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Originally published online as doi:10.1189/jlb.0407219 on August 28, 2007

Published online before print August 28, 2007
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(Journal of Leukocyte Biology. 2007;82:1466-1472.)
© 2007 by Society for Leukocyte Biology

Interactions of TANGO and leukocyte integrin CD11c/CD18 regulate the migration of human monocytes

Stephanie Arndt*, Christian Melle{dagger}, Krishna Mondal{ddagger}, Gerd Klein§, Ferdinand von Eggeling{dagger} and Anja-Katrin Bosserhoff*,1

* Institute of Pathology, University of Regensburg Medical School, Regensburg, Germany;
{dagger} Core Unit Chip Application, Institute of Human Genetics and Anthropology, Friedrich-Schiller-University, Jena, Germany;
{ddagger} Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany; and
§ University Medical Clinic, Section for Transplantation Immunology, Center for Medical Research, Tübingen, Germany

1Correspondence: Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. E-mail: anja.bosserhoff{at}klinik.uni-regensburg.de


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ABSTRACT
 
The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.

Key Words: cell adhesion • extravasation • interaction • integrin alpha X • p150.95 • CD11c/CD18


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INTRODUCTION
 
It has become evident that TANGO belongs to a gene family of four homologous proteins, TANGO, MIA, OTOR (FDP, MIAL), and MIA2. TANGO together with the homologous proteins define a gene family sharing important structural features, significant homology at both the nucleotide and protein level, and similar genomic organization [1 ]. The four members share 34–45% amino acid identity. TANGO encodes a mature protein of 103 amino acids in addition to a hydrophobic secretory signal sequence (accession number: gi:74054302). Sequence homology confirms the highly conserved SH3 structure present also in MIA, OTOR, and MIA2. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern previously determined for the other members of the MIA gene family. High levels of TANGO expression were observed both during embryogenesis and in adult tissues [1 ]. The only cells lacking TANGO expression are cells belonging to the hematopoietic system. Monocytes, as a part of the hematopoietic cell system are produced in the bone marrow from hematopoetic stem cell precursors, which circulate in the bloodstream for about 1 to 3 days and then typically move into tissues throughout the body to become macrophages. Monocytes, like all leukocytes, follow a defined series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion, and diapedes [2 ]. During extravasation, monocytes interact with a variety of extracellular matrix components. Many adhesion molecules on both leukocytes and endothelial cells have overlapping functions during the multiple steps of extravasation [3 4 5 ]. As leukocytes roll, they encounter chemokines and other stimuli that activate them to bind firmly to the endothelial cell through interaction of different integrins, including the leukocyte integrins CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (alphaX), and CD11d/CD18 (alphaD). Members of these integrins share the same β-chain (CD18) but are distinguished by their {alpha} chain [6 , 7 ]. CD11/CD18 integrins can interact with multiple ligands, including the Ig superfamily cell adhesion molecules (ICAMs, VCAMs) and matrix proteins such as fibrinogen, fibronectin, or collagens [2 , 8 9 10 ]. There are characteristic inserted domains (I-domains), which consist of ~200 amino acids in several types of {alpha} integrin subunits. I-domains are responsible for the binding of ligands and have a unique structure with 6 or 7 {alpha} helixes and 6 β sheets to form an independent structural and functional unit [11 , 12 ].

Recently, we discovered that MIA, a TANGO homologue, interacts with integrin {alpha} 4 β 1 and {alpha} 5 β 1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings suggest that MIA plays a role in tumor progression and the spread of malignant melanomas by mediating detachment of cells from extracellular matrix molecules by modulating integrin activity [13 ]. As MIA regulates attachment and migration of melanoma cells we hypothesized that TANGO could play a role in regulating attachment and migration of hematopoietic cells.

In the present study, we focused on the function of TANGO during adhesion of monocytic cells to endothelial cells in vitro, based on the fact that after adhesion, TANGO expression was induced in primary monocytes.


