5. Inhibition of NDRG2 in monocytes decreases ALCAM expression during DC differentiation. Monocytes were transfected with control or NDRG2 siRNA using a monocyte nucleofection kit (Amaxa). After differentiation into DC with GM-CSF and IL-4, inhibition of NDRG2 was analyzed by Northern blotting (A) on day 6 and immunoblotting (B) on days 3 and 6. C, Control; N, NDRG2. (C) ALCAM expression on day 6 was detected in DC, which were differentiated from control (Cont) or NDRG2 siRNA (NDRG2)-transfected monocytes using RT-PCR. The relative values of PCR products were calculated as follows: (mean intensityxband area) of NDRG2 or ALCAM/(mean intensityxband area) of β-actin. (D) After DC had differentiated from monocytes transfected with control or NDRG2 siRNA, cells were analyzed for surface antigen expression by immunostaining with their respective antibodies by flow cytometry on days 3 and 6 (thin line, control SMARTPool siRNA; bold line, NDRG2-specific siRNA).
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NDRG2 down-regulation affects the T cell stimulatory ability of DC
ALCAM has been demonstrated to interact in a homophilic manner in cis or in trans [18
, 32
] or in an heterophilic manner with CD6 mediated through CD6d3 and the N-terminal domain of ALCAM [14
]. We hypothesized that NDRG2 siRNA transfection affects the allostimulatory activity of DC, as the inhibition of NDRG2 during DC differentiation decreases ALCAM expression. To compare with DC, which have a decreased ALCAM expression, reduction of ALCAM expression was induced directly in monocytes using ALCAM-specific siRNA transfection. As predicted, ALCAM expression was decreased during DC differentiation of monocytes by a transfection with not only NDRG2-specific siRNA, but also ALCAM-specific siRNA (Fig. 6A
). NDRG2 or ALCAM silencing in monocytes did not change the expression level of DC-SIGN (CD209) that may be involved in interactions between T cells and DC. NDRG2 expression, however, was not affected in DC differentiated from ALCAM siRNA-transfected monocytes (Fig. 6B)
. Furthermore, ALCAM siRNA transfection in monocytes did not affect DC differentiation (data not shown). While the interference of NDRG2 expression with siRNA during DC differentiation did not affect CD54 expression on DC1a-positive DC or CD11a on CD3-positive T cells in the mixed lymphocyte reaction, it clearly decreased allogeneic T cell proliferation (Fig. 6C
and 6D)
. Interestingly, NDRG2 siRNA transfection induced more effective reduction of allogeneic T cell proliferation than ALCAM siRNA transfection, indicating the possibility that NDRG2 inhibition may induce diverse effects on DC function other than ALCAM reduction. However, as previously reported that ALCAM is essential for T cell proliferation [25
], DC transfected with ALCAM siRNA also showed a decreased induction in the T cell proliferation. Similarly, these findings were also confirmed in IL-2 level produced by allogeneic T cells (Fig. 6E)
. No significant difference in the cytokine production such as IL-12 and IL-10 between NDRG2 or ALCAM siRNA- and control siRNA-transfected DC was observed under these coculture conditions (data not shown). These results therefore suggest that although the involvement of soluble factors in the low level of stimulation may not be excluded completely, inhibition of NDRG2 during DC differentiation at least down-regulates ALCAM expression, which influences the ability of DC to stimulate allogeneic T cell proliferation.
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Figure 6. DC derived from NDRG2 siRNA-transfected monocytes show a decreased ability to induce allogeneic T cell proliferation. (A) Down-regulation of ALCAM 3 days after induction of DC differentiation from control siRNA- (filled histogram) and NDRG2 siRNA- or ALCAM siRNA-transfected (open histogram) monocytes. Cells were analyzed for surface antigen expression by immunostaining with their respective antibodies by flow cytometry. Numbers indicate mean fluorescence intensity (upper; filled histogram, lower; open histogram). (B) ALCAM siRNA transfection into monocytes does not affect the NDRG2 expression level of DC. After induction of DC differentiation from control, NDRG2, or ALCAM siRNA-transfected monocytes, NDRG2 expression in DC was detected by immunoblotting. (C) After induction of DC differentiation from control (filled histogram) or NDRG2 siRNA-transfected monocytes (open histogram), DC-SIGN expression was detected by flow cytometry. Expression of CD54 and CD11a is not affected by NDRG2 or ALCAM siRNA transfection. After DC differentiation from control, NDRG2, or ALCAM siRNA-transfected monocytes, DC (5x105) were incubated with allogeneic PBL (1x106) for 1 day. ICAM (CD54) expression was measured on CD1a-positive cells (R1), whereas LFA-1 (CD11a) expression was measured on CD3-positive cells (R2) by using flow cytometer (filled histogram, control SMARTPool siRNA; open histogram, NDRG2- or ALCAM-specific SMARTPool siRNA). (D) Different numbers of DC derived from monocytes transfected with control, NDRG2, or ALCAM siRNA were incubated with allogeneic PBL (1x105) for 4 days. Cell proliferation was measured by the incorporation of radioactive thymidine using a scintillation counter. Data are representative of at least two independent experiments. Mean ± SEM of triplicates is shown (Student’s t-test; *, P<0.05, compared with control siRNA). (E) DC (5x105) were incubated with allogeneic PBL (1x106), and IL-2 production on days 1, 2, and 3 was measured from supernatants using the ELISA kit. Results are means ± SD of two independent experiments (Student’s t-test; *, P<0.05; **P<0.5, compared with control siRNA).
