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The restricted or regulated entry of most blood-borne substances into the brain has been recognised for more than a century. The blood-brain barrier (BBB)-shielding function provided by endothelial cells is important in the treatment of neurological diseases because this exclusion of foreign substances also restricts entry of many potentially therapeutic agents into the brain. The recent identification of several neuroactive proteins of potential therapeutic value has highlighted the crucial need for effective and safe transcapillary delivery methods to the brain. One promising method is delivery through brain capillaries by augmentation of pinocytotic vesicles delivery systems that use this cellular mechanism are in development. Recent investigations in animal models show that large molecules of neurotherapeutic potential can be conjugated to peptidomimetic ligands, which bind to selected peptide receptors, and are then internalised and transported in small vesicles across the cytoplasmic brain capillary barrier. These conjugates have been shown to remain functionally active and effective in animal models of neurological disease.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that use 2 major pathways for antigen uptake: constitutive macropinocytosis and mannose receptor-mediated endocytosis. Efficient endocytosis is critical for DCs to fulfill their sentinel function in immunity. We investigated the influence of the immunosuppressive macrolide rapamycin on macropinocytosis of fluorescein isothiocyanate (FITC)-albumin and mannose receptor-mediated endocytosis of FITC-dextran by murine bone marrow-derived DCs by flow cytometry. The data show that (1) at a low, physiologically relevant concentration (1 ng/mL), rapamycin impairs macropinocytosis and mannose receptor-mediated endocytosis; (2) the effects are independent of DC maturation and can be demonstrated specifically in immature CD11c(+) major histocompatibility complex (MHC) class II(lo) DCs by 3-color flow cytometry; (3) inhibition of endocytosis is not related to apoptotic cell death; and (4) molar excess of the structurally related molecule FK506 inhibits the actions of rapamycin. The inhibitory effects of rapamycin on DC endocytosis were confirmed in vivo. To our knowledge, this is the first report that a clinically relevant immunosuppressant inhibits DC endocytosis.

Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-beta-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However, confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus, our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Previous studies introduced cytochrome c into intact cells via the disruptive techniques of microinjection or electroporation to provide support for the hypothesis that, in whole cells, cytochrome c release from mitochondria triggers caspase activation and other degradative changes. However, the types of measurements that could be undertaken with these techniques was limited. We used the simple and relatively gentle technique of pinocytic loading to demonstrate that, in intact cells, cytosolic cytochrome c specifically induced activation of caspase-3- and -9-like enzymes, and a loss of mitochondrial polarization coincident with an increase in mitochondrial permeability. Our results support the prediction from in vitro studies that activation of caspases-3 and -9 is downstream of cytochrome c release and provide the first direct evidence that, in whole cells, cytochrome c-dependent caspase-activation can exert a feedback effect to elicit mitochondrial permeabilization and collapse of the mitochondrial trans-membrane potential.

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Ibeta and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.

Activation of macrophages with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) leads to increased intracellular resistance to microbes and increased major histocompatibility complex class II-restricted antigen presentation, processes that both use the vacuolar compartment. Despite the requirement of the macrophage vacuolar compartment for microbicidal activities and antigen processing, the rates of endocytosis and membrane trafficking in activated macrophages are not clearly defined. In this study, vacuolar compartment dynamics were analyzed in murine bone marrow-derived macrophages activated with LPS and/or IFN-gamma, conditions that increased macrophage nitric oxide production and resistance to infection by Listeria monocytogenes. Relative to nonactivated cells, activated macrophages showed diminished rates of fluid-phase pinocytosis and phagocytosis and delayed progression of macropinosomes and phagosomes to late endosomes and lysosomes. In contrast to the slowing of membrane trafficking, rates of macropinosome acidification were similar between activated and nonactivated cells. One consequence of this slowed membrane trafficking in activated macrophages was a prolonged exposure of incoming molecules to an acidic nonlysosomal compartment, a condition which may facilitate microbicidal chemistries or antigen processing.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.

