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Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy.

Xing F, Wang J, Hu M, Yu Y, Chen G, Liu J - Nanoscale Res Lett (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China. tfyxing@jnu.edu.cn.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Morphologic changes of immature and mature BMDCs. (A, B) The morphology of the BMDCs treated with GM-CSF plus IL-4 was observed under a light microscope (magnification: ×100 (A) and ×400 (B)). (C, D) The morphology of the BMDCs stimulated with LPS was also done under a light microscope (magnification: × 100 (C) and × 400 (D)). (E, F) The images of the BMDCs treated with GM-CSF plus IL-4 were scanned by a scanning electron microscope (SEM) with different magnifications, including around ×1,200 (E) and ×5,000 (F); (G, H) SEM images of the BMDCs stimulated with LPS were recorded with different magnifications, i.e., around ×1,200 (G) and ×5,000 (H).
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Figure 1: Morphologic changes of immature and mature BMDCs. (A, B) The morphology of the BMDCs treated with GM-CSF plus IL-4 was observed under a light microscope (magnification: ×100 (A) and ×400 (B)). (C, D) The morphology of the BMDCs stimulated with LPS was also done under a light microscope (magnification: × 100 (C) and × 400 (D)). (E, F) The images of the BMDCs treated with GM-CSF plus IL-4 were scanned by a scanning electron microscope (SEM) with different magnifications, including around ×1,200 (E) and ×5,000 (F); (G, H) SEM images of the BMDCs stimulated with LPS were recorded with different magnifications, i.e., around ×1,200 (G) and ×5,000 (H).

Mentions: The bone marrow cells were cultured and induced in complete RPMI 1640 medium supplemented with a given dose of GM-CSF plus IL-4 for 6 days. Six days post induction of rmGM-CSF plus rmIL-4, the BMDCs appeared predominately round in loosely adhesive growth under a light microscope (Figure 1A,B) and SEM (Figure 1E,F). When observed at a high resolution, the BMDCs were ridgy in shape with a relative smooth membrane surface (Figure 1F), demonstrating that they are mostly in immature status. But the BMDCs with treatment of LPS (LPS-treated BMDCs) changed greatly under a light microscope (Figure 1C,D) and SEM (Figure 1G,H). After treatment of LPS, some of BMDC became significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia (Figure 1G,H), compared with the control (Figure 1E,F). The formation of roughness, protrusion, and ruffles on the cell membrane are considered to be associated with maturation of BMDCs. These results suggest that there exist obviously morphologic characteristics of mature BMDCs, consistent with previously reported data [15]. Generally speaking, it is considered that the morphologic change is the foundation of the phenotype and the function of BMDCs. MHC-II is one of activation molecules expressed on the surface of BMDCs, representing a phenotype of mature BMDCs. Flow cytometry analysis showed that the percentage of CD11c+MHC-II+ cells in LPS-treated BMDCs was twofold more than that in BMDCs (Figure 2A,D), indicating that some of the LPS-stimulated BMDCs become mature. This is supported by our finding that the percentages of CD11c+CD86+ cells, CD11c+CD80+ cells, and CD11c+CD40+ cells in LPS-treated BMDCs were 1.5-, 1.6-, and 2.5-fold more than those in BMDCs, respectively [16]. IL-12 release is a functional characteristic of DC maturation and also crucial for mature DCs to mediate Th1 differentiation so as to enhance immune responses. Mature DCs can direct differentiation of naïve CD4+ T cells into Th1 cells through IL-12 and interaction between DCs and the latter [17-19]. Therefore, we further examined whether BMDCs treated with LPS were of a functional feature of DC maturation. The amount of IL-12 in culture supernatants of BMDCs was assessed by ELISA. Compared with the control, LPS promoted significantly secretion of IL-12 by BMDCs (Figure 2E). In terms of previous reports, nuclear factor (NF)-kappaB plays a major role in regulation of DC maturation, and LPS-mediated activation of NF-kappaB in DCs leads to the production of IL-12 [20,21]. These results suggest that BMDCs acquire maturation after treatment of LPS, consistent with up-regulation of a co-stimulating molecule, MHC-II, on the surface of DCs. The forgoing findings from morphology, phenotype, and function of BMDCs indicate that there are distinct differences between both the immature and mature BMDCs. The confirmed immature and mature BMDCs have been successfully induced, isolated, and identified, being suitable further for a comparative study by AFM.


Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy.