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MATERIAL AND METHODS
 
Cell lines and cell culture conditions
Premonocytic cell lines, U937, HL-60, and THP-1 were used. All cells were maintained in DMEM supplemented with penicillin (400 U/ml), streptomycin (50 µg/ml), L-glutamine (300 µg/ml), and 10% heat-inactivated (1 h; 56°C) FCS (Sigma, Deisenhofen, Germany). Peripheral blood mononuclear cells (MNC) were isolated from leukapheresis concentrates of healthy donors by density gradient centrifugation over Ficoll/Hypaque. Human monocytes were separated from mononuclear cells, as described previously [14 ]. For in vitro differentiation of monocytes to macrophages purified monocytes were cultured adherently in Teflon bags for 7 days at a cell density of 106 cells/ ml in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2% pooled, human AB serum (Cambrex, Walkersville, MD, USA), penicillin (50 U/ml; GIBCO, Carlsbad, CA, USA), streptomycin (50 µg/ml; GIBCO) and L-glutamine (2 mM; GIBCO). In experiments to prevent adhesion, monocytes were cultured in continuously turning roller bottles with the same media as above except supplemented with recombinant human macrophage colony-stimulating factor (100 ng/ml; Cetus, Emmeryville, CA, USA). For in vitro differentiated dendritic cells, monocytes were cultured in Teflon bags for 7 days in RPMI media containing L-glutamine (2 mM), streptomycin (50 µg/ml), penicillin (50 U/ml), sodium pyruvate (1 mM; Gibco), minimum essential medium vitamins, nonessential amino acids (all Gibco), β-mercaptoethanol (0.05 M), IL-4 (6 pg/ml, Immunotools, Friesoythe, Germany), GM-CSF (50 pg/ml, Essex, Munich, Germany) and 10% heat-inactivated FCS.

The human microvascular endothelial cell line CDC/ EU.-HMEC-1 (HMEC) was kindly provided by the Centers for Disease Control and Prevention (Atlanta, GA, USA) and has been described previously [15 ].

RNA isolation and reverse transcription
Total cellular RNA was isolated from cultured and human blood cells using the RNeasy kit (Qiagen, Hilden, Germany). cDNAs were generated by reverse transcriptase reaction performed in 20 µl reaction volume containing 2 µg of total cellular RNA, 4 µl of 5 x first strand buffer (Invitrogen, Groningen, The Netherlands), 2 µl of 0.1 M DTT, 1 µl of dN6-primer (10 mM), 1 µl of dNTPs (10 mM) and DEPC-water. The reaction mixture was incubated for 10 min at 70°C, 200 U of Superscript II reverse transcriptase (Invitrogen) were added and RNAs were transcribed for 1 h at 37°C. Reverse transcriptase was inactivated at 70°C for 10 min, and the RNA was degraded by digestion with 1 µl RNase A (10 mg/ ml) at 37°C for 30 min.

Analysis of expression by quantitative PCR
Quantitative real-time PCR for TANGO was performed on a LightCycler (Roche, Mannheim, Germany). cDNA template (2 µl), 2.4 µl 25 mM MgCl2, 0.5 µl (20 mM) of forward and reverse primers (hTANGO for: 5'-ggctcttgaagatttcac-3'; hTANGO rev: 5'-atccgtctcatctgttgg-3') and 2 µl of SYBRGreen LightCycler DNA Master SYBR Green Mix in a total of 20 µl were applied to the following PCR program: 30 s at 95°C (initial denaturation); 20°C/s temperature transition rate up to 95°C for 15 s, 3 s at 60°C, 5 s at 72°C, 81°C acquisition mode single, repeated for 40 times (amplification). The PCR reaction was evaluated by melting curve analysis and checking the PCR products on 2% agarose gels. β-actin was amplified to ensure cDNA integrity and to normalize expression.

Expression of recombinant TANGO protein
A TANGO prokaryotic expression vector with a 15 amino acid Avi-tag peptide sequence, including a FXa cleavage site was constructed by overlap extension PCR (TANGO accession number: gi:74054302). The TANGO cDNA construct was cloned into the vector pIVEX2.3-MCS (Roche, Mannheim, Germany) [16 ]. The expression vector was used in the Rapid Translation System, a cell-free Escherichia coli-based protein transcription/translation system (Roche). By adding biotin, ATP and the E. coli biotin protein ligase BirA during the procedure, the protein was biotinylated at the introduced Avi-tag at the N terminus and called recombinant biotinylated TANGO. The correct expression of the TANGO protein was analyzed by SELDI-MS ProteinChip Technology; function was confirmed by analysis on tumor cells [16 ].

