Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy
© Xing et al; licensee Springer. 2011
Received: 6 March 2011
Accepted: 16 July 2011
Published: 16 July 2011
A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs.
Dendritic cells (DCs) are the most potent specialized antigen-presenting cells, which bridge the innate and adaptive immune response, controlling both immunity and tolerance. It is well known that DCs may be derived from bone marrow progenitors with two major developmental stages: immature and mature DCs . The development of immature DCs can be induced with using cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) , FMS-like tyrosine kinase 3 (FLT3) , or cytokine cocktails containing GM-CSF +/-IL-4  in vitro. After stimulation of lipopolysaccharide (LPS), poly I:C or thymic stromal lymphopoietin (TSLP), immature DCs can further differentiate into mature DCs, with increase of IL-12 and up-regulation of MHC-II, CD40, CD80, CD83, and CD86 molecules on the surface of DCs [5, 6]. The maturation status of DCs is relatively important for them whether to induce immune tolerance or to initiate immune response. It is well proved that the transition from immature DCs to mature DCs is accompanied by morphological changes to be suitable for requirement of immunological function changes of DCs. Scanning electron microscopy (SEM) is a conventional tool for imaging cell morphology, which requires a conductive surface and a high-vacuum condition . By contrast, atomic force microscopy (AFM), with continuously growing uses in investigating biomaterials, can be operated directly in air, vacuum, or physiological conditions with nanometer lateral resolution [7, 8]. Furthermore, AFM is capable of providing quantitative analysis of cell surface and adhesion force features. Although the morphology of DCs has early been observed by conventional optical microcopy, SEM, and transmission electron microcopy methods [7, 9], comparison of immature and mature DCs has not been, to date, carried out using AFM. Therefore, it is necessary to find out nanostructure of DCs, especially different nano-properties and adhesive force that cannot be discovered by optical and electron microscopy. In this study, AFM was exploited to reveal differences of the nano-features and adhesive force between both immature and mature bone marrow-derived dendritic cells (BMDCs). Obviously, this study would provide a novel insight into the nanostructure and force feature of immature and mature DCs.
Materials and methods
Preparation of bone marrow cells
Bone marrow-derived dendritic cells were generated according to Lutz's publication  with a little modification. In brief, cervical cords in female Balb/c mice with 6 to 8 weeks old (Sun Yat-sen University, Guangzhou, China) were mechanically dislocated to sacrifice them. After removing all muscle tissues from the femurs and tibias, intact bones were left in 70% ethanol for 2 to 5 min for disinfection and washed with phosphate-buffered saline (PBS). Then, both ends were cut with scissors and the marrow was washed with PBS through a syringe. Clusters within the marrow suspension were disintegrated by vigorous pipetting. The bone marrow cell suspension was centrifuged at 300 × g for 5 min. The cells were collected, suspended in PBS by addition of red blood cell lysate for depletion of erythrocytes, and incubated at 37.0°C for 8 min away from light. Then, they were washed with PBS at 300 × g for 5 min three times. At last, the cells were harvested and resuspended in RPMI1640 (Gibco BRL, Gaithersburg, MD, USA) complete culture medium containing 10% (v/v) fetal bovine serum (FBS) (Gibco BRL), 2 mmol/L L-glutamine, 10 μmol/L 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, and adjusted to 2 × 109/L.
Induction and separation of bone marrow-derived dendritic cells
The above cells were seeded into a 6-well plate to the end volume of 2 mL per well, and 10.0 μg/L of rmGM-CSF (PeproTech, Rocky Hill, NJ, USA) plus 10.0 μg/L of rmIL-4 (PeproTech) was added to the corresponding wells in the plate and cultured at 37.0°C in an incubator containing 5% CO2 to induce differentiation of bone marrow cells into bone marrow-derived dendritic cells. Then, the cells were fed once at the interval of 1 day with the identical dose of rmGM-CSF plus rmIL-4 for 6 days. At the end of the cell induction, all the cells expressing CD11c in the different wells were isolated respectively using the Mouse CD11c Positive Selection Kit (EasySep®Magnet, StemCell Technologies, Vancouver, Canada) according to the manufacturer's instruction and seeded into new wells with fresh medium. Finally, the CD11c-positive cells were treated with or without LPS (Sigma-Aldrich) at a dose of 1.0 mg/L for another 24 h in order to obtain mature BMDCs.
