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PubMed2005

Analysis of element accumulation in cell wall attached and intracellular particles of snow algae by EELS and ESI.

Snow algae frequently occur in alpine and polar permanent snow ecosystems and have developed adaptations to their harsh environment, where extreme temperature regimes high irradiation and low nutrient levels prevail. They live in a unique microhabitat, namely the liquid water between snow crystals. The predominant form appears as 'red snow' and in polar environment also 'green snow' frequently occurs. Light microscopy showed that most cells are densely covered by non-biotic particles of so far unknown composition. As snow normally contains very low amounts of nutrients, introduced mainly airborne like dust and precipitation, the inorganic particles at the surface of the snow algae may be important for their survival. By using electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI), we investigated element distribution in ultrathin sections of snow algae from different polar (Svalbard, 5 m a.s.l., 79 degrees N and maritime Antarctic, King George Island, 10 m a.s.l., 62 degrees S) and alpine habitats (2400-3100 m a.s.l. Tyrol) for the present study. It turned out that the main elements of the cell wall attached particles are Si, Al, Fe and O independently from the origin of the snow algae. Interestingly, the same elements were also found in vacuolar compartments inside the cells. These vacuoles contain electron dense granules or crystals and are frequently found to be connected to the cortical cytoplasm. This finding suggests an uptake mechanism of the respective elements by pinocytosis. Co-transport of toxic aluminium together with silicon may be unavoidable as the inorganic nutrient uptake of the snow algae is limited to the thin water layer between the ice crystals. However, formation of insoluble aluminium silicates may serve as detoxification mechanism.

Animals↗
PubMed2006

GCSF receptor regulates antigen uptake and expression of cytokines and costimulatory molecules in dendritic cells.

RNA interference (RNAi), a process that specifically silences target gene expression, is a powerful technique to modulate cellular functions. In this study, we identified two small interference RNA (siRNA) sequences that can specifically and efficiently silence the expression of the granulocyte colony-stimulating factor receptor (GCSF-R) gene and achieved stable knockdown of GCSF-R using pFIV lentivirus containing the GCSF-R siRNA. GCSF-R knockdown significantly reduces the expression of IL-lalpha, IL-lbeta, IL-6, IL-10, H-2Kb, I-Ab, CD80 and CD86, and increases PDL1 and PDL2 expression, while IL-12p35, TGFbeta, TNFalpha and CD40 expression is unaltered. Furthermore, GCSF-R knockdown significantly changes the endocytosis and micro-pinocytosis abilities as well as surface expression of antigens of DC2.4 cells.

Animals↗
PubMed1976

Mechanisms for localization of radiopharmaceuticals in neoplasms.

Localization of a radiopharmaceutical agent in a "tumor" is best conceptualized in terms of the altered regional physiology attendant to the presence of the "tumor". Such localization should be expected to occur in association with other disease states characterized by similar altered regional physiology. Neoplasms, areas of inflammation, and certain phases of infarct development are characterized by increased permeability of their capillary beds to macromolecules. This is largely due to neovascularization and the large intercapillary pores associated with new growth of capillary beds in these circumstances. Often, total perfusion to such lesions is increased in comparison to surrounding normal tissue. Thus, in all three clinical conditions, the entry of macro-molecules into the interstitial fluid space from the intravascular space is increased above that seen in normal tissue. Moreover, with neoplasms and inflammatory processes, there may be a delay in new lymphatic vessel growth adding to the residence time of the macromolecules in the interstitial fluid space. In all three conditions, the increased macrophage activity associated with tissue necrosis may result in ingestion of the labeled macromolecule by the macrophage. Pinocytosis may result in ingestion of the labeled macromolecule by other cells in the lesion, or there may be specific receptor sites on the cell membrane for the macromolecule, which may lead to fixation of the labeled macromolecule on the cell surface and possible intracellular translocation of the label itself. Radiolabeled macromolecules such as albumin, fibrinogen, or gamma globulins, and radionuclides that bind to macromolecules such as radiogallium, radioindium, and other radioelements, exhibit localizing behavior in tumors, inflammatory lesions, and during certain stages of infarcts. In the case of radiogallium and radioindium, the binding macromolecule is transferrin, and it is known that some cells have specific receptor sites for transferrin-bound iron on the cell membrane. It is possible that certain cells within these lesions have cell membrane receptor sites for radiogallium- and radioindium-labeled transferrin, and the cell erroneously accepts these radioelements from the transferrin in lieu of iron in attempting to engage in heme enzyme synthesis. Another mechanism that may be operative in the localization of agents in neoplasms, infarcts, and inflammatory lesions may be the altered cell permeability found in many cells of such lesions. It is known that many agents, such as supravital dyes, are excluded from entering normal cells by the selective permeability of normal cell membranes. When cell membrane permeability is altered, such as can be seen in traumatized, dying, or dead cells, the normally excluded agent may penetrate the abnormal cell membrane and bind, and consequently accumulate in intracellular constituents.