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NDRG2 belongs to the NDRG family, a new family of differentiation-related genes. This family is comprised of four members, NDRG1–NDRG4 [33 ]. NDRG2 is highly expressed in the adult brain, salivary gland, and skeletal muscle [29 , 34 ]. NDRG2 cDNAs, corresponding to four isoforms (Ndrg2a1, Ndrg2a2, Ndrg2b1, and Ndrg2b2), which differ in terms of their 5'-untranslated and N-terminal coding regions, have been reported in rat renal tissue [34 ]. NDRG2 has been implicated in cell growth [30 ], differentiation [28 ], and apoptosis [35 ] and is rapidly responsive to mineralcorticoid stimuli in the kidney [34 ]. Most recently, it has been shown that its inactivation is involved in the development of aggressive forms of cancer [36 ].
In this study, we demonstrated that interference of NDRG2 expression induced a down-regulation of ALCAM expression, and overexpression of NDRG2 strongly prevented cytokine-induced ALCAM reduction in monocytic cell lines. In parallel with this observation, NDRG2 siRNA transfection decreased the ability of DC to stimulate allogenic T cell proliferation. Unfortunately, it was not possible to compare the effect of siRNA directly with the neutralizing antibody capable of binding to ALCAM, as the commercially available form of the anti-ALCAM antibody was not effective for interfering with the ALCAM-CD6 interaction. However, it is worth noting that the inhibition of NDRG2 expression does not affect the DC differentiation phase and the morphological changes, which occur during their differentiation, although it seems to induce minimal changes in the growth rate of the NDRG2-overexpressing U937 cell line (data not shown). Originally, based on reports that NDRG2 is implicated in cell differentiation and growth, we expected that the inhibition of its expression could effectively result in the inhibition of differentiation. This was not the case, however, although RNA interference could not induce complete inhibition of its expression. Thus, our observations indicate that NDRG2 expression in DC may be involved in DC-related functions.
ALCAM is a member of a small subgroup of transmembrane glycoproteins, which belong to the Ig superfamily. ALCAM is expressed on a wide variety of cells and mediates homotypic and heterotypic cell-cell clustering through homophilic (ALCAM-ALCAM) and heterophilic (ALCAM-CD6) interaction [14 , 22 , 37 ]. However, it has been shown that the homophilic interaction has 100 times lower affinity than does the heterophilic interaction [23 ]. Gimferrer et al. [24 ] reported that in image analysis of antigen-specific T-APC conjugates, CD6 and its ligand (ALCAM) colocalize with TCR/CD3 at the center of the immunological synapse and that a soluble recombinant CD6 form reduces the number of mature antigen-specific T-APC conjugates significantly. Therefore, these results demonstrate the important role played by the interaction of ALCAM with CD6 during T cell activation and proliferation processes. In a more recently published work, it has been demonstrated that ALCAM-CD6 interactions are involved in the stabilization of DC-T cell contact and the proliferative phase of the T cell response [25 ]. Moreover, it shows that ALCAM and CD6 are actively recruited at the DC-T cell interface and localize all along the contact site. These findings indicate that the down-regulation of ALCAM expression in DC may profoundly affect the ability of DC to stimulate T cells. Therefore, it will be interesting to examine whether NDRG2 expression in APC is involved in the activation of CD6. It is interesting that NDRG2 inhibition in monocyte-derived DC was more effective in inhibiting allogenic T cell proliferation than ALCAM inhibition, whereas the transfection of NDRG2 siRNA directly into DC did not result in the reduction of ALCAM or the inhibition of T cell proliferation (data not shown), although ALCAM inhibition still induced significant down-regulation of proliferation. It is therefore worth considering that the inhibition of NDRG2 during DC differentiation might modulate other DC functions such as cytokine production. In fact, although we were not able to detect any significant difference of IL-12 or IL-10 production in DC-T cell cocultures, NDRG2-transfected U937 cells, when stimulated with GM-CSF and IL-4, could secrete IL-10 at a lower level than wild-type U937 cells (unpublished data). Therefore, we do not exclude the possibility that the effect of NDRG2 down-regulation on allogeneic T-cell stimulation may not be mediated solely by ALCAM reduction.
In conclusion, this work confirms and expands earlier observations of the specific expression of NDRG2 in differentiated cell lineages on a protein level and suggests that NDRG2 expression in DC may play a role in maintaining their ability to activate T lymphocytes by preserving the expression of costimulatory molecules.
Received May 14, 2007; revised September 2, 2007; accepted September 8, 2007.
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