Background: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells., Results: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis., Conclusions: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.

The authors previously showed that monocytes treated with calcium ionophore (CI) acquire characteristics of mature dendritic cells (DC) in part through a calcineurin-dependent pathway. In this study, the authors evaluated the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), and interleukin-12 (IL-12) alone or in combination with CI to induce DC characteristics in peripheral blood monocytes. Monocytes obtained by leukapheresis and countercurrent centrifugal elutriation were cultured with calcium, cytokines, or both, profiled by flow cytometry, and assessed for antigen uptake and sensitization of autologous CD8+ T cells to antigen. Monocytes treated with the combination of GM-CSF, IL-2, and IL-12 resulted in immunophenotypic and antigen uptake profiles typical of immature DC, including loss of surface CD14 expression, de novo low-level expression of B7.1, negligible CD83 expression, marked enhancement of CD40 and ICAM-1, and high major histocompatibility complex class I and II levels. A high level of antigen uptake by macro-pinocytosis was observed. In contrast, CI treatment significantly up-regulates B7.1, B7.2, CD40, CD54, and CD83 and substantially down-regulates CD14 and macro-pinocytosis, a profile consistent with mature DC. Many CI-induced modulations, but none resulting from cytokine treatment alone, were inhibited by the calcineurin phosphatase inhibitor cyclosporin A. Compared with monocytes treated with CI alone, combined treatment of monocytes with GM-CSF, IL-2, IL-12, and CI augmented B7.1 and CD83 expression and enhanced sensitization of autologous CD8+ T cells to melanoma-antigen-derived peptides. These results suggest that several independent pathways of DC activation can cooperatively enhance the function of monocyte-derived DC.

Immature dendritic cells (DCs) constitutively take up large volumes of fluid by macropinocytosis and concentrate the macrosolutes in the endocytic compartment. This concentration mechanism that is the basis of their high capacity to present soluble antigens requires that DCs be capable of rapidly exchanging water across their membranes. We report that two members of the aquaporin family, AQP3 and AQP7, are expressed in immature DCs and are downregulated after maturation. Treatment of DCs with p-chloromercuribenzenesulphonate (pCMBS), a mercuric drug that blocks aquaporins, inhibited uptake and concentration of macrosolutes taken up by fluid phase endocytosis and led to dramatic cell swelling. In contrast, pCMBS did not affect receptor-mediated endocytosis via the mannose receptor. These findings indicate that aquaporins represent essential elements of a volume control mechanism that allows DCs to concentrate macrosolutes taken up via macropinocytosis.

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcgamma receptor-mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcgamma receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcgamma receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, SIRPalpha (a member of the signal-regulatory protein family, also known as Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPalpha. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.

Treatment of the BAC1.2F5 macrophage cell line with Macrophage Colony Stimulating Factor (M-CSF) resulted in a rapid induction of vesiculation that was reminiscent of macropinocytosis. Time-lapse micrography showed that these vesicles initiated as small vesicles at the cell periphery, but grew in size and migrated with time to a perinuclear localisation after growth factor stimulation. Immunofluorescence showed that the M-CSF receptor (c-fms) associated with the small vesicles and also the larger phase-bright vesicles. Treatment with two distinct inhibitors showed that the rapid initiation of vesicle formation was not dependent on phosphatidylinositol-3' (PI-3) kinase activity; however, the subsequent maintenance, maturation and translocation of the large, phase-bright, c-fms-containing vesicles was dependent on PI-3 kinase activity. The inhibitors could also reverse the further maturation of preformed vesicles. The inhibition of vesicle trafficking and maturation correlated with ablation of M-CSF-induced PI-3 kinase activity associated with p110(alpha). These data demonstrate a role for PI-3 kinase in vesicle trafficking and maintenance. PI-3 kinase activity was also necessary for the macropinocytotic response in macrophages, a process that is essential for efficient antigen processing and presentation in macrophage-like cells.

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC(50)=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC(50)=8.4 to 10 m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC(50)=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs., (Copyright 2000 Academic Press.)

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-epsilon. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-epsilon, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

The influence of the calcium channel blocker nifedipine plus bicuculline-induced seizures on the permeability of the blood-brain barrier to protein was studied in rats. Evans-blue was used as a blood-brain barrier tracer. Four groups of rats were studied. Group I: control, Group II: Nifedipine, Group III: bicuculline-induced seizure, Group IV: Nifedipine + seizure. The mean value for Evans-blue dye in the brain was found to be 0.23+/-0.03 mg/g in control animals and 0.32+/-0.06 mg/g in the group of all rats during nifedipine-induced hypotension. This difference between control and hypotensive animals was not statistically significant (p < 0.5). Mean value for Evans-blue dye in the brain was found to be 1.4+/-0.3 mg/g in bicuculline-induced seizure, and 0.73+/-40.2 mg/g in the group of nifedipine plus bicuculline-induced seizures. This difference between Group III and Group IV was found statistically significant (p < .01). The calcium channel blocker nifedipine significantly prevents the blood brain barrier disruption during bicuculline-induced seizure.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.

Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10-8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated. Dex-DC expressed low levels of CD1a and, unlike untreated cells, high levels of CD14 and CD16. Molecules involved in Ag presentation (CD40, CD86, CD54) were also impaired. In contrast, molecules involved in Ag uptake (mannose receptor, CD32) and cell adhesion (CD11/CD18, CD54) were up-regulated. After exposure to TNF-alpha or CD40 ligand, Dex-DC expressed lower levels of CD83 and CD86 than untreated cells. Dex-DC showed a higher endocytic activity, a lower APC function, and a lower capacity to secrete cytokines than untreated cells. Overall, these results indicate that DC differentiated in the presence of Dex are at a more immature stage. Moreover, Dex also partially blocked terminal maturation of already differentiated DC. In conclusion, our data suggest that glucocorticoids may act at the very first step of the immune response by modulating DC differentiation, maturation, and function.

Lipopolysaccharide (LPS) is a mediator of inflammation and septic shock during bacterial infection. Although monocytes and macrophages are highly responsive to LPS, the biological effects of LPS in these cell types are only partially understood. We decided, therefore, to investigate the influence of LPS on macrophage pinocytosis and Fc receptor-mediated endocytosis, two prominent and related macrophage effector functions. We observed that LPS did not greatly influence endocytosis in either macrophages or monocytes, but did exert a dual action on pinocytosis: at lower concentrations (0.1 to 100 ng/mL), LPS caused a decrease in pinocytosis in both macrophages and monocytes, whereas at higher LPS concentrations, enhanced pinocytosis in macrophages was observed. Detoxified LPS was two orders of magnitude less potent in producing these effects. After inhibition of the LPS receptor CD14, the LPS-induced decrease in pinocytosis was absent, and stimulation of pinocytosis at lower LPS concentrations was unmasked. We conclude that LPS can influence pinocytosis via CD14-dependent and CD14-independent signaling pathways. Furthermore, as addition of LPS to macrophages effected pinocytosis but not Fc receptor-mediated endocytosis, these two processes are independently regulated in macrophages.

TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mphi cytoskeleton, and that the ability of TNF to inhibit Mphi chemotaxis may be due to decreased filopodium formation downstream of Cdc42.

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals >5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals >5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom.

Human low density lipoprotein (LDL) is prepared in the presence of antioxidants and is oxidized to different levels (measured by thiobarbituric acid reactive substance) with copper ion. The effects of unoxidized LDL and oxidized LDL (ox-LDL) on stress fiber formation, cell membrane ruffling, and pinocytosis (measured by [14C]sucrose uptake) in cultured human umbilical cord vein endothelial cells (EC) are compared. We show that at a concentration range of 100 to 200 microg cholesterol/ml, both unoxidized LDL and ox-LDL promote EC elongation and stress fiber formation, but the effect by the latter is more prominent when compared at the same dose range. In addition, ox-LDL also induces EC membrane ruffling and promotes pinocytosis. These effects are positively correlated with the extent of LDL oxidation and depend on the dose of ox-LDL. Ox-LDL-promoted membrane ruffling and pinocytosis are effectively blocked by brief preexposure of the cells to antioxidants. In contrast, stress fiber formation is not affected by antioxidant pretreatment. Although unoxidized LDL also promotes [14C]sucrose uptake, it is less potent than ox-LDL and significantly higher concentrations are required to produce a detectable effect. Unlike ox-LDL, unoxidized LDL-enhanced pinocytosis is not accompanied by the appearance of membrane ruffling; therefore, they may act via different mechanisms. Elevated pinocytosis may increase transcytotic activity of the endothelium, leading to an increased influx of plasma components such as LDL into the subendothelial space.

A series of fluorescein derivatized poly-D-lysine (FITC-PDL) probes were used to elucidate the role of fluorescein in receptor binding of fluorescein-conjugated macromolecules to J774 murine macrophages. Poly-D-lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D-isomer. This concept enabled the focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and accessibility. The results differed from those previously obtained with FITC-BSA in that saturating fluorescein densities on the poly-D-lysine polymer resulted in diminished rates of uptake by macrophages. Receptor-mediated endocytosis via clathrin-coated pits was concluded based on results that showed inhibition of FITC-PDL uptake by intracellular K+ depletion but not by the macropinocytosis inhibitor, amiloride. Further, FITC-PDL was found to inhibit the endocytic uptake of FITC-BSA suggesting competition between the two probes at the level of a macrophage receptor. Association rates (kon) for binding to the macrophage surface were measured for the various FITC-PDL probes based on fractional receptor occupancies. Results are discussed on the basis of receptor recognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility of the hapten moieties to facilitate receptor crosslinking.

To characterize possible differences between the fluid-phase endocytosis (pinocytosis) of bovine serum albumin and the receptor-mediated endocytosis of asialo-orosomucoid (AOM) in isolated rat hepatocytes, both probes were conjugated to radioiodinated tyramine-cellobiose, [125I]TC. The use of these conjugates made it possible to measure the uptake and intracellular distribution of the intact proteins as well as of their acid-soluble, membrane-impermeant degradation products. [125I]TC-albumin was taken up at a very low rate (0.5%/h) compared to [125I]TC-AOM (45%/h), suggesting that neither membrane adsorption nor membrane permeation compromised its suitability as a fluid-phase marker. Sucrose gradient analysis indicated that both probes sequentially entered light endosomes (1.11 g/ml), dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml), but [125I]TC-albumin traversed the endocytic compartments more rapidly than [125I]TC-AOM, and was partially degraded intralysosomally already after 15 min. The microtubule inhibitor, vinblastine, had a stronger inhibitory effect on the uptake and degradation of [125I]TC-AOM (80% and 95%, respectively) than on the uptake and degradation of [125I]TC-albumin (50% and 70%, respectively). In the presence of vinblastine, [125I]TC-AOM was retained both in light and dense endosomes, whereas [125I]TC-albumin was retained in dense endosomes only, suggesting that the early steps of fluid-phase endocytosis were less critically dependent on microtubular function than the early steps of receptor-mediated endocytosis. A perturbant of vacuolar pH, propylamine, inhibited the degradation of both probes strongly (75-100%), as would be expected from its lysosomotropic effect. Propylamine also inhibited endocytic uptake, with a stronger effect on [125I]TC-AOM uptake (95% inhibition) than on [125I]TC-albumin uptake (60% inhibition), probably reflecting a reduction in endosomal acidity, reduced receptor-ligand dissociation and diminished recycling of free asialoglycoprotein receptors to the cell surface in addition to a general trapping of membrane in swollen vacuoles. A protein phosphatase inhibitor, okadaic acid, strongly (80-100%) inhibited the uptake and degradation of both [125I]TC-albumin and [125I]TC-AOM. An inhibitor of lysosomal proteinases, leupeptin, strongly suppressed the degradation of both probes and moderately reduced the uptake of [125I]TC-AOM, whereas the uptake of [125I]TC-albumin was unaffected. In contrast, an inhibitor of autophagic sequestration, 3-methyladenine, reduced both the uptake and degradation of [125I]TC-albumin markedly (55% and 75%, respectively), with considerably less effect on [125I]TC-AOM (25% and 35%, respectively). As autophagy-inhibitory amino acid mixture did not share these effects, suggesting that 3-methyladenine may suppress endocytic fluid-phase uptake by an autophagy-independent mechanism. Fluid-phase and receptor-mediated endocytosis in hepatocytes thus appear to differ with respect to uptake mechanisms as well as in the kinetics by which endocytosed material traverses the endocytic-lysosomal pathway.

The effects of cis-diamminedichloroplatinum(II) [cisplatin], a potential anticancer drug, were studied on pinocytotic functions in the cellular slime mould Dictyostelium discoideum by administering FITC-dextran as a fluid phase marker. Cisplatin treatment at a concentration of 100 and 200 micrograms/ml for 1 h causes inhibition in pinocytotic uptake in growing Dictyostelium cells in a dose-dependent manner. Cisplatin treatment induced the association of more actin with the cell cortex, thereby presumably restricting the flexibility of the cell membrane and inhibiting the formation of pinosomes. Ultrastructural analysis of cisplatin-treated cells showed a lower number of pinosomes. These results have been discussed in the light of cisplatin's known actions that affect various cellular functions.

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

1. A polysaccharide-enriched fraction (CPPS) was prepared from Codonopsis pilosula root extract utilizing a procedure that entailed extraction with aqueous buffer and precipitation with ethanol. 2. After administration of CPPS in drinking water to C57BL/6 mice at a dosage of 10 mg/L for 4 weeks, the splenocytes exhibited lowered mitogenic responses to Concanavalin A (ConA) and lipopolysaccharide (LPS). The in vitro production of reactive nitrogen intermediates was inhibited. 3. However, when oral administration of CPPS was prolonged to 8 weeks, there was a potentiation of ConA-stimulated and LPS-stimulated mitogenic responses. 4. When tested under in vitro conditions, CPPS augmented the mitogenic response of splenocytes to ConA. However, there was no effect on the pinocytic activity of mouse macrophages, nor was there any proliferative activity on mouse melanoma B16 cells.

In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca2+ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20 degrees C and used to predict the amount of extracellular Ca2+ taken up by pinocytosis. This was compared with the measured rate of Ca2+ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca2+ + Mg2+)ATP-ase. Fluid uptake by pinocytosis was measured using [14C]sucrose. In hepatocytes incubated at 20 degrees C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca2+ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20 degrees C by measuring the uptake of 45Ca2+. The degree of stimulation of Ca2+ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn(2+)-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd3+. It was calculated that the process of pinocytosis accounts for at least 15% of Ca2+ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca2+ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca2+ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca2+ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca2+ inflow.

The physiological role of the beta-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (beta TC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3-4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting beta TC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these beta-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these beta TC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting beta-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting beta-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in beta TC6-F7 cells is not attributable to autocrine activation of beta-cell surface insulin receptors.

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40 degrees C but recovers its tyrosine kinase activity upon transfer to 34 degrees C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34 degrees C) as compared to non-transformed cells (Rat-1, Rat-1/tsLA29 kept at 40 degrees C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis. Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 microU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristic responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.