Binding Sites↗
PubMed2001

Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy.

Bafilomycin A(1) (BAF) and concanamycin A (ConcA) are selective inhibitors of the H(+)-ATPases of the vacuolar system. We have examined the effects of these inhibitors on different steps in endocytic pathways in rat hepatocytes, using [(125)I]tyramine-cellobiose-labeled asialoorosomucoid ([(125)I]TC-AOM) and [(125)I]tyramine-cellobiose-labeled bovine serum albumin ([(125)I]TC-BSA) as probes for respectively receptor-mediated endocytosis and pinocytosis (here defined as fluid phase endocytosis). The effects of BAF and ConcA were in principle identical, although ConcA was more effective than BAF. The main findings were as follows. (1) BAF/ConcA reduced the rate of uptake of both [(125)I]TC-AOM and [(125)I]TC-BSA. The reduced uptake of [(125)I]TC-AOM was partly due to a redistribution of the asialoglycoprotein receptors (ASGPR) such that the number of surface receptors was reduced approximately 40% without a change in the total number of receptors. (2) BAF/ConcA at the same time increased retroendocytosis (recycling) of both probes. The increased recycling of the ligand ([(125)I]TC-AOM) is partly a consequence of the enhanced pH in endosomes, which prevents dissociation of ligand. (3) It was furthermore found that the ligand remained bound to the receptor in presence of BAF/ConcA and that the total amount of ligand molecules internalized in BAF/ConcA-treated cells was only slightly in excess of the total number of receptors. These data indicate that reduced pH in endosomes is the prime cause of receptor inactivation and release of ligand in early endosomes. (4) Subcellular fractionation experiments showed that [(125)I]TC-AOM remained in early endosomes, well separated from lysosomes in sucrose gradients. The fluid phase marker, [(125)I]TC-BSA, on the other hand, seemed to reach a later endosome in the BAF/ConcA-treated cells. This organelle coincided with lysosomes in the gradient, but hypotonic medium was found to selectively release a lysosomal enzyme (beta-acetylglucosaminidase), indicating that even [(125)I]TC-BSA remained in a prelysosomal compartment in the BAF/ConcA-treated cells. (5) Electron microscopy using horseradish peroxidase (HRP) as a fluid phase marker verified that BAF/ConcA inhibited transfer of material from late endosomes ('multivesicular bodies'). (6) BAF/ConcA led to accumulation of lactate dehydrogenase (LDH) in autophagic vacuoles, but although the drugs partly inhibited fusion between autophagosomes and lysosomes a number of autolysosomes was formed in the presence of BAF/ConcA. This observation explains the reduced buoyant density of lysosomes (revealed in sucrose density gradients). In conclusion, BAF/ConcA inhibit transfer of endocytosed material from late endosomes to lysosomes, but do not at the same time prevent fusion between autophagosomes and lysosomes.

Animals↗
PubMed1982

Fine structure of human two-cell ova fertilized and cleaved in vitro.

The human two-cell ovum consists of two equally sized blastomeres, surrounded by the zona pellucida. Cumulus cells may be apposed to the exterior of the zona. The blastomere nuclei contain spherical nucleoli, made up of fibrillar matrix, and randomly dispersed chromatin. The cytoplasmic structures include mitochondria, smooth endoplasmic reticulum, annulate lamellas, the Golgi complex, free ribosomes, secondary lysosomes, cortical granules, and microtubules. The cell membrane bristles with microvilli and displays signs of pinocytosis except at the site of apposition of the two blastomeres. The fine structure of the two-cell ova fertilized and cleaved in vitro corresponds with the presumptive pattern with respect to the current knowledge of mammalian embryo preimplantation development. The ova seem to have been capable of further growth.

Adult↗
PubMed1999

Mechanisms for regulating platelet high density lipoprotein type3 binding sites: evidence that binding sites are downregulated by a protein kinase C-dependent mechanism.

The mechanisms for regulating platelet HDL3 binding sites were investigated. HDL3 binding was rapid (T(1/2) association=4 minutes) and completely reversible (T(1/2) dissociation=14.5 minutes) at 4 degrees C, 22 degrees C, and 37 degrees C, and kinetic analysis yielded forward and reverse constants of 7.3x10(-4) x s(-1) and 7.13x10(3) x s(-1) x M(-1), respectively. Nevertheless, neither inhibitors of binding sites recycling or of pinocytosis, such as ammonium chloride, chloroquine, monensin, colchicine, and sodium azide, modified the binding characteristics. Moreover, when platelets were loaded with cholesterol, binding sites were not regulated (up or down). However, when exposed to high concentrations of HDL3 (1.5 g/L), apoE-free HDL (1.5 g/L), HDL2 (0.5 g/L), apoE-rich HDL (0.5 g/L), and VLDL (0.3 g/L) there was rapid downregulation of the number of binding sites in isolated permeabilized platelets, as shown by the reduction of Bmax to 66%, 58%, 45%, 53%, and 51%, respectively. Downregulation was rapid, reversible, and dose and time dependent. In contrast, LDL (up to 2.0 g/L), IDL (up to 0.1 g/L), and chylomicrons (up to 0.5 g/L) had no effect. Protein kinase C inhibitors (150 nmol/L staurosporine, 100 micromol/L H-7, and 10 nmol/L bisindolylmaleimide) inhibited downregulation up to 62% (as average value). The role of the PKC activation in regulating the activity of HDL3 binding sites also was analyzed by determining the cytosol-to-membrane translocation of enzymatic activity. Downregulation mediated by HDL3 rapidly translocated PKC activity (21% +/- 11 of total PKC activity was membrane-associated in control platelets vs. 55+/-8% in downregulated platelets, mean+/-SEM, n=3). However, agents that block sequestration (0.30 g/L, concanavalin A), and other protein kinase inhibitors, such a cAMP-dependent protein kinase inhibitors (1 micromol/L, PKI), and beta2-adrenergic receptor kinase inhibitors (100 nmol/L, heparin) had no effect. The results show that neither endocytotic response nor cholesterol-dependent mechanisms participate in the modulation of platelet HDL3 binding sites. However, a new regulatory mechanism that involves PKC-dependent downregulation of the number of binding sites may be an important pathway to regulate the thrombogenicity of lipoproteins and their effects on platelet reactivity.

Binding Sites↗
PubMed1987

5'-Nucleotidase activity in cultured rat leptomeninges.

By means of a cytochemical procedure, 5'-nucleotidase activity was demonstrated in rat leptomeninges explanted to in vitro conditions. In organotypic type of culture, the enzyme activity was present on the membranes of cell bodies and their processes. High enzymatic activity was due to the cell-to-cell contact and also to pinocytosis and enhanced metabolism of nucleotides in meningeal cells in vitro.

5'-Nucleotidase↗
PubMed2001

In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts.

The cytotoxicity of dextran T40, methacrylated dextran (dex-MA) and hydroxyethyl-methacrylated dextran (dex-HEMA), dextran-based hydrogel discs and microspheres, and their degradation products, was studied by measuring the cell proliferation inhibition index (CPII) on human fibroblasts in vitro. In addition, during the 72 h incubation period light-microscopic observations were performed daily. After 24 h of incubation with dextran and dex-HEMA polymers, the cells showed elongated or spider-like forms, some lipid droplets and intracellular granula, indicative of pinocytosis and internalization of the polymers. During the next two days, the fibroblasts' appearance did not change. Methacrylic acid (MAA), formed by hydrolysis of dex-HEMA, did not influence the cell morphology. Dex-HEMA polymer solutions with a low and high degree of substitution (DS) at 100 mg/ml caused a CPII of 30-40% after 72 h. This is less than 10% growth inhibition per cell cycle and statistically not different from the CPII induced by 100 mg/ml dextran T40. Growth inhibition induced by MAA was also low. The various dex-MA hydrogel discs caused similar low growth inhibition. Interestingly, hydrogel microspheres of dex-MA and dex-(lactate-)HEMA caused a CPII of only 0-20% after 72 h. The results presented in this study demonstrate that methacrylate-derivatized dextran hydrogels show good biocompatibility in vitro making these degradable biomaterials promising systems for drug delivery purposes.

Biocompatible Materials↗
PubMedDate not supplied

Fluid shift across the placenta: II. Fetomaternal transfer of horseradish peroxidase in the guinea pig.

The fetal side of the isolated guinea-pig chorio-allantoic placenta has been perfused with a fluid containing horseradish peroxidase. Electron micrographs show that protein is transferred across the fetal capillary endothelium via micropinocytosis as well as by transport through intercellular clefts. Under normal hydrostatic pressure no pinocytosis or other signs of fetomaternal protein transfer across the trophoblast have been observed. Instead, macrophages ingest the proteins that have reached the interstitial space between fetal endothelium and trophoblast. When the fetal venous pressure is raised up to 20 mmHg about 30 to 50 per cent of the arterial perfusion fluid, including proteins, is shifted into the maternal lacunae. In the beginning of this process slender tubules, and later on bag-like channels, appear that cross the trophoblast and probably connect lacunae and interstitium. The inner surface of the channels and the maternal lacunar surface are labelled by peroxidase. This indicates a fetomaternal transfer of proteins conveyed by the fluid shift. Protein transfer and fluid shift return to their previous levels when the fetal venous pressure is reduced. The size and number of channels are reduced but their structure and the reversibility of the events refute the possibility that they are of traumatic origin. It cannot be decided whether the channels are pre-existent but unrecognizable, or de-novo formations.

Animals↗
PubMedDate not supplied

Placental accumulation of 57Co-vitamin B12 in mice studied by light- and electron-microscopic autoradiography.

Pregnant C57BL mice on day 18 of gestation were injected with 0.114 micrograms cyanocobalamin-57Co and killed either 40 min or 3 h later. Radioactivity measurements showed that the placenta was the organ with the highest concentration. Light- and electron-microscopic autoradiography of the placental labyrinth showed heavy accumulation of radioactivity in the second (middle) trophoblastic cell layer in the barriers between the maternal and fetal circulations. Silver grains were mainly associated with the folded plasma membrane deep in the cytoplasm or with vesicles, indicating a cellular uptake by pinocytosis. The cytosolic accumulations present may be derived from cellular metabolic sites, such as that of protein synthesis.

Animals↗
PubMed2000

The ins and outs of getting in: structures and signals that enhance BCR or Fc receptor-mediated antigen presentation.

