- Research article
- Open Access
Increased levels of soluble CD226 in sera accompanied by decreased membrane CD226 expression on peripheral blood mononuclear cells from cancer patients
- Zhuwei Xu†1,
- Tao Zhang†2,
- Ran Zhuang†1,
- Yun Zhang†1,
- Wei Jia1,
- Chaojun Song1,
- Kun Yang1,
- Angang Yang1 and
- Boquan Jin1Email author
© Xu et al; licensee BioMed Central Ltd. 2009
- Received: 23 April 2009
- Accepted: 02 June 2009
- Published: 02 June 2009
As a cellular membrane triggering receptor, CD226 is involved in the NK cell- or CTL-mediated lysis of tumor cells of different origin, including freshly isolated tumor cells and tumor cell lines. Here, we evaluated soluble CD226 (sCD226) levels in sera, and membrane CD226 (mCD226) expression on peripheral blood mononuclear cells (PBMC) from cancer patients as well as normal subjects, and demonstrated the possible function and origin of the altered sCD226, which may provide useful information for understanding the mechanisms of tumor escape and for immunodiagnosis and immunotherapy.
Soluble CD226 levels in serum samples from cancer patients were significantly higher than those in healthy individuals (P < 0.001), while cancer patients exhibited lower PBMC mCD226 expression than healthy individuals (P < 0.001). CD226-Fc fusion protein could significantly inhibit the cytotoxicity of NK cells against K562 cells in a dose-dependent manner. Furthermore, three kinds of protease inhibitors could notably increase mCD226 expression on PMA-stimulated PBMCs and Jurkat cells with a decrease in the sCD226 level in the cell culture supernatant.
These findings suggest that sCD226 might be shed from cell membranes by certain proteases, and, further, sCD226 may be used as a predictor for monitoring cancer, and more important, a possible immunotherapy target, which may be useful in clinical application.
- Peripheral Blood Mononuclear Cell
- K562 Cell
- Jurkat Cell
- Soluble CD226
- sCD226 Level
CD226, also named platelet and T cell antigen 1 (PTA1) or DNAX accessory molecule-1 (DNAM-1), is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. The CD226 molecule is mainly expressed on NK cells, T cells, NK T cells, and platelets, and is involved in cytotoxicity and cytokine secretion of T cells and NK cells and in platelet aggregation and activation [1–4]. It is highly conserved among human, gibbon, monkey, and mouse, suggesting this molecule may have important biological functions . Recently, CD226 has been identified as a receptor for CD112 and CD155 , and ligation of CD226 and leukocyte function-associated antigen-1 (LFA-1) with their respective ligands cooperates in triggering cytotoxicity and cytokine secretion by T and NK cells .
Recently, more attention has been paid to a putative role for CD226 in tumor development. The specific interaction between CD226 (on NK cells) and CD155 or CD112 (on tumor cells) plays an important role in the NK-mediated lysis of tumor cells [8–11]. Based on that, CD226 is thought to be one of the major activating NK receptors [12, 13] and involved in tumor immunosurveillance . Moreover, the abnormal expression of CD226 and its ligands in some tumors may be involved in the mechanisms of tumor escape, invasion, and migration [15, 16]. Although soluble CD155 has been detected in human serum and cerebrospinal fluid  and is anticipated to be one of the mechanisms of tumor immune escape , there has been no report on soluble CD226 (sCD226) or the levels of membrane CD226 (mCD226) and their relationship in tumors. Here, based on the mAbs and ELISA system established by our laboratory , we describe increased sCD226 levels in sera and lower mCD226 expression on PBMC from cancer patients compared with those of normal subjects, and demonstrate the possible function and origin of the increased sCD226. These findings may provide useful information for understanding the mechanisms of tumor escape and for immunodiagnosis.
Elevated sCD226 levels in sera and reduced mCD226 expression on PBMC in cancer patients
Inhibitory effect of CD226-Fc fusion protein on the cytotoxicity of normal PBMC against K562 cells
Regulation of sCD226 and mCD226 by protease inhibitors
Characterization of the sCD226
We have revealed that the sCD226 levels in sera of cancer patients is significantly enhanced compared with that in normal subjects, while there is a concomitant reduction in mCD226 expression on PBMC isolated from cancer patients. CD226-Fc fusion protein can notably inhibit the cytotoxicity of normal PBMC against K562 cells, indicating that the increased sCD226 might be one of the immune escape strategies used by tumor cells. Three protease inhibitors can decrease sCD226 level and increase mCD226 expression of PMA-stimulated PBMC and Jurkat cells, indicating that the increased serum sCD226 in cancer patients might be shed from the cell surface by several proteases, which are often up-regulated when tumor occurs. These findings suggest that sCD226 might be used as a predictor for monitoring cancer, and more important, a possible immunotherapy target, which may be useful in clinical application.