We have previously shown that ethanol administration impairs the processes of fluid-phase endocytosis (FPE) and receptor-mediated endocytosis (RME) in isolated rat hepatocytes after as early as 1 week of ethanol administration. The defects in RME were most prominent in the perivenule (PV) region of the liver lobule, the area wherein alcoholic liver disease has been shown to start and predominate. We undertook the present study to see if changes in FPE were likewise more apparent in the PV versus the periportal (PP) region of the liver. For these studies, we fed male Sprague-Dawley rats with an ethanol-supplemented liquid diet or an isocaloric control diet for 1 or 5 weeks. PV and PP hepatocytes were isolated using a digitonin-collagenase perfusion method. Internalization and efflux of the marker dye, Lucifer Yellow, was then examined in the cell populations. After as early as 1 week of feeding, cells from the PV region in ethanol-fed animals showed dramatic impairments in the net internalization of dye, compared with PV cells from the pair-fed controls, and these changes persisted throughout the 5-week feeding period. In contrast, internalization of Lucifer Yellow in cells from the PP region of the liver were not different between control and ethanol animals. Because net internalization represents the balance between uptake into the cells versus efflux from the cells, we examined these components individually. Early uptake of the dye into the cell was not altered by ethanol treatment. The decreased net internalization seemed to be caused by enhanced efflux of the dye, which was significantly increased in PV cells, compared with the same cell type in control animals. Cells from the PP region of the ethanol-fed animals did not exhibit altered efflux after either 1 or 5 weeks of feeding. These results indicate that ethanol-induced impairments in FPE are more dramatic in the PV region of the liver, and these impairments seem to result from an ethanol-induced enhancement of efflux.

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) +/- interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF +/- IL-4 suppressed T cell responses. Treatment of FSDC with IFN-gamma reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4+ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.

HEp-2 cells internalize non-pathogenic Escherichia coli bacteria by a low-efficiency internalization mechanism which is upregulated in Pho-derepressed strains (as shown by Sinai and Bavoil in 1993), and is independent of microfilament integrity but requires functional microtubules. Here, we further characterize the microtubule requirement of this pathway using various effectors of microtubule integrity and function. Furthermore, we show that internalization is enhanced upon treatment with monodansylcadaverine, a specific inhibitor of receptor mediated endocytosis, and is insensitive to brefeldin A, which promotes the microtubule-dependent reorganization of the endosome. An assay system is also described to directly evaluate the contribution of pinocytosis to this pathway based on the ability of the bacteria to cointernalize and consequently colocalize with the fluid-phase marker, Texas-red-conjugated dextran (TRD). Using this assay, Hoescht-stained bacteria were observed in TRD-containing vesicles in numbers that are consistent with their observed internalization rate. Overall, these data are strongly supportive of the existence of a low-efficiency macropinocytic mechanism of entry for these non-pathogenic bacteria. Moreover, the observed requirements for host tyrosine kinase and protein kinase C activities suggest that it is inducible.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.

In order to define the influence of contact allergens on the fluid-phase endocytosis (FPE) of soluble molecules of murine epidermal Langerhans cells (LC), we studied the internalization of FITC-labeled bovine serum albumin (FITC-BSA), TRITC-labeled dextrane (TRITC-DEX) as well as horseradish peroxidase by LC. A 3-parameter flow-cytometric technique was performed for quantification of internalized FITC-BSA in LC using quantum red-labeled reagents for detection of Ia-antigen expression by LC and propidium iodide for exclusion of dead cells from analysis. A temperature-dependent rapid accumulation of FITC-BSA was noticed in time-course studies reaching a plateau between 1 and 2 h of in vitro culture at 37 degrees C. The quantity of FPE under stimulation with phorbol 12-myristate 13-acetate (PMA), concanavalin A (Con A), staphylococcal enterotoxin B (SEB) and contact sensitizers (DNFB, Kathon CG, K2Cr2O7) as well as the irritant SLS was determined. Treatment of LC with PMA and Con A resulted in a significant increase of total FITC-BSA uptake. The contact sensitizers as well as SEB and SLS failed to mediate augmented fluid-phase endocytosis. By use of the pH-insensitive soluble marker, TRITC-DEX and a microscope photometer for evaluation these findings could be confirmed. This excluded any artificial influence of differences in pH values in endocytotic compartments which might have influenced the fluorescence intensity of the pH-sensitive fluorochrome FITC. For qualitative analysis of FPE, the intracellular distribution of internalized horseradish peroxidase in LC was studied. An aggregated pattern became apparent in untreated LC and did not change under stimulation with any of the substances used.(ABSTRACT TRUNCATED AT 250 WORDS)