Detection of protein interactions
Protein interactions were assessed in a modified procedure, as described elsewhere [17 ]. In brief, 30µl of streptavidin agarose beads (Molecular Probes; Eugene, OR, USA) were washed with PBS followed by incubation of recombinant biotinylated TANGO in PBS overnight at 4°C in an over-end-over mixer, or, as a negative control, streptavidin agarose beads that were incubated with recombinant biotinylated MIA underwent the same procedure. In parallel, 5x 106 HL-60 cells incubated with 50 ng/ml PMA were lysed in a buffer containing 100 mM sodium phosphate pH 7.5, 5 mM EDTA, 2 mM MgCl2, 3 mM 2-β-mercaptoethanol, 0.1% CHAPS, 500 µM leupeptin, and 0.1 mM PMSF. Afterward, the cell lysate was incubated with streptavidin agarose beads for depletion of proteins that bind nonspecifically to the streptavidin agarose beads. Pellets were discarded, and the precleared supernatant was incubated with 50 ng recombinant TANGO or MIA, respectively, and 30µl streptavidin agarose beads for 2 h at room temperature in an over-end-over mixer. Bound proteins were eluated from the beads by 30 µl 50% acetonitrile/ 0.5% trifluoroacetic acid and were gently vortexed for 5 min. Two microliters of the eluated samples were applied to the activated, hydrophobic surface of an H50 ProteinChip Array (Ciphergen Biosystems, Fremont, CA, USA) and dried on air. After washing with 3 µl aqua bidest, 0.5 µl sinapinic acid (saturated solution in 0.5% TFA/50% acetonitrile) was applied twice and the array was analyzed in a ProteinChip Reader (series 4000, Ciphergen), according to an automated data collection protocol.

Identification of interacting proteins
The identification of interesting proteins was performed as described [18 ]. Briefly, the eluates were reduced to a maximal volume of 10 µl by speed-vac and subjected to SDS-PAGE for separation of containing proteins followed by staining with Simply Blue Safe Stain (Enhanced Coomassie, Invitrogen). Afterward, interesting specific gel bands were excised, destained, and dried followed by rehydration and digestion with 10 µl of a trypsin solution (0.02 µg/ µl; Promega, Madison, WI, USA) overnight at 37°C. The supernatants of in-gel digestion were applied directly to NP20 ProteinChip arrays (Ciphergen). After addition of the matrix, peptide fragment masses were analyzed using the ProteinChip Reader, series 4000 instrument. A standard protein mix (all-in-1 peptide standard mix; Ciphergen), including Arg8-vasopressin (1082.2 Da), somatostatin (1637.9 Da), dynorphin (2147.5 Da), ACTH (2933.5 Da), and insulin β-chain (3495.94 Da) was used for calibration. Proteins were identified using the fragment masses searching in a publicly available database (http://129.85.19.192/profound_bin/WebProFound.exe).

Coimmunoprecipitation (CO-IP)
For coimmunoprecipitation, 1 x 106 HL-60 cells were incubated with 50 ng/ ml PMA for 24 h. One-hundred micrograms of cell lysates of HL-60 cells, treated with PMA, were precleared with 20 µl streptavidin-sepharose (Amersham, Biosciences, Piscataway, NJ, USA) for 4 h at 4°C in an over-end-over mixer. The lysate was then incubated with 50 ng/ml recombinant biotinylated TANGO protein and for further experiments additionally with an anti-human CD11c I-domain blocking antibody (clone 3.9, 10 µg/ml; American Diagnostica, Stamford, CT, USA), recombinant human ICAM-1/FC Chimera (5 µg/ ml; R&D Systems, Minneapolis, MN, USA) or fibrinogen (Biocat; 1 mg/ ml), respectively, over night at 4°C in an over-end-over mixer. As a negative control biotinylated recombinant MIA was used [13 ] or recombinant un-biotinylated TANGO protein. The next day, we added 50 µl streptavidin-sepharose to the protein mixture and incubated the cups again for 4 h at 4°C in an over-end-over mixer. Afterward, the beads were washed three times with PBS. The beads were resuspended in Laemmlis buffer, incubated at 95°C for 10 min, and separated on a 10% SDS-PAGE followed by Western blot analysis. For detection, we used anti-integrin {alpha} X (H-68) antibody (anti-CD11c, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against amino acids 613–680 mapping within an extracellular domain of anti-integrin {alpha} X.