Xing F, Wang J, Hu M, Yu Y, Chen G, Liu J - Nanoscale Res Lett (2011)

Morphologic changes of immature and mature BMDCs. (A, B) The morphology of the BMDCs treated with GM-CSF plus IL-4 was observed under a light microscope (magnification: ×100 (A) and ×400 (B)). (C, D) The morphology of the BMDCs stimulated with LPS was also done under a light microscope (magnification: × 100 (C) and × 400 (D)). (E, F) The images of the BMDCs treated with GM-CSF plus IL-4 were scanned by a scanning electron microscope (SEM) with different magnifications, including around ×1,200 (E) and ×5,000 (F); (G, H) SEM images of the BMDCs stimulated with LPS were recorded with different magnifications, i.e., around ×1,200 (G) and ×5,000 (H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3211875&req=5

Figure 1: Morphologic changes of immature and mature BMDCs. (A, B) The morphology of the BMDCs treated with GM-CSF plus IL-4 was observed under a light microscope (magnification: ×100 (A) and ×400 (B)). (C, D) The morphology of the BMDCs stimulated with LPS was also done under a light microscope (magnification: × 100 (C) and × 400 (D)). (E, F) The images of the BMDCs treated with GM-CSF plus IL-4 were scanned by a scanning electron microscope (SEM) with different magnifications, including around ×1,200 (E) and ×5,000 (F); (G, H) SEM images of the BMDCs stimulated with LPS were recorded with different magnifications, i.e., around ×1,200 (G) and ×5,000 (H).
Mentions: The bone marrow cells were cultured and induced in complete RPMI 1640 medium supplemented with a given dose of GM-CSF plus IL-4 for 6 days. Six days post induction of rmGM-CSF plus rmIL-4, the BMDCs appeared predominately round in loosely adhesive growth under a light microscope (Figure 1A,B) and SEM (Figure 1E,F). When observed at a high resolution, the BMDCs were ridgy in shape with a relative smooth membrane surface (Figure 1F), demonstrating that they are mostly in immature status. But the BMDCs with treatment of LPS (LPS-treated BMDCs) changed greatly under a light microscope (Figure 1C,D) and SEM (Figure 1G,H). After treatment of LPS, some of BMDC became significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia (Figure 1G,H), compared with the control (Figure 1E,F). The formation of roughness, protrusion, and ruffles on the cell membrane are considered to be associated with maturation of BMDCs. These results suggest that there exist obviously morphologic characteristics of mature BMDCs, consistent with previously reported data [15]. Generally speaking, it is considered that the morphologic change is the foundation of the phenotype and the function of BMDCs. MHC-II is one of activation molecules expressed on the surface of BMDCs, representing a phenotype of mature BMDCs. Flow cytometry analysis showed that the percentage of CD11c+MHC-II+ cells in LPS-treated BMDCs was twofold more than that in BMDCs (Figure 2A,D), indicating that some of the LPS-stimulated BMDCs become mature. This is supported by our finding that the percentages of CD11c+CD86+ cells, CD11c+CD80+ cells, and CD11c+CD40+ cells in LPS-treated BMDCs were 1.5-, 1.6-, and 2.5-fold more than those in BMDCs, respectively [16]. IL-12 release is a functional characteristic of DC maturation and also crucial for mature DCs to mediate Th1 differentiation so as to enhance immune responses. Mature DCs can direct differentiation of naïve CD4+ T cells into Th1 cells through IL-12 and interaction between DCs and the latter [17-19]. Therefore, we further examined whether BMDCs treated with LPS were of a functional feature of DC maturation. The amount of IL-12 in culture supernatants of BMDCs was assessed by ELISA. Compared with the control, LPS promoted significantly secretion of IL-12 by BMDCs (Figure 2E). In terms of previous reports, nuclear factor (NF)-kappaB plays a major role in regulation of DC maturation, and LPS-mediated activation of NF-kappaB in DCs leads to the production of IL-12 [20,21]. These results suggest that BMDCs acquire maturation after treatment of LPS, consistent with up-regulation of a co-stimulating molecule, MHC-II, on the surface of DCs. The forgoing findings from morphology, phenotype, and function of BMDCs indicate that there are distinct differences between both the immature and mature BMDCs. The confirmed immature and mature BMDCs have been successfully induced, isolated, and identified, being suitable further for a comparative study by AFM.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China. tfyxing@jnu.edu.cn.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus