Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model

Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood. The polystyrene particles interacted with the DC throughout the tested diameter range of 0.04-15 microm in a time- and concentration-dependent manner. The optimal particle diameter for fast and efficient acquisition by a substantial percentage of the DC was 0.5 microm and below. The surface of 1 and 0.1 microm polystyrene particles was covalently modified with different polyaminoacids/proteins, yielding particles with varying surface charge. Uptake of 1 microm particles was greatly enhanced when particles displayed a positive surface charge. In general, the present findings establish that particle diameters of 0.5 microm and below were optimal for DC uptake; however uptake of larger particles could be greatly enhanced by rendering the particle surface positive. Whether increased particle uptake is correlated with increased immune responses, remains to be established.     

Evaluation of phagocytic activity in human monocyte-derived dendritic cells.

Dendritic cells play a central role in initiation of primary T lymphocyte responses to foreign antigens. Their potency in antigen presentation vis-à-vis reported low or lack of ability to phagocytize particulate matter has limited our understanding of the role that they play in inducing immunity to particulate antigens. One hypothesis is that dendritic cells may possess a high phagocytic capacity when immature and located in peripheral tissues, which they lose on maturation. Our goal was to characterize the phagocytic capacity in human immature dendritic cells. The phagocytic capacity of human monocyte-derived immature dendritic cells was studied by morphological and morphometric means, and compared to that of professional phagocytes, human alveolar macrophages, their progenitors, the peripheral blood monocytes, and mature dendritic cells. Phagocytic index (proportion of phagocytic cells) was decreased by 42.8% (immature dendritic cells) and 74.2% (mature dendritic cells) with respect to monocytes. Similarly, the phagocytic index was decreased by 46.5% (immature dendritic cells) and 75.9% (mature dendritic cells) with respect to macrophages. Volume density of phagocytized particles was decreased by 76.1% (immature dendritic cells) and 96.7% (mature dendritic cells) with respect to the monocytes. However, volume density was decreased by 34.3% (immature dendritic cells) and 91% (mature dendritic cells) with respect to alveolar macrophages. These results show that human monocyte-derived immature dendritic cells possess a phagocytic capacity that is lower than that of peripheral blood monocytes and alveolar macrophages but higher than that of mature dendritic cells.     

Adhesion molecules in human dendritic cells

Dendritic cells are bone marrow-derived professional antigen-presenting cells that link innate and adaptive immunity, and they are essentially involved in the initiation of primary immune responses and in the establishment of peripheral tolerance. The existence of distinct functional states and subsets of dendritic cells is critical for the generation of pathogen-specific immune responses without the risk of autoimmunity or chronic inflammation. To fulfil their effector tasks in tissues and lymph nodes, dendritic cells must engage in multiple adhesive and migratory events. The molecular dissection of these adhesive interactions may provide new potential therapeutic targets to modulate immune responses and to improve current dendritic cell-based therapeutic cancer vaccines.     

In vitro uptake of gelatin nanoparticles by murine dendritic cells and their intracellular localisation.

The long term goal of this study is to develop an efficient nanoscopic vaccine delivery system, based on the biodegradable and natural polymer gelatin, to deliver therapeutic protein antigens along with adjuvants into dendritic cells (DCs). In this study, gelatin nanoparticles were tested for qualitative and quantitative uptake in murine DCs in vitro. A second aim of this study was to prove that the carrier system is able to deliver tetramethylrhodamine conjugated dextran (TMR-dextran), as a model drug into the DCs. The TMR-dextran was incorporated during the preparation of the gelatin nanoparticles. DCs were generated from murine bone marrow cells by an established ex vivo technique. Flow cytometry showed that 88% of the cells positive for the specific murine DC marker CD11c took up TMR-dextran loaded gelatin nanoparticles, whereas only 4% of the soluble form of TMR-dextran was taken up. Double color confocal laser scanning microscopy (CLSM) showed that gelatin nanoparticles were phagocytosed by DCs and the triple color CLSM showed that the TMR-dextran was localized mainly in lysosomes as expected, but partly also outside the lysosomes, presumably in the cytoplasm. An in vitro release study of TMR-dextran from gelatin nanoparticles demonstrated that there was hardly any release in phosphate buffered saline (PBS), but by trypsin-assisted degradation of gelatin nanoparticles resulted in the release of about 80% of the TMR-dextran from the particles. These results suggest that gelatin nanoparticles hold promise as a new biocompatible tool for vaccine delivery to DCs, with applications in cancer immunotherapy.     

Analysis of poly(D,L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro

Purpose. The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs). Methods. Parallel cultures of DCs and macrophages (M) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for M). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique. Cells were incubated with the nanospheres for 24 h. Confocal laser scanning microscopy was used to determine the intracellular location of the nanospheres and flow cytometry to measure the fraction of phagocytic cells in the culture and the amount of uptake per cell. After phagocytosis, cells were stained for MHC class II molecules, CD14, CD80, and CD86 to identify the phagocytic population. Results. DCs phagocytosed PLGA nanospheres as efficiently as M. Cell-surface marker expression conclusively established that the phagocytic cells were DC. Conclusions. DCs can take up PLGA nanospheres. Because DCs are the key professional antigen-presenting cells capable of stimulating naive T cells, our data suggest that PLGA nanospheres can be used as an efficient delivery system for vaccines designed to activate T cell-mediated immune responses.     

In vitro effects of trichothecenes on human dendritic cells.

The aim of this work was to study the in vitro effects of trichothecenes on human dendritic cells. Trichothecenes are mycotoxins produced by fungi such as Fusarium, Myrothecium, and Stachybotrys. Two aspects have been explored in this work: the cytotoxicity of trichothecenes on immature dendritic cells to determine IC 50 (inhibition concentration), and the effects of trichothecenes on dendritic cell maturation process. Two mycotoxins (T-2 and DON) known to be immunotoxic have been tested on a model of monocyte-derived dendritic cells culture. Cytotoxic effects of T-2 toxin and DON on immature dendritic cells showed that DON is less potent than T-2 toxin. The exposure to trichothecenes during dendritic cell maturation upon addition of LPS or TNF-alpha markedly inhibited the up-regulation of maturation markers such as CD-86, HLA-DR and CCR7. Features of LPS or TNF-alpha -mediated maturation of dendritic cells, such as IL-10 and IL-12 secretions and endocytosis, were also impaired in response to trichothecenes treatment. These results suggest trichothecenes have adverse effects on dendritic cells and dendritic cell maturation process.     

Maturation-dependent expression of C1q-binding proteins on the cell surface of human monocyte-derived dendritic cells

The expression and cell surface levels of many important receptors are dependent on the maturation stage of dendritic cells (DCs), and related to the unique function of immature and mature DCs. In this report, we show for the first time that human monocyte-derived DCs express two types of C1q-receptors, gC1qR and cC1qR. Furthermore, immature DCs secrete detectable amount of C1q into the culture supernatant. Immature DCs express higher cell surface levels of both C1qRs than mature ones, while the total C1qR protein and mRNA levels remain the same. The following experimental evidence supports this conclusion. (1) Inflammatory cytokines and LPS, which induce maturation of DCs, downregulate surface expression of both C1qR molecules. (2) Cytokines and drugs (IL-10, IFNalpha, dexamethasone) that keep DCs phenotypically and functionally immature significantly upregulate the cell surface expression of both C1qRs. (3) Neither of these treatments changed the intracellular gC1qR level nor the gC1qR mRNA levels measured by real-time RT-PCR. The elevated surface expression of C1qRs on DCs has been found not to be due to increased apoptosis or cell death as the result of DC treatment. Taken together, these data show that human monocyte-derived DCs express gC1qR and cC1qR, their expression on the cell surface is maturation dependent and imature DCs secrete C1q. These data strongly suggest the role of C1qRs in immature DC function and in the regulation of immune processes.     

Maturation-dependent expression of C1q binding proteins on the cell surface of human monocyte-derived dendritic cells

The expression and cell surface levels of many important receptors are dependent on the maturation stage of dendritic cells (DCs), and related to the unique function of immature and mature DCs. In this report, we show, for the first time, that human monocyte-derived DCs express two types of C1q receptors, gC1qR and cC1qR. Furthermore, immature DCs secrete detectable amount of C1q into the culture supernatant. Immature DCs express higher cell surface levels of both C1qRs than mature ones, while the total C1qR protein and mRNA levels remain the same. The following experimental evidence support this conclusion: (1) Inflammatory cytokines and LPS, which induce maturation of DCs, downregulate surface expression of both C1qR molecules. (2) Cytokines and drugs (IL-10, IFN-alpha, Dexamethasone), which keep DCs phenotypically and functionally immature, significantly upregulate the cell surface expression of both C1qRs. (3) Neither of these treatments changed the intracellular gC1qR level nor the gC1qR mRNA levels measured by real time RT-PCR. The elevated surface expression of C1qRs on DCs has been found to be not due to increased apoptosis or cell death as the result of DC treatment. Taken together, these data show that human monocyte-derived DCs express gC1qR and cC1qR, their expression on the cell surface is maturation dependent, and immature DCs secrete C1q. These data strongly suggest the role of C1qRs in immature DC function and in the regulation of immune processes.     

Effect of piceatannol in human monocyte-derived dendritic cells in vitro

Piceatannol is an anti-inflammatory, immunomodulatory, and antiproliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Dendritic cells (DCs) play a pivotal in the initiation of T-cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. This study investigated the effect of piceatannol on the phenotypic and functional maturation of human monocyte-derived DCs in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days, followed by another 2 days in the presence of piceatannol or LPS. DCs harvested on day 8 were examined using functional assays. The expression levels of CD1a, CD80, CD83, and CD86 as expressed by mean fluorescence intensity (MFI) on DCs differentiated from immature DCs after culture with 1 muM of piceatannol for 2 days were enhanced and decreased endocytic activity. Piceatannol-treated DCs also displayed enhanced T-cell stimulatory capacity in a MLR, as measured by T-cell proliferation. Similar results were obtained with DCs differentiated with LPS from immature DCs. However, piceatannol did not inhibit phenotypic and functional maturation induced by LPS from immature DCs. Piceatannol-treated DCs induced the differentiation of naive T cells towards a helper T-cell type 1 (Th1) response at DCs/T (1:5) cells ratio depending on IL-12 secretion. These results demonstrate that piceatannol may be used on DC-based vaccine for cancer immunotherapy.     

Cantharidin modulates development of human monocyte-derived dendritic cells

Cantharidin (CTD), a naturally occurring small molecule isolated from a medicinal insect, possesses anti-cancer and pro-inflammatory properties. We aimed to examine the effect of CTD on human myeloid dendritic cells (DCs) by examining immature DCs differentiated and maturated from CD14⁺ monocytes. CTD added into a culture of starting CD14⁺ monocytes markedly and dose-dependently reduced viability of harvested DC. Mature DCs differentiated in the presence of CTD had much fewer, shorter membranous projections than those without CTD. Changes in morphological features characteristic of necrotic cells were also evident. Furthermore, CTD affected DC differentiation and maturation phenotypes including down-regulation of surface CD1a, CD83 and DC-SIGN. DCs derived in the presence of CTD possessed an impaired allostimulatory activity on naive CD4⁺CD45⁺RA⁺T cell in terms of proliferation and interferon-γ production. It suggests that CTD may redirect DC differentiation toward a less mature stage and that this effect is not solely due to its cytotoxicity. Whether this effect refers to immune suppression or tolerance to disease treatments with unwanted immune reactions needs further evaluation.     

Pharmaceutical excipients influence the function of human uptake transporting proteins

Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.     

Effects of anti-lymphocytes and anti-thymocytes globulin on human dendritic cells

Dendritic cells (DC) were implicated as the mediators of primary stimulation of the immune response against donor antigen. Anti-lymphocytes globulin (ALG) and anti-thymocytes globulin (ATG) antibodies have been widely used as an immunosuppressive agent in the treatment and prevention of rejection after organ transplantation. In this study, we attempted to determine whether ATG and ALG are able to bind and interfere in human dendritic cell function. ALG and ATG bound to human DCs at least in part by recognising CD1a, MHC I, MHC II, CD11a, CD86, CD32, CD4, CD11b, CD29 and CD51/61. This binding, that was more relevant in mature DCs, induced complement-mediated lysis. ALG and ATG did not influence either the macropynocytosis of Lucifer Yellow (LY) or the receptor-mediated endocytosis of FITC-Dextran. In MLR assay, ATG and ALG were able to significantly inhibit T-cell proliferation by binding on T cell but not on dendritic cells. CONCLUSIONS: we demonstrate that ALG/ATG is able to interfere in the activation of T cells by dendritic cells in two different ways: by inhibiting the capacity of lymphocyte to proliferate after the DC stimulation and by inducing a complement-mediated lysis of DC.     

Platonin modulates differentiation and maturation of human monocyte-derived dendritic cells

Platonin is a photosensitizer with NF-kappaB inhibitory activity that activates macrophages and suppresses lymphocyte response. In this study, we tested the effect of platonin on differentiation and maturation of human myeloid dendritic cells (DC) from CD14+ monocytes. Triggering of DC differentiation by GM-CSF and IL-4 resulted in typical immature DCs that were further stimulated to maturation by combination of cytokines. When platonin (2 to 50 ng/mL) was added to the culture, the resulting DCs had thicker and blunter protruding projections, lower CD83 expression, greater CD80 expression, and less stimulatory activity on allogeneic naive CD4+CD45RA+ T cells in terms of their proliferation and interferon-gamma production. This suggests that platonin redirects DC differentiation toward an intermediate stage of maturation, whereby the DCs have uniquely enhanced expression of CD80 which may confer some degree of immune tolerance.     

The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells.

Chimeric oligodeoxynucleotides with terminal nonionic methylphosphonate analogue sections and internal phosphodiester regions offer several advantages as antisense effectors over either structure alone. These include enhanced biological stability relative to all-phosphodiester molecules, increased activity in directing ribonuclease H mediated destruction of target RNA, increased specificity and reduced non-specific toxicity. However, another important parameter, the ability of these molecules to enter intact mammalian cells, has not previously been investigated. Therefore, oligodeoxynucleotides were tagged at their 5'-termini with fluorescein reporter groups and a detailed study of uptake kinetics in human leukaemia MOLT-4 cells undertaken by calibrated flow cytometry. Baseline measurements with all-phosphodiester and all-methylphosphonate molecules confirmed that uptake of oligodeoxynucleotides by intact cells is a highly inefficient process. The kinetic data were in agreement with previous reports of mechanisms of cell uptake involving receptor mediated endocytosis in the case of phosphodiester molecules and simple diffusion for methylphosphonates. Chimeric oligodeoxynucleotides exhibited saturable cell uptake kinetics similar to all-phosphodiester oligodeoxynucleotides, suggesting that uptake was receptor-mediated and distinct from concentration-dependent uptake of all-methylphosphonate molecules. Similarly, chimeric molecules were apparently confined to the endosomal compartment within cells. These results imply that reversible masking of the negative charges of the phosphodiester sections of chimeric oligodeoxynucleotides may be required to change the uptake mechanism back to simple diffusion and allow intracellular delivery to the site of the target RNA.     

Enhanced uptake and anti-maturation effect of celastrol-loaded mannosylated liposomes on dendritic cells for psoriasis treatment

Psoriasis is an autoimmune skin disease in which dendritic cells (DCs) trigger the progression of psoriasis by complex interactions with keratinocytes and other immune cells. In the present study, we aimed to load celastrol, an anti-inflammatory ingredient isolated from Chinese herbs, on mannosylated liposomes to enhance DC uptake as well as to induce DC tolerance in an imiquimod-induced psoriasis-like mouse model. Mannose was grafted onto liposomes to target mannose receptors on DCs. The results demonstrated that compared with unmodified liposomes, DCs preferred to take up more fluorescence-labeled mannosylated liposomes. After loading celastrol into mannose-modified liposomes, they effectively inhibited the expression of maturation markers, including CD80, CD86 and MHC-II, on DCs both in vitro and in vivo. Additionally, after intradermal injection with a microneedle, celastrol-loaded mannose-modified liposomes (CEL-MAN-LPs) achieved a superior therapeutic effect compared with free drug and celastrol-loaded unmodified liposomes in the psoriasis mouse model in terms of the psoriasis area and severity index, histology evaluation, spleen weight, and expression of inflammatory cytokines. In conclusion, our results clearly revealed that CEL-MAN-LPs was an effective formulation for psoriasis treatment and suggested that this treatment has the potential to be applied to other inflammatory diseases triggered by activated DCs.     

Fluorescein uptake by a monocarboxylic acid transporter in human intestinal Caco-2 cells.

The fluorescein transport characteristics of the human intestinal epithelial Caco-2 cell line were examined in monolayer cultures. The initial uptake rate was concentration-dependent and saturable; the Michealis constant and the maximum velocity were 0.40 mM and 1.32 nmol/min/mg protein, respectively. A protonophore, carbonyl cyanide m-chlorophenyl-hydrazone, reduced uptake significantly. The replacement of extracellular sodium ions by lithium ions did not alter the initial uptake rate. These facts imply that the transport is driven by a proton gradient. The initial uptake rate was strongly dependent upon extracellular pH, and the uptake was optimal at approximately pH 5.5. Based on the protolytic constants, the main species of fluorescein in the pH range of 5.5 to 6.0 was calculated to be a monoanion, suggesting that fluorescein was taken up by Caco-2 cells as a monocarboxylate. The following findings support this conclusion: the uptake was inhibited significantly by monocarboxylate compounds such as salicylate and pravastatin, but not by di- or tricarboxylic acids or by acidic amino acids. Furthermore, salicylate-preloaded cells showed remarkably enhanced uptake of fluorescein, indicating that monocarboxylates and fluorescein share a common transport carrier. The transporter has a wide spectrum of substrate recognition and seems likely to be different from MCT1.     

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of ¹⁰⁹Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd²⁺ species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.     

Cyclosporine uptake by cultured human proximal tubule cells

Characteristics of cyclosporine A (CsA) uptake in cultured normal human proximal tubule cells (PTC) have been studied. The uptake was very rapid, concentration dependent, and reached a steady state level within 10 min. Kinetic analysis yielded an apparent Km of 5.0 μM and V_max of 66.7 pmoles/min μg DNA. Verapamil, a calcium channel blocker, at 0.1, 0.5, and 1.0 mM had no inhibitory effect on CsA uptake. Subcellular distribution of CsA following 1, 2, 5, and 10 min incubation of PTC suspension was determined by digitonin differential solubilization. The cytosol fraction contained 26% and the nuclear fraction contained 74% of CsA at all time periods studied. These studies demonstrate that in normal human renal PTC, uptake of CsA is rapid and saturable. Most of the CsA, once taken up by the human PTC, is concentrated in the nucleus.