Antigen-presenting cells internalize antigen by fluid-phase pinocytosis or by endocytosis via surface receptors such as the B cell receptor (BCR) and Fc receptors for IgG, IgA and IgE (FcR). While both modes of internalization lead to antigen presentation it is recognized that receptor-mediated endocytosis greatly enhances the efficiency of processing and antigen presentation. Receptors facilitate the entry of antigen into the endocytic pathway by interaction of their internalization motifs with the endocytic machinery. These motifs include tyrosine-based, dileucine and casein kinase-like motifs. However these structures appear insufficient to support processing of cryptic epitopes, leading to a limited immune response. Cryptic epitope processing appears dependent on receptor signaling which is mediated by immunoreceptor tyrosine activation motifs (ITAMs). The signaling cascade which follows receptor crosslinking promotes reorganization and acidification of the late endocytic compartment or MIIC. Signaling events downstream of Syk, in particular calcium flux and protein kinase C activation, are necessary for MIIC induction. PI(3) kinase is also involved at multiple steps in antigen presentation, including production of PIP3 and transport of cathepsins. PIP3 is crucial both as a binding substrate for proteins implicated in vesicle transport and for the recruitment of signaling molecules to the plasma membrane. Among PIP3 activated molecules, protein kinase B (PKB) has been linked to endocytic function. We observe association of activated PKB with the MIIC after signaling through antigen presentation-competent receptors, but not mutant, presentation-defective receptors.

Amino Acid Sequence↗
PubMed2003

Immunophenotype and functional properties of feline dendritic cells derived from blood and bone marrow.

Dendritic cells (DCs) are a heterogeneous population of cells of fundamental importance in initiating innate as well as specific immune responses. The identity and function of DCs in the cat are unknown, although they are likely pivotal in the response to infection. In this study, feline DCs were derived by 3-10-day culture of adherent blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) in the presence of IL 4 and GM-CSF. BMMC consistently yielded a greater number of DCs than PBMC, and there were fewer macrophages than DC from both compartments. DCs expressed a distinct constellation of surface molecules, which included CD1a, CD1b, and CD1c, CD11b, CD14, and 2-3-fold higher levels of MHC class I and II molecules than co-cultured macrophages or fresh blood monocytes. DCs displayed typical cytoplasmic processes, limited non-specific esterase activity, and acquired antigen by phagocytosis, pinocytosis, and binding to specific receptors. Cytokine-exposed cells induced proliferation of allogeneic lymphocytes. Thus, the cells derived by these culture conditions had markers and functions analogous to immature myeloid DCs. Availability of feline DCs will enable investigation of their role in infectious disease and their potential therapeutic application.

Animals↗
PubMed1999

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4.

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.

Animals↗
PubMed1998

Extravasation of macromolecules.

Macromolecules can extravasate across the normal endothelium by transcapillary pinocytosis as well as by passage through interendothelial cell junctions, gaps or fenestrae. The main biological factors that control extravasation of a solute include regional differences in the capillary structures, the disease state of the organ or tissue, and the rate of blood and lymph supply. Physicochemical properties that are of profound significance in the extravasation of macromolecules are molecular size, shape, charge and hydrophilic/lipophilic balance (HLB) characteristics. Extravasation of small drugs, proteins, oligonucleotides and genes can be controlled by conjugating or forming complexes with macromolecular carriers. This requires a thorough understanding of the relationship between the chemical structures, physicochemical properties and the pharmacokinetics of both carrier and active molecules. This review article discusses the extravasation of macromolecules from the view points of pharmacokinetics and drug delivery systems, with the main emphasis on the extravasation across the liver, kidney and tumor capillaries.

Journal Article↗
PubMed1995

Differential sensitivity to antigenic competition in antigen-specific and -nonspecific antigen presentation by B cells.

We have previously shown that a specific Ag presentation by B cells is different from a nonspecific one in the sensitivity to protein synthesis inhibition. In the present study we have compared the sensitivity of these Ag presentations to antigenic competition. A20-HL cells expressing TNP-specific IgM were pulsed with anti-mouse IgM goat IgG (aMGG) or trinitrophenylated goat IgG (TNP-NGG) as an Ag internalized through Ag receptor or NGG as an Ag internalized by fluid-phase pinocytosis. The pulsed cells induced IL-2 production by NGG-specific cloned T cells. The presence of dog IgG during pulsing A20-HL cells severely inhibited the presentation of NGG but not of aMGG or TNP-NGG. The presence did not decrease the internalization of 125I-NGG into A20-HL cells, suggesting that the inhibition was localized into the complex formation of antigenic peptides with MHC class II molecules. Thus, a specific Ag presentation by A20-HL cells is different from a nonspecific one in its sensitivity to antigenic competition.

