A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism

Liposome-Coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8+ T Cells

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity

Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates.

<p>A, DQ-OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages expressing DM-DsRed (class II) or labeled with red fluorescein (class I), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage DM or class I); cell only, macrophages without co-culture with DQ-OVA-coupled liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig. 1A. Asterisk, significant (<i>p</i><0.01) difference of samples.</p

Intracellular localization of liposomal antigens taken up by macrophages.

<p>A, DQ-OVA was coupled to oleoyl liposomes and added to the culture of cloned macrophages of which endosomal marker EEA1-positive compartments, or lysosomal marker LAMP-1-positive compartments were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage EEA1 or LAMP-1); cell only, macrophages without co-culture with DQ-OVA liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig, 4A. Asterisk, significant (<i>p</i><0.01) difference of samples.</p

Influence of inhibitors for uptake of OVA coupled to oleoyl liposomes by macrophages.

<p>Alexa- or DQ-labeled OVA was coupled to oleoyl liposomes and added to the culture of macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Treatment of macrophages with cytochalasin B or DMA was done 60 minutes prior to the addition of OVA-liposome conjugates.</p

IFN-γ production by splenic CD4/CD8⁺ T cells of mice immunized with OVA after co-culture with CD11c⁺ cells pulsed with OVA coupled to oleoyl liposomes.

<p>Splenic CD4/CD8⁺ T cells were taken from mice immunized with OVA and were cultured with CD11c⁺ cells pulsed with OVA coupled to oleoyl liposomes with or without inhibitors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. IFN-γ production of T cells in the supernatants in the absence of inhibitors was normalized to 100%. Data represent the mean values ± SD of triplicate culture. Asterisk, significant (<i>p</i><0.01) difference as compared with the ‘no inhibitor’ group.</p