bDLE was produced by the Laboratory of Immunology and Virology, Faculty of Biological Sciences, University Autonomous of Neuvo León (UANL) (San Nicolás de los Garza, Mexico). bDLE is a mixture of low-molecular weight substances (cut-off of 10–12 kDa) obtained from the dialysis of disintegrated bovine spleens in water, subsequently lyophilized and determined to be free of pyrogens using the Limulus amoebocyte lysate assay (Endotoxin Detection kit; MP Biomedicals, LLC, Santa Ana, CA, USA), and confirmed to be free of bacterial contamination by culturing in various culture media as well as in vivo mouse inoculation. bDLE obtained from 75×10⁸ leukocytes is defined as five units (5 U). For the subsequent assays, bDLE was suspended in RPMI-1640 (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The suspension was filtered with a 0.2 µm-diameter filter (EMD Milipore, Billerica, MA, USA).

The K562 cell line was originally established from the pleural effusions of a patient with CML in terminal blast crisis. The cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.), at 37°C in a humidified incubator with 5% CO₂. To determine the cytotoxic effect and induction of cell differentiation by bDLE in K562 cells, cells were seeded onto 6-well plates at a density of 1×10⁵ cells/well and treated with bDLE (0.07, 0.14, 0.21, 0.28, 0.35, 0.5, 0.75 and 1 U/ml). PMA (10 ng/ml; Sigma-Aldrich; EMD Millipore) and dimethyl sulfoxide (DMSO;1.5% v:v; Sigma-Aldrich; EMD Millipore) were used as positive controls for the induction of cell differentiation in the K562 cell line. All treatments (bDLE, PMA and DMSO) were suspended in RPMI-1640 medium supplemented with 10% FBS, and plates were incubated for 96 h at 37°C in a 5% CO₂ atmosphere. After trypsinization, adherent and non-adherent cells were collected. The MOLT-3 (T acute lymphoblastic leukemia) cell line was obtained from American Type Culture Collection and was used as a cytotoxic control of the T cell lineage.

A total of 5 male BALB/c mice (6 weeks old; 24–26 g) were obtained from the Laboratory of Immunology and Virology of the University Autonomous of Nuevo León) and maintained in a controlled environment at 25°C (12 h light/dark cycles) with free access to food and water. The mice were sacrificed by cervical dislocation, and resident peritoneal macrophages were obtained through repeated sterile ice-cold RMPI-1640 lavages within the peritoneal cavity, according to protocols approved by the Institutional Animal Care and Use Committee of the Laboratory of Immunology and Virology of the University Autonomous of Nuevo León. Furthermore, human monocytes were isolated from peripheral blood obtained from normal donors, the blood was diluted with PBS at a ratio of 1:1 (vol/vol) and subsequently the blood was centrifuged on a Ficoll-Paque gradient for 30 min at 500 × g at room temperature. The interphase layer consisted of peripheral blood mononuclear cells and was washed three times with culture medium. Murine peritoneal macrophage and human monocyte cells (2×10⁶ cells/ml) were seeded in 6-well plates and incubated at 37°C in an atmosphere of 5% CO₂ for 3 h to allow cell adherence to the plastic. Subsequently, the non-adherent cells were removed, and adherent cells were obtained by trypsinization. Cell supernatants were centrifuged at 500 × g at 25°C for 10 min and seeded at 1×10⁵ cells/well. Cells were treated with bDLE (0.5, 0.75 and 1 U/ml) and incubated at 37°C in a 5% CO₂ atmosphere for 96 h, followed by evaluation of cell death by propidium iodide (PI) staining.

To evaluate cell death, cells were centrifuged at 500 × g at 25°C for 10 min and washed with PBS, resuspended in 0.2 ml PBS and PI (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 min at room temperature (25°C) in the dark, followed by analysis with an Accuri™ C6 flow cytometer (BD Biosciences).

K562 cells previously treated for 96 h with bDLE, PMA or DMSO were harvested, centrifuged at 500 × g at 25°C for 10 min and resuspended in 0.2 ml PBS. Cell proliferation percentage was estimated by cell counting following trypan blue staining for each group. Furthermore, to assess the proliferative capacity of the differentiated cells, following 96 h of incubation with bDLE, PMA or DMSO, the cells were collected, centrifuged at 500 × g at 25°C for 10 min, washed with RPMI-1640 and seeded at 1×10⁵ cells/well. Cells were subsequently incubated at 37°C in an atmosphere of 5% CO₂ for 10 days, and the cell count was evaluated by trypan blue staining and proliferation was estimated.

Cell cycle progression analysis was performed with the Cycle Test™ Plus DNA Reagent kit (BD Biosciences) according to manufacturer's protocol. In brief, 1×10⁵ cells/well previously treated for 96 h (with bDLE, PMA or DMSO) were washed with PBS and fixed with 1 ml buffer solution (containing sodium citrate, sucrose and DMSO). Subsequently, 250 µl of solution A (containing trypsin in a spermine tetrahydrochloride detergent buffer) was added, and the samples were incubated for 10 min at room temperature. Following incubation, 200 µl of solution B (trypsin inhibitor and RNase buffer) was added, followed by the addition of 200 µl of cold (2–8°C) solution C (containing propidium iodide staining solution). Samples were gently mixed and incubated for 10 min in the dark on ice. Data acquisition was performed on a BD Accuri C6 flow cytometer (10,000 events were analyzed) and data analysis was performed using ModFit LT version 4.0 software (Verity Software, Inc., Topsham, ME USA).

To determine the effects of treatment on the size and morphological characteristics of K562 cells, the cells were harvested and fixed with 1% formaldehyde for 1 min at room temperature (20–25°C). Slides were dried, and Romanowsky staining solution was added, rinsed with deionized water, air dried and observed under a Nikon inverted light microscope (Eclipse TE300; Nikon Corporation, Tokyo, Japan) at magnification, ×40.

For assessment of monocytic differentiation induced by treatments, K562 cells were harvested and incubated for 30 min at room temperature simultaneously with phycoerythrin-conjugated anti-CD14 (cat. no. 340585; 1:20; Invitrogen; Thermo Fisher Scientific, Inc.), fluorescein isothiocyanate (FITC)-conjugated anti-CD68 (cat. no. 562117; 1:20; BD Biosciences) and allophycocyanin-conjugated anti-CD163 (cat. no. 556018; 1:20; BD Biosciences). To evaluate megakaryocytic lineage the cells were incubated with peridinin chlorophyll protein complex-conjugated anti-CD42a (cat. no. 340537; 1:20; BD Biosciences) in PBS with 1% FBS and 0.1% sodium azide for 30 min at 25°C. Following incubation, samples were washed and resuspended in PBS and 10,000 events were recorded by flow cytometry (BD Accuri C6 flow cytometer). The CD14⁺ cell population was gated and the CD68⁺ and CD163⁺ populations were evaluated.

K562 cells previously treated for 96 h (with bDLE, PMA or DMSO) and washed three times with PBS, were incubated with FITC-Dextran (0.1 mg/ml; molecular weight 70,000; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 60 min. As a negative control, cells were incubated with FITC-Dextran at 4°C for 60 min. To stop phagocytosis the cells were washed twice with cold PBS supplemented with 1% FBS. Phagocytosis was assessed by flow cytometry (10,000 events were analyzed) and quantified as a percentage of cellular uptake of FITC-Dextran.

The supernatants of each treatment were used to determine NO production using the nitrate-nitrite colorimetric assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's protocol. Briefly, 40 µl of supernatants were mixed with 40 µl of assay buffer, 10 µl of enzyme cofactor and 10 µl of nitrate reductase, and incubated at room temperature for 3 h (to allow the conversion of nitrate to nitrite). Following 10 min of incubation in Griess reagent at room temperature, the absorbance was measured at 560 nm in a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Cytokine production was analyzed using a BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines kit (BD Biosciences). For the evaluation, cell supernatants were harvested following 96 h incubation, and stored at −20°C until analysis according to manufacturer's protocol. The cytokines evaluated were interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α and the evaluation of chemokine production included: Chemokine (C-X-C motif) ligand (CXCL)8/IL-8, chemokine (C-C motif) ligand (CCL)5/regulated on activation, normal T cell expressed and secreted (RANTES), CXCL9/monokine induced by gamma interferon, CCL2/monocyte chemoattractant protein-1 (MCP-1) and CXCL10/interferon gamma-induced protein 10. Cytokine and chemokine production were measured by flow cytometry (BD Accuri C6) according to the manufacturer's protocol, and CBA analysis was performed using FCAP Array™ software version 1.0 (Soft Flow, Inc., St. Louis Park, MN, USA).

All experiments were performed in triplicate, and statistical analysis was performed using analysis of variance followed by post hoc Dunnett's tests. SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

bDLE decreased the viability of K562 [0.5 U/ml (33.0%), 0.75 U/ml (93.2%) and 1 U/ml (96.8%)] and MOLT-3 [0.21 U/ml (18.8%), 0.28 U/ml (62.0%), 0.35 U/ml (67.1%), 0.5 U/ml (79.8%), 0.75 U/ml (87.5%) and 1 U/ml (94.7%)] leukemia cell lines in a dose-dependent manner (P=0.03), demonstrating that bDLE treatment induced a highly cytotoxic effect on the MOLT-3 cell line (Figs. 1 and 2). The relative viability of the primary human monocytes and murine peritoneal macrophages treated with bDLE remained at 75–80% (Fig. 3). PMA treatment weakly affected the viability of K562 cells, and did not affect the MOLT-3 cell line (P=0.75). DMSO treatment did not affect the leukemia cell line viability (P=0.85; Figs. 1 and 2).

bDLE treatments (0.07–0.35 U/ml) induced alterations in cell cycle progression, as shown in Fig. 4 and Table I. bDLE caused S phase arrest at all doses tested [0.07 U/ml (57.28%), 0.14 U/ml (59.62%), 0.21 U/ml (64.45%), 0.28 U/ml (58.68%) and 0.35 U/ml (58.64%)]. PMA or DMSO treatments decreased the percentage of cells in the S phase (16.09 and 45.77%, respectively), compared with the untreated cells (52.34%). bDLE or PMA treatments increased the percentage of cells in the G₂/M phase [0.07 U/ml (5.11%), 0.14 U/ml (9.57%), 0.21 U/ml (8.19%), 0.28 U/ml (9.46%), 0.35 U/ml (8.27%) and PMA (33.07%)]. Untreated cells and cells treated with DMSO showed no affect on the percentage of cells in the G₂/M phase (3.24 and 4.81%, respectively). Furthermore, a significant decrease in the percentage of cells in the G₀/G₁ phase was detected following bDLE treatment [0.07 U/ml (37.61%), 0.14 U/ml (30.80%), 0.21 U/ml (27.36%), 0.28 U/ml (31.86%) and 0.35 U/ml (33.09%)] compared with PMA (50.84%), DMSO (49.42%) and untreated cells (44.43%). Furthermore, bDLE treatment for 96 h significantly inhibited the K562 cell proliferation rate in a dose-dependent manner (P=0.05) [0.07 U/ml (1,550,000 cells/ml), 0.14 U/ml (1,125,000 cells/ml), 0.21 U/ml (775,000 cells/ml), 0.28 U/ml (550,000 cells/ml) and 0.35 U/ml (400,000 cells/ml]. Treatment with PMA (75,000 cells/ml) and DMSO (1,000,000 cells/ml) obtained similar results when compared with untreated cells (1,650,000 cells/ml), in which the cellular proliferation was the highest (P=0.05) (Fig. 5). Subsequently, the K562 cells previously differentiated by treatments with bDLE, PMA or DMSO for 96 h were incubated for 10 days, and it was observed that the bDLE treatment decreased the rate of cell growth in a dose-dependent manner (P=0.03) compared with the control. The cellular growth was significantly decreased following PMA and DMSO treatment (P=0.005) compared with the control (Fig. 6).

