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International Immunology Advance Access originally published online on June 1, 2006
International Immunology 2006 18(7):1159-1169; doi:10.1093/intimm/dxl050
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Binding of multivalent CD147 phage induces apoptosis of U937 cells

Nutjeera Intasai1,2,3, Sabine Mai2, Watchara Kasinrerk4,5 and Chatchai Tayapiwatana4

1 Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
2 Department of Physiology, Manitoba Institutes of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg R3E 0V9, Canada
3 Division of Clinical Microscopy and
4 Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
5 Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand

Correspondence to: C. Tayapiwatana; E-mail: asimi002{at}chiangmai.ac.th and W. Kasinrerk; E-mail: watchara{at}chiangmai.ac.th


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
CD147 is a broadly expressed cell-surface molecule and serves as a signaling receptor for extracellular cyclophilins. CD147 also appears to interact with immune cells, but its counter-receptor on these cells has not been clearly described. In the present report, we displayed multiple copies of the CD147 extracellular domain (CD147Ex) on VCSM13 phage to study the interaction of CD147 with its ligand. Recognition of phage containing fusion protein of CD147Ex and gpVIII (CD147Ex phage) by four different anti-CD147 mAbs indicated that at least parts of the CD147 are properly folded. Specific binding of CD147Ex phage to various cell types was demonstrated by flow cytometry. Morphological changes, however, were observed only in U937, a monocytic cell line, after 24 h incubation with multivalent CD147Ex phage. After 48 h, U937 cell propagation ceased. Staining with annexin V and the presence of cleaved caspase-3 indicated that many of the CD147Ex phage-treated cells had lost viability through apoptotic cell death. The above results suggest that CD147 induces apoptosis in U973 cells and that at least a portion of this cell death program involves a caspase-dependent pathway.

Keywords: caspase-3, cell signaling, ligand-receptor interaction, phage display, prokaryotic expression


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The human leukocyte surface molecule CD147 is a 50- to 60-kDa type 1 transmembrane glycoprotein that belongs to the Ig superfamily (1). CD147 is composed of two Ig domains in the extracellular region, a single transmembrane domain and a short cytoplasmic domain (2–4). The extracellular region contains three glycosylation sites (4), but the glycan portion of the molecule differs according to cell and tissue types. Thus, the different glycosylation patterns of the 27-kDa core protein results in its variable molecular weight (2). The CD147 molecule, also known as EMMPRIN (2), basigin (3, 5) and M6 Ag (6), is broadly expressed on both hematopoietic and non-hematopoietic cells. Expression of CD147 on T cells depends on the differentiation state: it is expressed at higher levels on the cell surface of immature thymocytes than on mature peripheral T cells (7). CD147 expression is up-regulated on activated T cells (2, 3). High expression of CD147 is also observed on human tumor cells (8–10) and seems to be responsible for stimulating fibroblast matrix metalloproteinases (MMPs) production leading to extracellular matrix degradation, which results in tumor growth and metastasis (11, 12). Some CD147 mAbs inhibited homotypic aggregation of the estrogen-dependent breast cancer cell line, MCF-7, as well as MCF-7 cell adhesion to type IV collagen, fibronectin and laminin (1). Certain CD147 mAbs also induced homotypic cell aggregation of a monocytic cell line, U937 (13). This activation depends upon the activation of protein kinases and re-organization of the cytoskeleton (14). Treatment of immature fetal thymocytes by CD147 mAbs caused aggregation of their CD147 molecules and inhibited their further development into mature T cells (15). Enhancement of mixed lymphocyte responses in CD147 knockout mice indicates a negative regulatory function of CD147 in T cells (16). Triggering of CD147 molecules on T cells by an inhibitory mAb resulted in modulation of lipid rafts, impaired signaling and resulted in impaired expression of the IL-2R {alpha} chain CD25 (17).

Most functional studies of CD147 have relied on specific mAbs to induce the cells of interest (11–17). The use of mAbs to mimic native ligand-receptor signaling, however, did not verify the role of CD147 in binding to a membrane ligand to initiate signal transduction. Recently, purified native CD147 was shown to induce the production of secreted MMP-1 by human dermal fibroblasts as well as MMP-2 by a breast carcinoma cell line (MDA-435) (18). The binding affinity of CD147 for its ligand is presumably weak, as is typical for Ig superfamily proteins (18).

Phage display technology is a powerful technique for presenting proteins or peptides and has been widely used to identify ligands that bind to a protein of interest (19, 20). However, glycosylation and oligomerization of expressed proteins are not achieved by this technique, and some domains may not fold properly. In an attempt to study the CD147-ligand interaction, we first generated phage-displayed CD147 fused to the minor coat protein, gpIII (21). However, this phage did not bind to cells (unpublished results), indicating a lack of receptor activity. Expression of gpIII fusion protein is restricted to less than one molecule per phage particle (22). Since many molecules of the major coat protein, gpVIII, are present on the phage particle, we have now constructed a multivalent phage displaying the CD147 extracellular domain (CD147Ex) via fusion with the major coat protein, gpVIII (CD147Ex phage) (23). Multiple CD147 molecules on phage particles may enhance binding avidity by multivalent ligand interactions and mimic the ligand–receptor interaction in functional studies of CD147.