Scanning electron microscopy
After the stimulation of LPS, the CD11c-positive cells were rinsed with PBS containing 0.5 mM MgCl2 and 1 mM CaCl2, made naturally subside to the glutin-coated glass for 10 min, then fixed at 4°C for 30 min with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and post-fixed for 30 min with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4. The glass was gradually dehydrated in ethanol (30%, 50%, 70%, 90%, and twice in 100% for 5 min at each step) and subjected to critical point drying using carbon dioxide as transitional medium. The samples were stored in a vacuum exsiccator to prevent putative deterioration by air humidity. Then, they were connected to stub holders with liquid silver paint to improve electrical conductivity and imaged in SEM (ESEM-30, Philips, Mahwah, NJ, USA) with a field emission electron gun operating at standard high-vacuum settings.
The CD11c-positive cells were harvested after the selection of immunomagnetic beads and the stimulation of LPS as described above. After being centrifuged, they were washed with PBS at 300 × g for 5 min and resuspended in PBS. Then, the cells were stained with both 0.25 μg anti-CD11c-FITC and 1.0 μg anti-MHC-II-PE (eBioscience, USA) per million cells in a 100μl total volume. After being mixed gently on a vortex machine, they were placed at 4.0°C in the dark for 30 min, and then rinsed with PBS for two times and centrifuged at 300 × g for 5 min. The expression level of CD11c on the surface of the cells was analyzed by flow cytometry (FAC-Scalibur, Becton Dickinson, Franklin Lakes, NJ, USA). A total of 5 × 103 events were analyzed for each determination and calculated by CellQuest software (Becton Dickinson).
The above selected BMDCs were treated with or without LPS at a concentration of 1.0 mg/L for 24 h. Their culture supernatant was collected. The level of IL-12 in the supernatant was determined via enzyme linked immunosorbent assay (ELISA) with the IL-12 ELISA Kit (Bender MedSystems, Burlingame, CA, USA) according to the manufacturer's protocol. Absorbance value was measured at 450 nm in 680 type microplate reader (Bio-Rad, Berkeley, CA, USA). The concentration of IL-12 was quantified according to a standard curve.
AFM observation was performed according to the reported method [11, 12]. In brief, the mica carrying the BMDCs was fixed for 15 min in 2% glutaraldehyde phosphate buffer at pH 7.4, washed gently with distilled water three times, and dried naturally. Then, contact mode scanning was immediately performed using a commercial AFM (AutoProbe CP Research, Thermomicroscopes, Sunnyvale, CA, USA) in air at room temperature. The curvature radius of the silicon nitride tip (UL20B, Park Scientific Instruments) was around 10 nm, and a force constant about 2.8 N/m was used. To obtain high resolution, we scanned samples at rate of 0.3 Hz. All of the AFM images were flattened with provided software (Thermomicroscopes Proscan Image Processing Software Version 2.1) to complete quantitative analysis.
An autoprobe CP AFM was used in a contact mode in air to perform the topography images at room temperature according to the publications [11–14]. AFM-based force spectroscopy was used to perform the force detection. The same silicon nitride tip was applied for measurement of all the force-distance curves at the same speed. Force-distance curves were obtained through standard retraction between the tip and cell surface. Two hundred fifty-six force-distance curves were recorded for every cell (n = 10 cells for each group). All force-distance curve experiments were performed at the same loading rate.
where N is a total quantity of measured spots, Z n means a height of any spot, and represents an average height of all the spots. The calculated R rms and R a refer only to the area shown in the top central part of the cells.
Numerical data obtained from each experiment were expressed as mean ± SD, analyzed by SPSS 10.0 statistical package. The Student's t test was followed for data comparison and a P value of less than 0.05 was considered statistically significant.