The body distribution of poly(vinyl alcohol) (PVA) with molecular weights (MW) from 14,800 to 434,000 Da was investigated after intravenous administration and compared with that of other water-soluble polymers such as poly(ethylene glycol) (PEG), gelatin, dextran, and pullulan. The half-life of PVA in the circulation was prolonged from 90 min (MW 14,800 Da) to 23 h (MW 434,000 Da), similar to that of PEG which had a half-life of 30 min (MW 6000) and 20 h (MW 170,000). However, the half-life of PVA was much longer than that of other polymers when compared at a similar molecular weight. PVA was located in most organs but with very small accumulation. An insignificant interaction of PVA with cell components, such as macrophages and blood cells, was observed. Similar to PEG, the excretion rate of PVA at the glomeruli was rapidly reduced around 30,000 Da, as the molecular weight increased. These results indicate that the half-life of intravenously injected PVA in the blood was mainly determined by the permeation characteristics of the kidney.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.

The results suggest that protein kinase C (PKC) plays a pivotal role in the control of F-actin levels, locomotion, pinocytosis, and cell shape in lymphocytes. The PKC inhibitor Ro 31-8220 elicits a high proportion of polarized (ED50 = 1.5 x 10(-6) M) and locomoting cells and reduces the relative amount of F-actin (by 29% at 10(-5) M) in initially resting cells. Phorbol myristate acetate (PMA) counterbalances the polarizing effect of Ro 31-8220. This indicates that the spherical shape and the F-actin content of resting cells are maintained by constitutive PKC activity. PMA-induced increases in fluid pinocytosis, F-actin content, and formation of nonpolar cells with surface protrusion are suppressed by Ro 31-8220 (IC50 = 2-4 x 10(-7) M). Spherical cells and, at higher concentrations (ED50 = 3.3 x 10(-6) M), polarized cells are formed instead. As a result, lymphocyte function switches from fluid pinocytosis to cell polarity and locomotion. The data indicate that PKC is instrumental in selectively switching lymphocyte function between resting state, locomotor activity, and fluid pinocytosis. Ro 31-8220 is extremely potent in stimulating lymphocyte polarity and locomotion (B and T cells). It acts faster and/or produces a higher proportion of polarized lymphocytes than other available agonists. It may thus be used as a tool in further experiments requiring locomoting lymphocytes.

Electropermeabilization is a technique which allows free access of molecules to cytosol. In the present study, we report on results dealing with the penetration of macromolecules. Under electric conditions that allow maintenance of cell viability at a high level, i.e., at low electric field intensity but long time duration (ms time range), all the pulsed cells become permeable to macromolecules. By loading beta-galactosidase the electro-transferred activity in the cells is maintained over 24 hours. Transfer mediated during the pulse occurs by free diffusion into the cytoplasm, while post pulse transfer takes place by a different pathway. When added a few minutes after application of the electric field, the enzyme enters the cell via a macropinocytosis-like process. This is a new long-term effect of the electric field pulse on the membrane.

An attempt was taken to incorporate Ca2(+)-dependent photoprotein obelin into young brown adipocytes using the three methods: 1) osmotic lysis of pinocytotic vesicles; 2) electroporation in a high electric field; 3) hypoosmotic shock. The young adipocytes were isolated from rat interscapular brown fat. The maximum incorporation of obelin into these cells was achieved using the hypoosmotic shock technique. A constant luminescence of intact cells loaded with obelin following hypoosmotic shock was observed in three independent experiments.