In the present study, the function of CD147 was investigated using phage display of CD147. We found that multivalent CD147Ex phage bound to, and induced the apoptotic cell death of, the monocytic cell line, U937. We address here the possible role of the CD147-ligand interaction on the induction of apoptosis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cells and cell lines
The human monocytic cell line (U937), T cell lines (Jurkat and Molt4) and a B cell line (Daudi), acute myelogenous leukemic cell line (KG1a) and T cell lymphoma (HUT78) were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO), 40 µg ml–1 gentamicin and 2.5 µg ml–1 amphotericin B in a humidified atmosphere of 5% CO2 at 37°C. The BW5147 mouse thymoma cell line was maintained in the same medium and culture conditions.

PBMCs were obtained from healthy donors by Ficoll–Hypaque density gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden).

Multivalent CD147Ex phage preparation
A phage displaying multiple copies of the CD147Ex via the major coat protein, gpVIII, was produced as previously reported (23). Briefly, a DNA encoding CD147Ex fragment was amplified from the mammalian expression vector, pCDM8-CD147 (6). After amplification, the 550-bp PCR product of CD147Ex was inserted into the pComb8 phagemid (kindly provided by C. F. Barbas, Scripps Institute, USA) to construct pComb8-CD147Ex, a phagemid expressing the CD147Ex. The pComb8-CD147Ex was transformed into TG1 Escherichia coli host strain [supE hsd{Delta}5 thi{Delta}(lac-proAB) F'(traD36proAB+, laclq lacZ{Delta}M15)] (kindly provided by A. D. Griffiths, MRC Cambridge, UK). The pComb8-CD147Ex-transformed E. coli were grown at 25°C with simultaneous isopropyl-ß-D-thiogalactopyranoside stimulation and VCSM13 phage infection. Phage particles containing CD147 were detected by sandwich ELISA (23). Four different anti-CD147 mAbs (M6-1B9, IgG3; M6-1E9, IgG2a; M6-1D4, IgM and M6-2F9, IgM) (13, 14) were used as capture antibodies, and HRP-conjugated anti-gpVIII mAb (Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to detect these phage particles.

Identification of CD147Ex phage binding to various cell lines by flow cytometry
Fifty microliters of 6 x 1011 tu ml–1 multivalent CD147Ex phages or VCSM13 control phages in RPMI 1640 were mixed with 50 µl of 107 cells ml–1 for each cell line tested, and the mixture was incubated on ice for 30 min. The incubated cells were centrifuged at 12 000 rpm for 10 s and the supernatant was discarded. Cells were re-suspended in 50 µl RPMI 1640 containing anti-gpVIII mAb (10 µg ml–1; Amersham) and incubated on ice for 30 min. After incubation, cells were centrifuged and the supernatant was discarded. Cells were then stained with FITC-conjugated anti-mouse Ig (Silenus, Melbourne, Australia) in RPMI 1640 and incubated on ice for 30 min. After centrifugation and aspiration of the supernatant, the cells were re-suspended in PBS. Flow cytometric analysis was carried out on a FACSCalibur (BD Biosciences, San Jose, CA, USA) using CellQuest software (BD Biosciences).

Confirmation of CD147Ex phage binding to U937 cells by immunocytochemistry
Three milliliters of 1010 tu ml–1 CD147Ex phage or VCSM13 phage in RPMI 1640 were added to 3 ml of 3 x 105 U937 cells ml–1 in RPMI 1640–20% FBS, and the mixture was incubated at 37°C for 24 h in a 5% CO2 atmosphere. After incubation and low-speed centrifugation and re-suspension in a smaller volume, 100 µl of 1 x 106 cells ml–1 phage-incubated U937 cells were placed on a silane-coated slide, air dried, and fixed with 3.7% formaldehyde in PBS containing 50 mM MgCl2 for 10 min. Slides were washed three times in PBS containing 50 mM MgCl2 and blocked with 1% BSA in saline sodium citrate (SSC) at room temperature for 5 min. Anti-gpVIII mAb (Amersham) was added and incubated at room temperature for 1 h. After washing, Alexa680-conjugated goat anti-mouse Ig (Molecular Probes, Eugene, OR, USA) was added to the slide and incubated at room temperature for 30 min. After washing, U937 nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes). Image analysis was carried out using the Spectra Cube (Applied Spectral Imaging, Carlsbad, CA, USA) on a Zeiss Axiophot 2 microscope (Carl Zeiss Canada Ltd., Toronto, Ontario, Canada) and the SKYVIEW 2.1.1 software (Applied Spectral Imaging). After spectral image acquisition, the acquired spectral image was classified using the combinatorial table containing the fluorochrome combinations for the above analyses (24). This classified image was generated to delineate the cleaved caspase-3 positive area (classified in red). Inverted DAPI images represent nuclei.