Patients' and control sera
Samples of patients' sera were obtained from 259 patients with various kinds of tumors, before treatment, within 1 week after entrance to Xijing Hospital, the Fourth Military Medical University. The series included a total of 13 esophageal carcinoma (7 male and 6 female), 45 gastric carcinoma (25 male and 20 female), 24 colorectal carcinoma (11 male and 13 female), 23 liver cancer (13 male and 10 female), 16 breast cancer (female), 15 cervix/ovarian/endometrial cancer (female), 46 lung cancer (28 male and 18 female), 28 leukemia/lymphoma (14 male and 14 female), and 49 others (including tumor in central nervous system, in kidney, in bladder, in parotid gland, in thyroid gland, in submaxilary gland, in suprarenal gland, in prostate gland, cholangiocarcinoma, melanoma, nasopharyngeal carcinoma, laryngocarcinoma, soft tissue sarcoma, and osteogenic sarcoma). Normal sera were isolated from 129 healthy volunteers (69 male and 60 female). Both the patients and the volunteers were 25–65 years old. They had no chemotherapy and had no significant signs or symptoms of microbial infection at the time of entrance. This study was approved by the institutional review board of the University and all individuals provided written and/or oral informed consent. The sera were stored at -20°C until use.
100 μL of the anti-CD226 mAb, LeoA1  (2.5 mg/L in 0.05 M sodium carbonate buffer, pH9.5) was added to each well of an ELISA plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. After three washes, serum samples or standard CD226 serially diluted with PBS containing 1% BSA and 0.1% (v/v) Tween-20 were added to the wells and incubated for 1 h at 37°C. After extensive washing with PBS containing 0.1% (v/v) Tween-20 (PBS/Tween), the wells were reacted with another anti-CD226 mAb  which was conjugated with HRP and diluted in PBS containing 3% PEG for 1 h at 37°C. Color development was performed by adding 100 μL TMB (eBioscience, CA, USA) for 10 min at 37°C and stopped by 2 M H2SO4. The absorbance at 450 nm was determined with a microplate reader (BioRad, CA, USA).
Flow cytometry analysis
PBMC were freshly isolated from peripheral blood of healthy adults or cancer patients by Isopaque-Ficoll (Hao Yang, Tianjin, China) gradient centrifugation and incubated with LeoA1 or control IgG1 (BD BioSciences, CA, USA) at 4°C for 30 min, followed by washing and incubation with FITC-labeled goat anti-mouse Ig (Dako, Glostrup, Denmark) at 4°C for 30 min and then analyzed on a FACScan (BD BioSciences). Expression of CD155 and CD112 on K562 cells were analyzed similarly.
Cell culture and stimulation
Freshly isolated normal PBMC or Jurkat cells were cultured in RPMI 1640 (Hyclone, UT, USA) supplemented with 10% FCS, 100 U/mL penicillin/streptomycin, 10 mM HEPES, and 50 μM β-mercaptoethanol at 37°C in 5% CO2. The cells (1×106 cells/mL) were stimulated with PMA (50 ng/mL, Sigma-Aldrich, MO, USA) for 6 h, washed twice, and co-cultured with or without different protease inhibitors (Sigma-Aldrich, INHIB-1 and 131377) for 6 h. Although the concentration of the 15 protease inhibitors was not all the same, the optimized concentration for 3 effective protease inhibitors (1–10-Phenanathroline, AEBSF, and N-Ethylmaleimide) was all 1 mM. Then sCD226 level in the supernatants was detected by the ELISA mentioned above, and mCD226 expression on the cells was analyzed by flow cytometry.
Characterization of molecular weight of sCD226
Twelve ml of serum from cancer patients and healthy adults, respectively, and 30 ml of culture supernatant from normal PBMC and Jurkat cells treated with PMA (50 ng/mL) for 24 h were used to characterize the molecular weight of sCD226 by immunoprecipitaion and Western blot analysis. Briefly, LeoA1 or normal mouse IgG was covalently coupled to CNBr-activated Sepharose 4B (GE Healthcare, London, the United Kingdom) according to the manufacturer's instructions. After preclearing, the sera or supernatant samples were aliquoted and incubated with 100 μL of LeoA1-Sepharose 4B or normal mouse IgG-Sepharose 4B (negative control for immunoprecipitation) at 4°C overnight. After 4 times washing (20 min incubation for each washing) with Washing Buffer (10 mMTris-HCl, 140 mM NaCl, 0.5 mM MgCL2, 0.5 mM CaCL2, 0.02% NaN3, pH7.4) and following the last centrifugation, the supernatant was aspirated and 40 μl of 2 × loading buffer was added to the bead pellet, vortexed, heated (90–100°C, 5 min), and centrifuged. The supernatant was gently collected, without disturbing the pellet, and then loaded onto 12% SDS-PAGE as the order in Figure 4, and transferred to one complete nitrocellulose membrane (Millipore, Massachussets, USA). The membrane was blocked with 5% (w/v) bovine skim milk in PBS at room temperature for 1 h, then was cut into individual lanes using scissors, and incubated with anti-SED mAb (negative control for Western blot) or anti-CD226 mAb FMU4  at 4°C overnight. Then the membranes were washes six times with PBS-T and incubated with HRP labeled goat anti-mouse IgG secondary antibody (DAKO) at room temperature for 1 h. After four times washes with PBS-T, the individual membrane lanes were put together one by one and enhanced chemiluminescence (ECL) reagent (GE Healthcare) was applied to the membranes according to manufacturer's introductions, which were then exposed to an x-ray film (Kodak, Rochester, NY, USA).
The Mann-Whitney U test or Kruskal-Wallis test was used to determine the significance of the differences of sCD226 and mCD226 expression between the groups. And the paired-samples T test was used to compare percentage of cytotoxicity between the groups. A P value < 0.05 was regarded as significant. Analysis was performed with SPSS (SPSS Inc., Chicago, IL, USA).
This study was supported by National Natural Science Foundation of China (No. 30500219 and No. 30801003) to Y.Z. and R.Z., respectively.
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