Microscopic examination by Romanowsky staining in the K562 cells treated with bDLE (0.07–0.35 U/ml) for 96 h, revealed morphological changes characteristic of monocytic/macrophage differentiation occurring in a dose-dependent manner. The cells developed pseudopodia extensions, increased cell size and cytoplasm to nuclear ratio, as well as attachment to the culture dishes, compared with untreated cells. These morphological changes were similar to the effects obtained by PMA treatment in terms of induced monocyte/macrophage differentiation in K562 cells (Fig. 7). In order to confirm the monocytic/macrophage differentiation induced by the bDLE and inducer positive controls, the treated cells were monitored for expression of CD14⁺ monocytic marker. Flow cytometry histograms revealed an increased expression of CD14⁺ on K562 cells following treatment with bDLE in a dose-dependent manner: 0.07 U/ml (14.9%), 0.14 U/ml (24.4%), 0.21 U/ml (31.0%), 0.28 U/ml (35.2%) and 0.35 U/ml (39.7%), compared with the untreated cells that expressed 6.1%, DMSO (3.3%) or PMA (29.6%) treatments (Fig. 8). When the CD14⁺ cell population was gated the surface markers CD68⁺ (M1-like phenotype) and CD163⁺ (M2-like phenotype), characteristic of macrophage polarization, were analyzed, it was determined that bDLE treatment at various doses induced cells toward an M2-like phenotype, increasing the CD163⁺ surface marker levels in a significant manner (P=0.05) [0.07 U/ml (33.0%), 0.14 U/ml (47.6%), 0.21 U/ml (50.5%), 0.28 U/ml (51.7%), 0.35 U/ml (49.5%) compared with the untreated cells (12.0%), PMA (26.6%) or DMSO (8.7%) treatments]. No difference (P=0.15) was identified between treatments when the CD68⁺ surface marker was evaluated. The double positive population of CD68⁺/CD163⁺ increased slightly with bDLE treatment at doses of 0.07 U/ml (4.2%), 0.14 U/ml (7.1%), 0.21 U/ml (4.8%), 0.28 U/ml (4.1%) and 0.35 U/ml (5.3%), with high levels of expression following PMA treatment (27.5%), and no effect on the percentage of marker-positive cells following DMSO treatment compared with untreated cells (0.3%) (P=0.35) (Fig. 9). It is well-known that differentiated macrophages possess increased phagocytic capacity (11). The present study observed that the bDLE treatments (0.07–0.35 U/ml) significantly increased (P=0.05) in a dose-dependent manner the ability to uptake FITC-Dextran reagent: 0.07 U/ml (13.7%), 0.14 U/ml (17.7%), 0.21 U/ml (21.3%), 0.28 U/ml (31.4%) and 0.35 U/ml (38.3%), similar to PMA treatment (32.7%), when compared with untreated cells (5.3%). DMSO treatment (7.9%) exerted a similar effect to that observed in the untreated control cells (Fig. 10). Furthermore, megakaryocytic differentiation was assessed by the expression of the CD42a⁺ marker, and it was observed that PMA had the potential to induce the expression of this surface marker in 24.8% of cells and bDLE treatment induced lower expression levels in a dose-dependent manner (P=0.05) [(0.07 U/ml (4.8%), 0.14 U/ml (7.6%), 0.21 U/ml (8.9%), 0.28 U/ml (13.1%) and 0.35 U/ml (13.9%)]. No difference (P=0.38) was observed between the DMSO treated (0.2%) and untreated cells (0.9%). The results of the present study demonstrated that bDLE had the potential to induce monocytic/macrophage differentiation (Figs. 7–10), and had a lower capacity to induce megakaryocytic differentiation (Fig. 11).

To determine if the previously differentiated cells remained sensitive to treatment with bDLE, cells were treated with cytotoxic doses. It was observed that treatment at 1 U/ml induced cell death in 84.4% (Fig. 12). Furthermore, murine peritoneal macrophages and primary human monocytes treated with the an identical dose of bDLE demonstrated 17.4 and 27.9% cell death, respectively (Fig. 3).

The results of the present study demonstrated that bDLE treatment significantly decreased (P=0.05) NO production in a dose-dependent manner in K562 cells following 96 h of incubation [0.07 U/ml (57.12 M), 0.14 U/ml (50.72 M), 0.21 U/ml (44.32 M), 0.28 U/ml (30.72 M), and 0.35 U/ml (13.12 M)] compared with untreated cells (60.64 M) and PMA treated cells (51.68 M). DMSO treatment significantly decreased NO production (17.2 M), similar to the effects caused by 0.35 U/ml bDLE (Fig. 13). Furthermore, cytokine (IL-1b, IL-6, IL-10, TNF-α and IL-12 p70) and chemokine production (CCL2/MCP-1, CCL5/RANTES and CXCL8/IL-8) were evaluated in the K562 cell line treated with the inducers of differentiation. Untreated cells demonstrated expression of IL-6, IL-8 and TNF-α, and IL-1b and IL-12p70 were not expressed (Table II); all chemokines evaluated in the present study were observed to be expressed in untreated cells (Table III). PMA treatment induced overexpression of the chemokines (Table III) and cytokines (Table II) evaluated, but IL-12p70 was not expressed. bDLE treatment at doses of 0.07, 0.14, 0.21 and 0.28 U/ml increased the expression of all cytokines evaluated, except IL-1b and IL-12 p70. bDLE at a dose of 0.28 U/ml decreased TNF-α expression. At doses of 0.35 U/ml the expression levels of IL-6 and TNF-α were the lowest compared to the control and PMA treatment groups, and IL-8 expression was elevated. bDLE increased the expression of chemokines CCL2/MCP-1 in a dose-dependent manner. At doses of 0.07 and 0.14 U/ml increased expression of CCL5/RANTES was observed, and there was no difference in expression between the doses evaluated compared to the control. All the doses evaluated increased the expression of CXCL8/IL-8 (Table III). DMSO treatment reduced the expression of all cytokines (Table II) and chemokines evaluated (Table III).