Western blot analysis
Five x 106 cells were washed with 1 xPBS, and protein lysate was prepared as described [16 ]. Forty micrograms of protein were denatured, loaded, separated on SDS-PAGE, and subsequently blotted onto PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking for 1 h with 3% BSA/PBS, the membrane was incubated for 16 h with a primary antibody [anti-TANGO antibody generated by BioGenes (Berlin, Germany) 1:200 or anti-integrin {alpha} X, 1:20 (Santa Cruz Biotechnology)]. The peptide sequence recognized by the TANGO antibody reads as follows: SNRFPDDEDAQEETE. For loading control we used β-actin antibody (Sigma; 1:5000). After washing the membrane was incubated for 1 h with an alkaline phosphate-coupled secondary antibody (Chemicon, Temecula, CA, USA). Immunoreactions were visualized by NBT/BCIP (Zytomed, Berlin, Germany) staining.

Attachment of premonocytic cells to fibrinogen, ICAM-1, or to endothelial cells coated on plastic
Ninety-six-well high binding, type I certified polystyrene flat-bottom stripwell plates (Costar, Corning, Corning, NY) were coated with human fibrinogen (Biocat, 1 mg/ ml) or recombinant human ICAM-1/FC Chimera (5 µg/ ml; R&D Systems) and incubated at 4°C overnight. The coating puffer was discarded, and the plates were dried for 1 h in the cell culture hood. 3 x 106 HL-60 cells were incubated with 500 µl PBS and 500 µl 2 µM CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester) staining solution for 4 min by room temperature in the dark. All of the following steps were performed in the dark. The staining was stopped by adding 12 ml PBS/ 10% FCS, and the pellet was incubated again with DMEM medium with PMA (50 ng/ ml). Forty-thousand stained cells were incubated with recombinant biotinylated TANGO (50 ng/ ml) or CD11c I-domain blocking antibody (clone 3.9, 10 µg/ ml; American Diagnostica), incubated for 10 min in the dark, and subsequently added to each well coated with fibrinogen, ICAM-1, or to plates, which were already preincubated with a confluent layer of HMECs. Afterward, the plates were incubated for four hours in a 37°C incubator. Fluorescence was measured with the Fusion fluorescence reader from Packard Bio Science Company and displayed in %. Experiments were repeated at least three times.

Transmigration through fibrinogen or endothelial cell layer
Migration assays were performed using Boyden chambers containing polycarbonate filters with 5-µm pore size (Neuro Probe), as described [16 ]. Filters were coated with fibrinogen (1 mg/ml; Sigma) or with a confluent layer of HMECs, respectively. The lower compartment was filled with Mel Im-conditioned medium, used as a chemoattractant and for activation of HMECs. HL-60 cells were washed and resuspended in DMEM without FCS at a density of 3 x 105 cells/ ml and placed in the upper compartment of the chamber. Fifty nanograms recombinant TANGO was added to the upper compartment and for control without recombinant TANGO. After incubation at 37°C for 30 min, the filters (three per experiment) were collected, and the cells adhering to the lower surface were fixed, stained, and counted. All assays were repeated at least three times.

Statistical analysis
Results are expressed as means ± SD (range) or percent. Comparison between groups was made using the Student’s paired t test. A P value < 0.05 was considered statistically significant. All calculations were performed using the GraphPad Prism 4 software (GraphPad, San Diego, CA, USA). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, not significant.