Results and discussion
Morphologic and functional characteristics of BMDCs
Nano-structural comparison of immature and mature BMDCs
Regarding the enhancement of the mature DC height and volume, it is associated with the differentiation and maturation of DCs induced by LPS. It is well known that LPS can activate Toll-like receptor 4 on the surface of immature DCs. The activation of a Toll-like receptor 4 signaling pathway finally causes nuclear translocation of the nuclear factor (NF)-kappaB transcription factor. The inhibition of NF-kappaB activation blocks maturation of DCs, followed by down-regulation of major histocompatibility complex and co-stimulatory molecules, which indicates that the activated NF-kappaB signaling pathway may be responsible for DC maturation. Simultaneously, it is found that LPS activates the extracellular signal-regulated kinase1/2 (ERK1/2) in DCs. The specific inhibition of MEK1, an upstream kinase of ERK1/2, abrogates the ability of LPS to prevent apoptosis but does not impact the DC maturation, which suggests that ERK1/2 signaling pathway may mainly maintain DC survival . Ardeshna's research group showed that LPS activated the p38 mitogen-activated protein kinase (p38 MAPK), ERK1/2, phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and NF-kappaB pathways in the process of DC maturation. PI3 kinase/Akt signaling pathways are important in maintaining survival of LPS-stimulated DCs. Inhibiting p38 MAPK prevented activation of the transcription factor ATF-2 and CREB, and significantly reduced the LPS-induced up-regulation of co-stimulatory molecules . It is also demonstrated by another research group's results that ERK1/2, p38MAPK, c-jun N-terminal kinase (JNK), and NF-kappaB signaling pathways are implicated in the events of DCs maturation . The differentiation and maturation of DCs require more synthetic materials and energy production, with enhancement of the whole cellular or subcellular metabolism and function. Morphological changes of cells are foundation of their metabolism and function changes, adapting to the need of the both latters. The big increase of subcellular organelles in LPS-stimulated mature DCs, especially including lysosome, mitochondrium, and endoplasmic reticulum with enrichment of cytoplasm, can be observed under a transmitted electronic microscope, finally resulting in the augmentation of the DC height and volume. The increase of mature DC surface area may be helpful for the expression of co-stimulatory molecules and relevant receptors on the surface of mature DCs, promoting intercellular interaction of mature DCs and other associated cells. Of course, these morphological changes of mature DCs may be regulated by the foregoing different and sometimes overlapping pathways.
Adhesive force comparison of immature and mature BMDCs
It should be pointed out that the AFM tip is going to be contaminated at the first touch and continue with the following touches, and this contamination can influence the next interaction of the tip with the cells. Therefore, contamination control of AFM tips is very important for reliable AFM imaging and surface/interface force measurements. Most contaminants may result in poor imaging quality either by causing tip effects and/or noise . Tip effects reflect the increase in tip size as the contaminants add to the tip apex . A noisy AFM image can be a result of uncontrollable interaction (such as sudden bridging or breaking) between the tip and the sample surface mediated by interspersed sticky contaminants. Nie et al. considered that such a contaminant confined on the tip apex displays an uncontrollable variation in the oscillation amplitude of the cantilever, causing noise in the AFM images the contaminated tip collects, but such a contaminant may be removed from the apex by pushing the tip into a material soft enough to avoid damage to the tip . According to our experience, cell samples should be gently washed with the buffer at least three times for removing debris attachment from cell culture media and themselves before AFM determination. We think that a contact mode for the determination may be replaced by a tapping mode in order to reduce the contamination and cell damage if serious contamination occurs. Actually, traditional cleaning methods for the tip, including plasma, UV-ozone, solvent treatments, and so on, have been abroad applied, but there still are some shortcomings. Recently, Gan et al. reported that calibration gratings with supersharp spikes could be employed to scrub away contaminants accumulated on a colloidal sphere probe by scanning the probe against the spikes at high load at constant-force mode. This method may be superior to traditional cleaning methods in several aspects . Anyway, control of AFM tip contamination is an extremely common issue and remains to be further studied.
Taken together, the above results first reveal the characterization of the surface nanostructure and adhesion force of the immature and mature BMDCs, providing profoundly understanding structure/function relationship of BMDCs.
This project was supported by the National Natural Science Foundation of China (no. 30471635, no. 30971465), the Natural Science Foundation of Guangdong Province in China (04010451, 5006033), the Fundamental Research Funds for the Central University (21610608), and the "211" project grant.
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