Animals↗
PubMedDate not supplied

Effect of aging on the blood-brain barrier.

Aging is commonly associated with progressive deterioration in central nervous system (CNS) function. Nutritional factors or environmental toxins have important effects on CNS degenerative changes. The blood-brain barrier (BBB) is a major modulator of nutrient delivery to the CNS. The tight junctions and the paucity of pinocytosis or fenestrations in brain capillary endothelium act as an effective barrier between the CNS and the circulating toxic agents. Senescence is associated with significant, though often subtle, changes in BBB. Conditions which are commonly associated with aging, such as hypertension and cerebrovascular ischemia, aggravate the age-related alterations in BBB function. The histologic changes in brain vasculature with aging is region selective and species specific. The common age-related histologic changes include loss of capillary endothelial cells, elongation of the remaining endothelial cells, and decreased capillary diameter in rat cortex, but not in the monkey or human cortex, and a decrease in the number of mitochondria in endothelial cells of the brain capillaries in the monkey but not in the rat. The age-related alterations in BBB transport function include a decrease in BBB choline transport with aging and decreased brain glucose influx. The BBB neutral amino acid transport appears to be unaltered in the aged mice. Most of the studies reported so far have failed to show a significant age-related alteration in BBB permeability to water-soluble substances and high molecular weight solutes in the absence of neurological disease. A more profound change in BBB permeability appears to be associated with Alzheimer's disease. Immunohistological studies have demonstrated the presence of serum proteins in the cerebrovascular amyloid in patients with Alzheimer's disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1984

Experimental brain tumors and edema in rats. II. Tumor edema.

Experimentally induced brain tumors in rats with the morphological characteristics of low differentiated gliomas were investigated with special consideration of the distribution of extravasated serum proteins within the tumor itself. Protein extravasation across tumor vessels occurs by pinocytotic vesicles which enclose serum proteins at the luminal surface of the endothelial cells. These vesicles move to the abluminal cell surface where proteins are released after fusion between vesicles and the cell membrane. Serum proteins then spread through the extracellular space from where they are taken up in tumor cells by pinocytosis. They are then transmitted to and digested by lysosomes in which acid phosphatase activity could be demonstrated. Tracer studies (human serum albumin, horseradish peroxidase) revealed that proteins reach the lysosomes 15 min after systemic application. The cellular content of tracers is variable even when systemic circulation has lasted 120 min.

Animals↗
PubMed1997

The efficiency of antigen delivery from macrophage phagosomes into cytoplasm for MHC class I-restricted antigen presentation.

Macrophages can present exogenous antigen in association with MHC class I molecules. Indirect evidence indicates that antigens internalized by phagocytosis can enter cytoplasm before following the conventional MHC class I presentation pathway. However, little is known about how common such entry is, or to what extent it depends on the kind of particle ingested. This study reports quantitative and morphological characterization of antigen delivery from phagosomes into cytoplasm for MHC class I-restricted antigen presentation. Ovalbumin (OVA) was associated with polystyrene particles (PS), biodegradable poly-e-caprolactone particles (PCL), and sheep red blood cells (SRBC), and its delivery into macrophage cytoplasm, via phagocytosis was assessed with a T hybridoma assay for MHC class I-restricted presentation of OVA-derived peptides. Although direct introduction of antigen into cytoplasm by scrape-loading produced the most efficient presentation, comparable signals could be obtained after phagocytosis of PCL or PS. Phagocytosis of OVA-loaded SRBC, and OVA internalized by pinocytosis, did not deliver efficiently. MHC class I-restricted presentation of phagosome-derived OVA required cytoplasmic processing, as it was inhibited by proteasome inhibitors and brefeldin A. Morphological studies showed that biotinylated OVA originating in PCL phagosomes could be delivered into the cytoplasm of 90% of the macrophages. These results indicate that phagocytosis per se is not sufficient to deliver antigen into cytoplasm, but that phagocytosis of solid, synthetic polymeric particles delivers antigen efficiently into cytoplasm for MHC class I processing.

Animals↗