Effect of CD147Ex phage on various cell types
One hundred microliters of 1010 tu ml–1 CD147Ex phage in RPMI 1640 was added to 100 µl of 3 x 105 cells ml–1 of each cell type in RPMI 1640–20% FBS in the wells of a 96-well flat-bottom tissue culture plate (NUNC, Roskilde, Denmark) and the mixture was incubated at 37°C in a 5% CO2 incubator. VCSM13 phage and phage-displayed green fluorescent protein were used as controls. Live cultures were observed at 1, 2, 3, 4, 8, 24 and 48 h of incubation. Images were taken under a Zeiss Axiovert 100 microscope (Zeiss).

Antibody neutralization of CD147Ex phage in U937 cell death induction
One hundred microliters of 1010 tu ml–1 CD147Ex phage in RPMI 1640 were pre-incubated with 10 µg ml–1 anti-CD147 mAbs, M6-14D and M6-2F9, at 37°C for 30 min. The mixture was added to 100 µl of 3 x 105 cells ml–1 of each cell type in RPMI 1640–20% FBS in a 96-well flat-bottom tissue culture plate (NUNC) and incubated at 37°C in a 5% CO2 incubator. The cultures were observed at 1, 2, 3, 4, 8, 24 and 48 h of incubation. Images were taken under a Zeiss Axiovert 100 microscope (Zeiss).

Cytotoxicity assay
CD147Ex phage-induced U937 cell death was investigated by mixing 3 ml of 1010 tu ml–1 multivalent CD147Ex phage or VCSM13 phage re-suspended in RPMI 1640 with 3 ml of 3 x 105 U937 cells ml–1 in RPMI 1640–20% FBS, and incubating for 48 h at 37°C in a 5% CO2 incubator. After washing, cells were stained using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes) according to the manufacturer's instructions. Stained cells were washed twice and cytospun onto microscope slides at a density of 105 cells per slide. Nuclei were counterstained with DAPI (Molecular Probes). Images were taken using a Zeiss Axioplan2 microscope (Zeiss) and FITC (excitation at 494 nm, emission at 518 nm), Texas red (excitation at 595 nm, emission at 620 nm) and DAPI (excitation at 359 nm, emission at 461 nm) filters (Zeiss). The fluorescence intensity was analyzed by Northern Eclipse 5.0 (Empix Imaging Inc., Mississauga, Ontario, Canada) using the NucDen function.

Annexin V assay
U937-CD147Ex phage incubation was performed as described for the cytotoxicity assay. VCSM13 phage and phage-displayed survivin (SVV) were included as controls. After incubation, incubated U937 cells were washed with cold PBS and re-suspended in 100 µl of binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). One hundred microliters of 106 cells ml–1 of phage-incubated U937 cells were incubated with 5 µl annexin V conjugated with FITC (BD Biosciences) for 15 min at room temperature in the dark. Two microliters of propidium iodide (PI, 50 µg ml–1) were added to the incubated cells. Flow cytometric analysis was carried out within 1 h on a FACSSort (BD Biosciences) using CellQuest software (BD Biosciences).

Examination of cleaved caspase-3 by flow cytometry
Incubation of U937 cells with CD147Ex phage was performed as described previously in the cytotoxicity assay. VCSM13 phage and cisplatin (Sigma–Aldrich, St Louis, MO, USA) were included as controls. After incubation, U937 cells were harvested, washed and re-suspended in PBS. Methanol-free formaldehyde was then added to the re-suspended cells to a final concentration of 0.25%, incubated at 37°C for 10 min and placed on ice for 1 min. The cells were then permeabilized with ice-cold 90% methanol and incubated on ice for 30 min. One million permeabilized cells were washed twice with 0.5% BSA in PBS and re-suspended in the same buffer. Cells were subsequently incubated with rabbit anti-cleaved caspase-3 (ASP175) (5A1) mAb (Cell Signaling, Beverly, MA, USA) at room temperature for 30 min. Cells were then stained with Alexa488-conjugated anti-rabbit Ig antibody (Molecular Probes) at room temperature for 30 min. After incubation, cells were washed and re-suspended with 500 µl of 0.5% BSA in PBS. Flow cytometric analysis was performed on a Beckman Coulter EPICS ALTRA (Beckman Coulter, Fullerton, CA, USA).

Detection of cleaved caspase-3 by immunocytochemistry
U937 cells were incubated with CD147Ex phage for 48 h as described in the cytotoxicity assay. VCSM13 phage and cisplatin (Sigma–Aldrich) were included as controls. After 48 h incubation, cells were fixed for 10 min with 3.7% formaldehyde in PBS containing 50 mM MgCl2. Fifty microliters of 1 x 106 cells ml–1 fixed cells were placed on a silane-coated slide and air dried. The slides were fixed with 3.7% formaldehyde in PBS containing 50 mM MgCl2 for 10 min. Following washing, cells were permeabilized with 0.2% Triton-X 100 for 12 min. Slides were then washed in PBS containing 50 mM MgCl2 and blocked with 1% BSA in SSC at room temperature for 5 min. Then, the fixed cells were incubated with rabbit anti-cleaved caspase-3 (ASP175) (5A1) mAb (Cell Signaling) at 4°C overnight. After washing, cells were blocked and then incubated with Alexa680-conjugated goat anti-rabbit IgG antibody (Molecular Probes) at room temperature for 30 min. U937 nuclei were counterstained with DAPI. Image analysis was carried out using the Spectra Cube (Applied Spectral Imaging) on a Zeiss Axiophot 2 microscope (Zeiss) and the SKYVIEW 2.1.1 software (Applied Spectral Imaging). Spectral, classified and inverted images are generated as described previously.

Analysis of nuclear translocation of apoptosis-inducing factor by western blot
U937 cells were incubated for 24 h with CD147Ex phage as described previously. VCSM13 phage and phage-displayed SVV were used as controls. Nuclear and cytoplasmic fractions were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Perbio Science, France). Protein content was quantified by a modified Lowry assay. Eighty micrograms of total protein from cell lysates were resolved on 12.5% SDS-PAGE under reducing conditions, and then electroblotted onto a polyvinylidene fluoride membrane (PALL, East Hills, NY, USA). The membrane was blocked at room temperature for 2 h in 5% skimmed milk in Tris-buffered saline (TBS) pH 7.6, and then incubated with rabbit anti-apoptosis-inducing factor (AIF) antibody (Cell Signaling), which recognizes both a precursor form of AIF at 67 kDa and a mature form of AIF at 57 kDa, at 4°C overnight on a shaking platform. On the following day, the blot was washed with 0.1% Tween 20 in TBS pH 7.6 and incubated with goat anti-rabbit Ig–HRP at room temperature for 1 h. After washing, the immunoreactive bands were then visualized by the chemiluminescent detection system (Amersham).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Multivalent CD147Ex phage binds to various cell types
Phage expressing multivalent CD147 ectodomain were produced and assayed for display of the CD147 molecules by sandwich ELISA using four separate anti-CD147 mAbs. Similar to our previous report (23), the CD147Ex phage was recognized by all these anti-CD147 mAbs (data not shown).

To study the interaction of CD147Ex with molecules on the cell surface of various cell types, CD147Ex phage was incubated with each cell type and binding was determined by flow cytometry. The CD147Ex phage bound to all tested human hemotopoietic cell lines including U937, Daudi, Jurkat, Molt4, KG1a and HUT78 as well as BW5147 mouse thymoma cells (Fig. 1). A population of peripheral blood lymphocytes was also shown to exhibit positive reactivity (Fig. 1). In contrast, wild-type phages did not bind to any cell line tested. To confirm the flow cytometric results by a different method, U937 cells were incubated with CD147Ex phages and control phages for 24 h, stained with anti-gpVIII mAb and followed by goat anti-mouse Ig–Alexa680. Binding of CD147Ex phage on U937 cells was also demonstrated by immunocytochemistry (Fig. 2). Interestingly, the CD147Ex phage-binding cells harbored apoptotic bodies (Fig. 2B, arrow).


Figure 1
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  		Fig. 1 Binding of CD147Ex phages on various cell types. The cells were incubated with CD147Ex phages (solid lines) and VCSM13 control phages (dotted lines). Cell lines or PBMCs were stained with anti-gpVIII mAb and FITC-conjugated anti-mouse Ig antibody. Peripheral blood lymphocytes were gated according to their size and granularity and analyzed for phage binding. The figure shows the percentages of CD147 phage bound cells. Results from one representative experiment of three are shown.

 

Figure 2
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  		Fig. 2 Detection of CD147Ex phage binding on U937 cells by immunocytochemistry. U937 cells were incubated with recombinant phages or wild-type phages for 24 h, stained with anti-gpVIII mAb followed by goat anti-mouse Ig–Alexa680. The nuclear morphology was verified by DAPI staining. A and D represent spectral images, B and E represent inverted DAPI and C and F represent classified images in which Alexa680 is designated in red. Black arrow shows the apoptotic bodies and white arrow indicates the binding of CD147Ex phages on U937 cells.

 
CD147Ex phage induces morphological changes and inhibits growth of U937 cells
We tested the effects of CD147Ex phages and controls on a variety of cell lines. These included U937, Jurkat, Daudi and Molt4. Of these tested cell lines, only U937 cells exhibited morphological changes after incubation with CD147Ex phage. Morphological characteristics of apoptotic cell death such as cytoplasmic blebbing and nuclear fragmentation (25) were observed after 24 h of incubation with CD147Ex phages, but not with control phages (Fig. 3A, arrows and Fig. 3B and C, respectively). After a 48 h of incubation, the cell density of U937 cells in wells containing CD147Ex phage (Fig. 3D) was noticeably lower than in wells containing wild-type phages or phage-displayed irrelevant protein (Fig. 3E and F). The U937 cell concentration at the beginning of the experiment was 3 x 105 cells ml–1. By 48 h, the CD147Ex phage-incubated cells remained at a similar concentration, 2.9 x 105 ± 0.7 x 105, while the wild-type- and irrelevant protein expressing phage-incubated cells increased to 9.5 x 105 ± 0.4 x 105 and 8.4 x 105 ± 0.8 x 105 cells ml–1, respectively.


Figure 3
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  		Fig. 3 CD147Ex phages induced morphological change and growth arrest of U937 cells. U937 cells were incubated with (A) CD147Ex phages, (B) VCSM13 phages or (C) phage-displayed green fluorescent protein (GFP) and observed under an inverted microscope (200x). Morphological change of U937 cells was observed after 24 h incubation (arrows). The cell density of U937 cells in wells containing (D) CD147Ex phages was significantly lower than those in the presence of (E) VCSM13 phages and (F) phage-displayed GFP after 48 h of incubation (100x). Morphological change of CD147Ex phage-incubated U937 cells at 24 h of incubation was substantially reduced when pre-incubated CD147Ex phages with anti-CD147 mAbs, (E) M6-1D4 and (F) M6-2F9.

 
To demonstrate the effect of CD147Ex displayed on phage particles in triggering cell death, anti-CD147 mAbs, M6-1D4 and M6-2F9 were pre-incubated with CD147Ex phages before incubating with U937 cells. Morphological changes of U937 cells were significantly decreased compared with those induced by CD147Ex phages in the absence of CD147 mAb (Fig. 3G and H).

Cytotoxic effect of CD147Ex phage on U937 cells
Since the morphological changes of CD147Ex phage-incubated U937 cells suggested that cell death had occurred, vitality and cytotoxicity staining was performed. In the method employed, intracellular esterase activity in live cells converts the non-fluorescent cell-permeable calcein AM to the intensely fluorescent calcein, which is retained within live cells. Ethidium homodimer (EthD-1) enters the permeabilized plasma membranes of dead cells and undergoes a 40-fold increase in red fluorescence after binding to nucleic acids. Therefore, the nuclei of the dead cells appear red under the fluorescence microscope. Thus, cells with an active metabolism (calcein positive) that allow EthD-1 to penetrate and stain their nucleic acids most likely correspond to late-state apoptosis (26). We found double staining with calcein and EthD-1 in many recombinant phage-incubated U937 cells (Fig. 4A–C). In contrast, wild-type phage-incubated U937 cells were positive only for calcein (Fig. 4D–F). One hundred cells were examined in three dependent experiments for the fluorescence intensity of calcein and EthD-1. Mean EthD-1 fluorescence intensity of U937 cells when incubated with multivalent CD147Ex phages was 4.4 times higher than that with wild-type phages, while the mean calcein fluorescence intensity from both conditions were not significantly different.


Figure 4
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  		Fig. 4 Cytotoxicity of CD147Ex phages on U937 cells. U937 cells were incubated with (A–C) CD147Ex phages or (D–F) VCSM13 phages for 48 h and stained with LIVE/DEAD Viability/Cytotoxicity reagent. Green arrows indicate live cells and pink arrows represent dead cells. Results are representative of three separate experiments.

 
CD147Ex phage induces apoptosis of U937 cells
To confirm that the death of multivalent CD147Ex phage-incubated U937 cells is due to apoptosis, the annexin V assay was employed. In the presence of CD147Ex phages, more annexin V-positive cells were observed compared with un-induced and those treated with the wild-type phage and phage-displayed irrelevant protein (Fig. 5). These results suggested that multivalent CD147Ex phage induces apoptotic cell death in U937 cells (27–29).


Figure 5
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  		Fig. 5 Annexin V assay of CD147Ex phage-induced U937 cells. U937 cells were incubated with VCSM13 phages, phage-displayed SVV, CD147Ex phages or cisplatin for 48 h. Cells were incubated with annexin V–FITC and PI and analyzed by flow cytometry. Viable U937 cells are negative for both annexin V and PI; early apoptotic U937 cells are positive for annexin V while being negative for PI; late apoptotic U937 cells are positive for both annexin V and PI and necrotic U937 cells are positive for PI while being negative for annexin V (27–29).

 
Involvement of caspase-3 activation in the mechanism of cell death of U937 cells incubated with CD147Ex phages
To assess the mechanism of apoptotic cell death in CD147Ex phage-incubated U937 cells, cleaved caspase-3 was analyzed by flow cytometry. The level of cleaved caspase-3 observed in recombinant phage-incubated U937 cells (Fig. 6, thin line) was higher than wild-type phage-treated U937 cells (Fig. 6, filled histogram). However, the level of cleaved caspase-3 induced by CD147 phage was not as high as that triggered by cisplatin (Fig. 6, thick line). There was no difference in the level of cleaved caspase-3 between wild-type phage-induced U937 cells and un-induced U937 cells (data not shown).


Figure 6
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  		Fig. 6 CD147Ex phage-induced U937 cell death resulted in caspase-3 activation. U937 cells were incubated with CD147Ex phages (thin line), VCSM13 control phages (filled histogram) or cisplatin (thick line) for 48 h. Cleaved caspase-3 was examined by flow cytometry after intracellular staining with rabbit anti-cleaved caspase-3 mAb. Results are representative of two separate experiments.

 
The intracellular cleaved caspase-3 assay was also determined by immunocytochemistry. Un-treated U937 cells and wild-type phage-treated U937 cells were negative for cleaved caspase-3, whereas induction of U937 cells by cisplatin and CD147Ex phage resulted in positive staining (Fig. 7). We noted that of the CD147Ex phage-treated cells that had apoptotic nuclei, some were positive (Fig. 7L) and some were negative (Fig. 7O) for anti-cleaved caspase-3, whereas all cisplatin-treated cells with apoptotic nuclei were positive for anti-cleaved caspase-3.


Figure 7
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  		Fig. 7 Apoptotic cell death of U937 cells in CD147Ex phage induction showed both positive and negative of cleaved caspase-3. U937 cells were incubated with RPMI 1640, VCSM13 phage, cisplatin or CD147Ex phages. Cleaved caspase-3 was determined by intracellular staining using rabbit anti-cleaved caspase-3 mAb and goat anti-rabbit IgG–Alexa680. A, D, G, J and M represent spectral images; B, D, H, K and N represent inverted DAPI images and C, F, I, L and O represent classified images, in which Alexa680 is designated in red. Arrows indicate cleaved caspase-3.

 
AIF is not involved in the death of U937 cells after incubation with CD147Ex phage
AIF is a mitochondrial protein that, upon mitochondrial transmembrane potential ({Delta}{Psi}m) loss, is released from mitochondria and translocated into the nucleus (30). To determine the involvement of AIF in the induction of nuclear apoptosis of CD147Ex phage-treated U937 cells, nuclear and cytoplasmic fractions of CD147Ex phage-treated as well as VCSM13 phage-treated and phage-displayed SVV-treated U937 cells were separated and the western blot analysis was carried out to detect AIF. In un-induced cells, AIF was present primarily as its 67-kDa proform and processed to a 57-kDa mature form when caspase-independent cell death is activated. Nuclear translocation of the 57-kDa AIF did not appear to increase in CD147Ex phage-treated cells after 24 h incubation (Fig. 8). In contrast, it appeared to be less than in the un-induced cells (Fig. 8).


Figure 8
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  		Fig. 8 Analysis for nuclear translocation of AIF in CD147Ex phage-treated U937 cells. U937 cells were incubated with RPMI 1640 (un-induced), VCSM13 phages, phage-displayed SVV or CD147Ex phages for 24 h. Nuclear (N) and cytoplasmic proteins (C) were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents. Western blot analysis was performed as described in Methods by using the antibody against AIF.

 
Correlation between binding of CD147Ex phage to U937 cells and apoptotic nuclei
The binding of CD147Ex phage to individual U937 cells that harbor apoptotic nuclei was examined by immunocytochemistry. Recombinant phage adhering to individual U937 cells was visualized on classified image which is shown in red (Fig. 2A–C). Sixty percent of CD147Ex phage-incubated U937 cells were positively stained with anti-gpVIII mAb, while 42% of CD147Ex phage-incubated U937 cells had apoptotic nuclei. In contrast, anti-gpVIII mAb did not bind to wild-type phage-incubated U937 cells (Fig. 2D–F).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
After the discovery of the CD147 protein, there was no concrete evidence linking CD147 with the triggering of membrane bound molecules. Although recombinant CD147 could be expressed on the surface of other cell types in vitro, this was not suitable for studying CD147 function in triggering its cell-surface ligands and leads the signal into the cells, as other pairs of membrane protein interactions may counteract or interfere with the signal originating from CD147 induction. To overcome this problem, phage display technology is introduced for this purpose. Hence, we previously generated phage-displayed CD147Ex via gpIII and used it for studying the CD147 function and characterization of CD147 ligands (21). However, the binding of these recombinant phages to cells could not be detected by immunofluorescence. This is most likely due to the level of heterologous CD147 proteins displayed on phage gpIII being restricted to less than one molecule per phage particle (22). High levels of heterologous protein display can be achieved on gpVIII, as each phage particle contains ~2000 copies of the protein (31, 32). Moreover, multivalent CD147 molecules expressed on the 930-nm length filamentous phage might overcome the limitation of CD147 binding to low levels of its ligands on the cell surface. Thus, we decided to generate phage that displayed multivalent CD147Ex on its surface (23). With this new approach, the present study demonstrated the binding of multivalent CD147Ex phages to various cell types and a new function of CD147 has been uncovered for U937 cells.

The physical association between CD147 and ß-1 integrin in the membrane has been reported (33, 34). Berditchevski et al. (33) suggested that association of {alpha}3ß1 integrin and {alpha}6ß1 integrin with CD147 is in lateral fashion, similar to the interaction of ß-3 integrins with CD47, another Ig superfamily protein (35, 36). It has been reported that CD147 also serves as a signaling receptor for extracellular cyclophilin A (37–39). Recently, a cyclophilin-binding region in the CD147 molecule has been identified as the transmembrane domain (40). The proline residue located at position 211 in the transmembrane domain was the critical residue responsible for intracellular interaction of cyclophilins and CD147 (40). Interaction between lactate transporter, MCT1 and CD147 has also been reported (41, 42). Association between MCT1 and fusion protein of extracellular domain of CD2 and transmembrane or cytoplasmic domains of CD147 suggested that transmembrane and cytoplasmic domains of CD147 are important for lateral interaction between MCT and CD147 (41). None of these studies have reported interactions of the CD147 external domain with any cell-surface molecule. With our novel approach, only the external domain of CD147 is expressed on phage particles; thus, it is unlikely that integrins, cyclophilins or MCTs are the CD147 ligand. Recently, the possibility that CD147 could be an autoreceptor has been suggested (18). The first extracellular Ig domain of CD147, which contains a glycosylated site, is required for homophilic counter-receptor binding activity (18, 43). In contrast, the CD147 displayed on phage surface was in non-glycosylated form. Thus, it should not interact with the naive CD147 on the cell membrane. This finding suggests further study of the interaction of CD147 with its surface ligand conveying the molecular signals.

Using the tool we developed in this study, we were able to show that triggering U937 cells with multivalent CD147Ex phages resulted in morphological changes, that is, apoptotic nuclei, in contrast to other cell types. Engagement of cell-surface receptors by multiple ligands might be necessary for signaling. A similar phenomenon has been reported in a study that showed IgE dimers were less effective than larger IgE oligomers in stimulating cellular responses (44). Dependency of cellular responses on receptor aggregates also has been observed for related receptors, such as the B cell receptor (45, 46) and TCR (47, 48). Thus, signal transduction by individual binding of monovalent CD147 may differ from the cross-linking of cell-surface ligands with multivalent phage carrying CD147Ex. Herein, the interaction of multivalent phage displaying CD147Ex and its counter-receptors on U937 cells mediated the morphological changes characteristic of apoptotic cell death. In addition, pre-incubation of CD147Ex phages with anti-CD147 mAbs, M6-1D4 and M6-2F9, significantly reduced the death of U937 cells compared with U937 cells induced by CD147Ex phage only. These mAbs may hinder the epitope that play an important role in death induction of U937 cells. Thus, interactions between CD147Ex on the phage particle and its ligands on U937 cells might consequently lead to death signals. The death of multivalent CD147Ex phage-incubated U937 cells did not result from chemical toxicity of the phage preparation because this phenomenon was not observed when using wild-type phage or phage expressing irrelevant protein. In addition, cell death was specifically observed in U937 cells but not in other tested cell lines. In addition to multivalent CD147Ex phage-induced apoptotic morphological change of U937 cells, growth arrest of U937 cells was observed after 48 h of incubation.