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RESULTS
 
Induction of TANGO after monocyte adhesion
The only cells lacking TANGO expression are cells belonging to the hematopoietic system. One of the major differences between fully differentiated hematopoietic cells and all other tissue cells is the lack of adhesion until these cells leave the bloodstream. To analyze the regulation of TANGO expression in the hematopoietic system, we concentrated on blood monocytes. RT-PCR studies using RNA isolated from monocytes at several stages of differentiation indicated different expression levels of TANGO (Fig. 1A ). Freshly isolated blood monocytes (D0), monocytes attached to each other, for one (N1) and two days (N2), and cells attached to Teflon for 1 day (A1) did not express TANGO on the mRNA level. High TANGO expression was observed in adherent monocytes after two days of incubation (A2). TANGO expression was enhanced after 7 days of culturing the cells on Teflon (A7) or on culture plates (data not shown). Interestingly, when we incubated the cells in suspension in a roller bottle and inhibited attachment to substrate or other cells, TANGO was not expressed after 1 day (R1). Furthermore, the addition of macrophage colony stimulating factor (M-CSF) to the roller bottle cells, which leads to differentiation of monocytes to macrophages (R1+ and R7+) in the absence of adhesion, also failed to induce TANGO. Therefore, we suggest that TANGO expression is regulated by adhesion. This is further supported by the finding that after incubation of monocytes adherently for 7 days on Teflon bags (A7) and then for one additional day as suspension cells in roller bottles (A7-> R1), the expression of TANGO was lost on mRNA level (Fig. 1B) and on protein level (Fig. 1C) . These data suggest that TANGO is only present when cells are cultured adherently. Interestingly, we failed to detect TANGO mRNA expression in in vitro monocyte-generated dendritic cells (Fig. 1D) . Taken together, our data indicate that TANGO expression is related to monocyte adhesion and apparently to adhesion-dependent macrophage but not dendritic cell differentiation.


Figure 1
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  		Figure 1. Expression of TANGO during monocyte to macrophage differentiation. (A) TANGO mRNA expression pattern at different stages of monocyte differentiation. D0, freshly isolated blood monocytes; N1, N2, monocytes attached to each other, for 1 and 2 days; A1, cells attached to Teflon for 1 day; A2, A7, adherent monocytes after 2 and 7 days of incubation; R1, cells incubated in suspension in a roller bottle for one day; R1+,R7+, addition of M-CSF to the roller bottle cells for 1 and 7 days. β-actin was used for normalization. TANGO mRNA expression analysis (B) and protein analysis of A7->A1 (C), incubation of monocytes for 7 days on Teflon bags, detachment of cells by cold-shock and incubation for one additional day on Teflon bags; A7->R1, incubation of monocytes for 7 days on Teflon bags, cold-shock detachment of the cells and incubation for one additional day as suspension cells in roller bottles. (D) Analysis of TANGO expression in monocytes, macrophages, and in vitro monocyte-generated dendritic cells. *, P < 0.05; **, P < 0.01.

To further analyze the regulation of TANGO transcription in myeloid cells, we used premonocytic cell lines e.g., HL-60, U937, and THP-1, which basically express no TANGO mRNA and detected a concomitant attachment of the cells and an increase of TANGO expression after incubation with phorbol myristate acetate (PMA) (Fig. 2 ).


Figure 2
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  		Figure 2. Expression of TANGO in premonocytic cell lines TANGO mRNA expression in HL-60, U937, and THP-1 cell lines before and after incubation with phorbol myristate acetate (PMA).

Colocalization of TANGO with CD11c (integrin alphaX)
To analyze the functional relevance of TANGO expression after cell attachment, we aimed to screen for interacting proteins. Coimmunoprecipitation experiments followed by staining with silver or Coomassie detected specific gel bands marked with 1, 2, 3, and 4 in Fig. 3A 3B . The gel bands of the Coomassie-stained SDS-gel were excised, destained, and dried followed by rehydration and digestion with trypsin. The supernatants of in-gel digestion were applied directly to NP20 ProteinChip arrays and measured. Peptide fragment masses were analyzed using a ProteinChip Reader and the proteins were identified using the fragment masses searching in a publicly available database. The fragment masses of the four protein bands shown in Fig. 3A 3B belong to the integrin CD11c. Additionally, we analyzed coimmunoprecipitation lysates on Western blot analysis using a CD11c specific antibody (Fig. 3C) . Interestingly, we detected the same four protein bands on Western blot than on Coomassie- and silver-stained gels, which suggested a controlled fragmentation of CD11c.


Figure 3
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  		Figure 3. Interaction of TANGO with CD11c Co-immunoprecipitation of TANGO and CD11c analyzed by silver staining (A), Coomassie staining (B), and Western blot analysis (C) followed by digestion and analyses of gel bands (marked with 1, 2, 3, and 4) from Coomassie staining by SELDI-TOF mass spectrometry. As a negative control, MIA was used for coimmunoprecipitation.

Influence of TANGO on cellular attachment
Next, we aimed to analyze TANGO/ integrin CD11c interaction in detail. Miller et al. [19 ] described an induction of CD11c expression in different premonocytic cell lines stimulated with PMA. We could confirm these findings on mRNA and protein level (data not shown). Results of TANGO expression analysis in macrophages let us assume that TANGO is involved in processes after adhesion. First, we incubated premonocytic cells with recombinant biotinylated TANGO on 96-well cell culture plates and found no changes in cell attachment to plastic of those cells (data not shown). Next, we used fibrinogen, ICAM-1 (known ligands of CD11c), and HMEC-coated plates to analyze whether TANGO may have an influence on cellular attachment of premonocytic cells. When we incubated the coated plates with HL-60 cells treated with recombinant TANGO, we detected a significantly reduced cellular attachment of the cells to fibrinogen (Fig. 4A ), to ICAM-1 (Fig. 4B) and to HMECs (Fig. 4C) , respectively. Attachment to fibrinogen or ICAM-1 was also reduced by CD11c I-domain blocking antibody as reported previously. Next, we were interested whether TANGO competes for CD11c ligand binding by binding to the ligand binding site in the I-domain of CD11c or binds to a different region of CD11c. In coimmunoprecipitation experiments in the presence of recombinant biotinylated TANGO and the CD11c I-domain blocking antibody, neither ICAM-1 nor fibrinogen reduced the binding between CD11c and TANGO (Fig. 4D) . Therefore, we suggest that the binding site of TANGO to CD11c is different from the binding site of fibrinogen and ICAM-1.


Figure 4
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  		Figure 4. Influence of TANGO protein and CD11c I-domain blocking antibody on cellular attachment The attachment of activated premonocytic HL-60 cells to fibrinogen (A), ICAM-1 (B), and HMECs (C) was decreased after treatment with recombinant TANGO protein or CD11c I-domain blocking antibody. To analyze whether TANGO competes for CD11c ligand binding by binding to the ligand binding site in the I-domain of CD11c, we performed coimmunoprecipitation experiments in the presence of recombinant biotinylated TANGO. Neither the CD11c I-domain blocking antibody nor ICAM-1 or fibrinogen reduced the binding between CD11c and TANGO (D).

TANGO promotes transmigration of premonocytic cells
The fact that TANGO reduced cellular attachment allowed us speculate that TANGO may participate in the process of migration. Transmigration of HL-60 cells was performed through fibrinogen (Fig. 5A ) and HMECs (Fig. 5B) -coated Boyden Chamber assays, respectively. Incubation with recombinant TANGO in the upper compartment showed a significant increased transmigration of HL-60 cells compared with the control cells without TANGO treatment. Therefore, we assume that TANGO is a regulator of migration of monocytic cells.


Figure 5
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  		Figure 5. TANGO promotes migration of premonocytic cells. Migration of premonocytic HL-60 cells after recombinant TANGO incubation through fibrinogen (A) or HMEC layer (B) was performed in a Boyden chamber assay. **, P < 0.01; ***, P < 0.001.


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DISCUSSION
 
Transendothelial migration of monocytes initially involves tethering of cells to the endothelium, followed by rolling along the vascular surface, firm adhesion to the endothelium, and diapedesis between the tightly apposing endothelial cells. As all migratory events, this transendothelial migration process has to be controlled by defined steps of adhesion and loss of adhesion of cell-cell or cells-matrix contacts. Monocyte adhesion/trafficking was of current interest in our study due to primary results that show that TANGO is involved in these processes. We revealed that TANGO is not expressed in primary human blood monocytes and in THP-1, HL-60, and U937 premonocytic cells. TANGO, however, was inducible after treatment of premonocytic cells with PMA, which is known to induce predominantly attachment of those cells. This result was the first indication that TANGO expression is regulated by adhesion. Interestingly, we failed to detect TANGO mRNA expression in in vitro monocyte-generated dendritic cells. Therefore, TANGO expression might be related to monocyte adhesion and apparently to adhesion-dependent macrophage but not dendritic cell differentiation.

Leukocyte extravasation from the bloodstream through the endothelial barrier during inflammatory reaction involves a coordinated interaction of a variety of adhesion receptors at the surface of leukocytes and the endothelial cells [3 , 20 ]. We discovered that TANGO directly binds to the leukocyte-specific β2 integrin CD11c/CD18, also named integrin {alpha} X or p150.95. CD11c/CD18, together with LFA-1 and MAC-1, are cell surface glycoproteins involved in leukocyte adhesive interactions [21 ]. Miller et al. described that U937 and HL-60 cells do not express Mac-1 and CD11c but LFA-1 [19 ]. After stimulation of U937 and HL-60 cells with PMA, the expression of Mac-1 and CD11c was induced showing more CD11c than Mac-1 expression. We could support these data with regard to CD11c and suggest that the simultaneous induction of CD11c and TANGO after PMA treatment has implication for function. Firm adhesion of the leukocyte to the endothelial surface is a prerequisite for migration across the endothelium and is mediated by endothelial ICAM-1 and VCAM-1. Whereas VCAM-1 has only been demonstrated to play a role in the migration of monocytes across endothelium [22 ], there is general agreement that the β2-integrins and their endothelial ligand ICAM-1 are essential for transporting all leukocytes across the endothelium [23 24 25 26 ]. CD11c/CD18 is expressed on activated monocytes and neutrophils and on certain activated lymphocytes and functions as an adhesion molecule in cell-cell and cell-substrate interactions. It has been shown that CD11c binds to complement fragment iC3b [27 , 28 ], matrix molecules, such as fibrinogen [29 , 30 , 9 ] and the Ig superfamily of ICAM-1, ICAM-2, and VCAM-1 [31 , 8 ]. CD11c includes an I-domain, which has been implicated in ligand binding [32 , 33 ]. After revealing direct binding of TANGO to CD11c, we were interested to see whether TANGO could influence the adhesion process of monocytic cells to endothelial cells, ICAM-1, or fibrinogen in vitro. To analyze this, we incubated premonocytic cells seeded on fibrinogen or ICAM-1-coated plates or on a confluent layer of HMECs with recombinant TANGO protein. A decrease in attachment after TANGO incubation let us speculate that TANGO might be a potential inhibitory ligand for CD11c. Results showing that TANGO binding to CD11c cannot be inhibited by fibrinogen or ICAM-1, however, revealed that TANGO inhibitory function is not due to competition for the I-domain binding site. Therefore, we speculate that TANGO modifies integrin CD11c activity.

A similar mechanism of integrin blocking is known for melanoma inhibitory activity, MIA, which shows high homology to TANGO. Previous studies revealed direct interaction of MIA with several matrix proteins such as fibronectin or laminin [34 , 35 ]. Fibronectin type I and type II domains are known to interact with integrin {alpha} 5 β 1. Recently, we discovered that MIA interacts with integrin {alpha} 4 β 1 and {alpha} 5 β 1 and promotes detachment and invasiveness of melanoma cells by regulating integrin activity [13 , 34 ].

Subsequent assays revealed that TANGO, by blocking attachment via CD11c, positively modulates migration. As migration is a controlled process of adhesion and loss of adhesion of cells, we speculate, in analogy to MIA, that TANGO is involved in these processes.

In summary, we revealed that TANGO interacts with integrin CD11c and is simultaneously induced with integrin CD11c expression after adhesion of premonocytic cells. TANGO reduces the attachment and promotes the migration capacity of premonocytic cells in vitro potentially by modulating integrin CD11c/CD18 activity. Further investigations have to be performed to clearly determine the CD11c binding site of TANGO modulating integrin activity and to understand the mechanisms leading to down-regulation of integrin activity.


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ACKNOWLEDGEMENTS
 
We are indebted to Susanne Wallner and Sibylla Lodermeyer for technical assistance. The work was supported by the DFG.

Received April 12, 2007; revised July 24, 2007; accepted August 7, 2007.


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