The morphological features indicating apoptotic cell death, that is, double staining with calcein and EthD-1 and double staining with annexin V and PI of U937 cells incubated with the multivalent CD147Ex phage, suggest a possible involvement of caspase activation. To address this question, the presence of cleaved caspase-3 in U937 cells incubated with multivalent CD147 phage was examined by flow cytometry and immunocytochemistry. The level of cleaved caspase-3 in U937 cells incubated with multivalent CD147Ex phages was higher than in un-induced or wild-type phage-incubated U937 cells. However, it was not as remarkable as levels of cleaved caspase-3 seen in cisplatin-induced U937 cells. When individual U937 cells were analyzed by immunocytochemistry, the percentage of recombinant phage-induced U937 cells harboring apoptotic nuclei was 74.3%, but with only 12.8% containing high level of cleaved caspase-3. Therefore, it is possible that the apoptotic cell death of CD147Ex phage-incubated U937 cells is partially explained by an enhancement in cleaved caspase-3 level. The reasons for this differential reaction of U937 cells to CD147Ex phage are currently unknown. This may be resulted from different kinetics of apoptotic induction and/or other pathways of program cell death (PCD). Initiation of caspase-dependent and -independent cell death in response to a given stimulus has been recently reported (49, 50). Triggering of HLA class I molecules on Jurkat T lymphoblasts results in apoptotic cell death induction by two parallel pathways, caspase-dependent and -independent pathways (50). According to current knowledge, one central molecule in caspase-independent cell death is AIF (30). Upon induction of apoptosis, AIF translocates from mitochondria to the nucleus and results in DNA fragmentation and marginal chromatin condensation (30). Thus, analysis of AIF localization in both nuclear and cytoplasmic fractions of U937 cells by western blot was performed earlier than caspase-3 analysis. In this study, we showed that nuclear translocation of AIF in CD147Ex phage-treated U937 cells was not detected, which indicates that a caspase-independent pathway via AIF is not involved in cell death mediated by multivalent CD147. Since caspase-independent pathway is not involved in this study, alternative pathways of PCD may play roles in cleaved caspase-3 negative apoptotic cell death. This may be apoptosis-like or necrosis-like cell death as described for other molecules, that is, bNIP3 (51–53). The cell death resembled apoptosis, as evidenced by outer membranes phosphatidyl serine exposure and apoptotic nuclei, but was cleaved caspase-3 as well as AIF independent. Therefore, it is suggested by our study that the caspase-dependent pathway may play a major role of apoptotic cell death in U937 cells after incubation with CD147Ex phage.

Under physiological conditions, interaction of CD147 with its counter-receptor may have a very low affinity with an extremely fast dissociation rate, as has been described for a variety of cell adhesion receptors, such as CD2 with its counter-receptors CD48 and CD58 (54, 55). The functional consequences of the physical association of these cell adhesion molecules with their ligands may also require high polyvalent ligands that are regulated by their expression levels. Thus, it is tempting to speculate that the fast and strong up-regulation and patching of CD147 upon activation of T cells (6, 56) controls its interaction with its counter-receptor. Following CD147 engagement, firm cell contact was established by ß-1 and ß-2 integrins, which were characterized as physical and functional partners of CD147 (13, 33, 34). Evidence for CD147 as a ß-2 integrin amplifier is given by the enhanced adhesiveness of leukocyte function-associated antigen-1 upon CD147 triggering by mAbs (13). The cooperation between CD147 and integrins is conserved among species as lack of CD147 impairs the integrin-dependent cellular architecture of Drosophila cells (57). Of physiological relevance, we suggest that CD147 may play a fundamental role in the negative regulation of immune responses (17, 58) by induction of apoptosis in the target cells expressed its counter-receptor. This is also consistent with the finding that lymphocytes isolated from CD147–/– knockout mice better proliferate in a mixed lymphocyte reaction than those observed in lymphocytes derived from wild-type mice (59).

In conclusion, we introduce here a useful strategy, applying the phage display technique to study the interaction of cell-surface molecules. A multivalent CD147Ex phage was generated and used in a functional study of the CD147 molecule. The results clearly demonstrated that a CD147 ligand exists on various cell types. A function of CD147 in triggering apoptotic cell death in U937 cells was identified. Since this phenomenon specifically occurred with U937 cells, which is a monocytic cell line, the involvement of CD147 in immune regulation of specific lineage and developmental stages will be the focus of future studies.


   Acknowledgements
 
We gratefully acknowledge Edward Rector for his kind guidance in detection of cleaved caspase-3 by flow cytometry. We are thankful to Sawitree Chiampanichayakul, Umpa Yasamut, Khajornsak Tragoolpua and Wutigri Nimlamool for their technical assistances and Mark E. Peeples for his helpful discussions and critical reading of the manuscript. We are especially grateful to Suttichai Krisanaprakornkit for valuable suggestion in detection of AIF by western blot. This work was supported by the scholarship under the Commission on Higher Education Thai Staff, the Thailand Research Fund and the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand and Canadian Institutes of Health Research Strategic Training Program ‘Innovative Technologies in Multidisciplinary Health Research Training’, Canada.


   Abbreviations
 
AIF, apoptosis-inducing factor
CD147Ex, CD147 extracellular domain
CD147Ex phage, phage containing fusion protein of CD147Ex and gpVIII
DAPI, 4'6-diamidino-2-phenylindole
EthD-1, ethidium homodimer
FBS, fetal bovine serum
MMP, matrix metalloproteinase
PCD, program cell death
PI, propidium iodide
SSC, saline sodium citrate
SVV, survivin
TBS, Tris-buffered saline

   Notes
 
Transmitting editor: D. Wallach

Received 13 July 2005, accepted 27 April 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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