High-affinity receptor-mediated internalization and degradation of interleukin 2 in human T cells

Receptor-mediated internalization and degradation of IL-2 were investigated in cell lines carrying human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I) and PHA-treated normal PBL. The HTLV-I-carrying cell lines ILT-Yan and TL-Mor, and the PBL expressed both high- and low-affinity IL-2-R. However, another HTLV-I-carrying T cell line, MT-1, expressed mainly low-affinity receptors. Greater than 50% of the IL-2 bound to high-affinity receptors was internalized within 10 min when these cells were incubated at 37 degrees C. The internalized IL-2 was rapidly degraded and the products were excreted into the culture fluid. The t1/2 of IL-2 degradation in these cells was estimated as 60-80 min at 37 degrees C. The internalization and degradation of IL-2 were both temperature dependent. Light-microscopic autoradiography with 3H-labeled IL-2 confirmed the internalization of IL-2, and suggested that some IL-2 might be carried to the nucleus.     

Evidence for a 5'-nucleotidase in human leukemic leukocytes

Fifteen preparations of leukocytes isolated from patients with chronic leukemia, 9 lymphogenic (CLL) and 6 myelogenous (CML), were tested for 5'-nucleotidase activity.The presence of 5'-nucleotidase was demonstrated in 5 cases: four CLL and one CML. The properties of this enzyme were similar to those of 5'-nucleotidase from other tissues: its activity was dependent on Mg²⁺, and was not inhibited by tartrate, could not be solubilized by freezing and thawing and the enzyme was active at alkaline pH.On the other hand, an enzyme active on 5'-AMP in acid medium, found in all preparations, shared several characteristics with acid phosphatase. It was inhibited by tartrate, was not affected by the absence of Mg²⁺ and was partially solubilized by freezing and thawing.     

Iron binding, internalization, and fate in human alveolar macrophages

Chronic inflammation in such diseases as rheumatoid arthritis has been associated with the accumulation of iron in mononuclear phagocytes. Cigarette smoking, which also produces chronic pulmonary inflammation, may be associated with iron accumulation in alveolar macrophages (AM). We have examined the total iron content in human AM and found it to be 43.0 +/- 7.7 (mean +/- SEM) and 12.8 +/- 1.3 nmol/1 X 10(6) cells (P less than 0.01) from smokers and nonsmokers, respectively. Because the higher iron content in smokers' macrophages may reflect increased internalization, the binding and uptake of iron-saturated transferrin was examined in cells from smokers and nonsmokers. However, no significant differences were found between the two groups. The smoking-related alteration in iron content may instead reflect differences in the fate of internalized iron. Iron internalized by AM as iron 59 initially bound to transferrin was distributed to a cytoplasmic, largely ferritin-associated, pool more slowly in smokers than in nonsmokers, during a 24-hour incubation in vitro. Significantly less newly internalized iron was returned to the culture medium by AM from smokers, which by 24 hours had released 11.0% +/- 3.7% of the initially internalized 59Fe compared with 36.0% +/- 2.3% for nonsmokers (P less than 0.01). The increased accumulation of iron by AM in the alveolar space of smokers may modulate hydroxyl radical production in the microenvironment of these cells.     

Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation

Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B(2) receptor (B(2)R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B(2)R. B(2)R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B(2)R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B(2)R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B(2)R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B(2)R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.     

Aggregated C-reactive protein binds to human polymorphonuclear leukocytes and potentiates Fc receptor-mediated chemiluminescence

Aggregated C-reactive protein (CRP), an acute phase reactant, binds to and influences the functional activities of human mononuclear leukocytes. Our purpose was to examine the potential interactions between CRP and human polymorphonuclear leukocytes (PMNL). Binding of CRP to PMNL was examined by flow cytometry after cell incubation with fluorescein-labeled heat-aggregated CRP (Agg-CRP) or Agg-CRP and fluorescein-conjugated F(ab')2 anti-CRP antibodies. It was determined that, at an optimal dose of Agg-CRP (100 micrograms), approximately 36% of PMNL were fluorescence positive. This was in contrast to the 70% of monocytes and 8% of lymphocytes that expressed Agg-CRP binding sites. Although less than half of resting PMNL bound Agg-CRP, up to 93% of PMNL activated with 1.0 micrograms/ml of phorbol myristate acetate (PMA) expressed binding sites for Agg-CRP. Exposure to PMA similarly enhanced the amount of Agg-CRP bound per PMNL as determined by mean channel fluorescence. To evaluate whether Agg-CRP binding to PMNL could induce or modify a biologic response, respiratory burst activity was assessed by measuring luminol-enhanced chemiluminescence. Whereas Agg-CRP alone did not elicit a chemiluminescence response, Agg-CRP synergistically enhanced the chemiluminescence response induced by heat-aggregated IgG (Agg-IgG). Although this enhancing effect of Agg-CRP could be observed at both optimal and suboptimal concentrations of Agg-IgG, no enhancement of PMA or serum-opsonized zymosan-induced CL was detected. These data demonstrate that aggregated CRP binds to and selectively modulates the response of PMNL to Fc receptor-mediated activation.     

Insulin receptor function in fibroblasts from patients with leprechaunism. Differential alterations in binding, autophosphorylation, kinase activity, and receptor-mediated internalization.

Insulin receptor function was examined in cultured skin fibroblasts from three patients with leprechaunism (Ark-1, Minn-1, and Can-1), a rare syndrome of severe insulin resistance and neonatal growth retardation. All three patients cell lines demonstrated insulin binding less than 15% of control. This was primarily due to reduced affinity of the receptor in Can-1 and due to reduced number of receptors in the other two cell lines (Ark-1 and Minn-1). When expressed as a fraction of total insulin bound, the percentage of cell-associated insulin internalized and degraded did not differ between the patient cell lines and the controls. However, chloroquine, which inhibited degradation by 50% in the control cells, had no effect in the cells from the patients. When normalized to insulin binding, insulin receptor autophosphorylation was normal in cells from Can-1, but reduced in those of Ark-1 and Minn-1. In contrast, the receptor-associated tyrosine kinase activity toward exogenous substrates was decreased in all three patient cell lines. These results suggest that leprechaunism is a biochemically heterogenous disease associated with a variety of alterations in receptor function. Cells from Ark-1 and Minn-1 exhibit parallel alterations in receptor autophosphorylation and kinase activity. Cells from Can-1 demonstrate normal receptor autophosphorylation but reduced kinase activity, thus displaying a unique form of a mutant insulin receptor. Despite reduced kinase activity, all three cell lines exhibit normal rates of insulin internalization, but decreased lysosomal-mediated degradation. Our data imply that receptor autophosphorylation and kinase activity may be regulated separately and that kinase activity may be linked to insulin degradation, but not necessarily internalization.     

Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10- fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.     

Characteristics of high-affinity folate binding in human leukocytes

High-affinity binding of [3H]folate in leukocytes from normal subjects was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). Binding displayed positive cooperativity, and the binding affinity increased with decreasing concentration of the binding protein. Both phenomena could be interpreted in terms of ligand binding to a polymerizing system where the affinity of ligand for the oligomer is greater than its affinity for the polymer prevailing at higher concentrations of the binding protein.     

Evidence for endotoxin binding capacity of human Gc-globulin and transferrin

In the present paper the ability of Gc-globulin and transferrin to bind endotoxin of Escherichia coli 0 111: B 4 is demonstrated. This conclusion is based on four lines of evidence. By affinity chromatography using lipopolysaccharide of E. coli 0 111: B 4 two endotoxin-binding proteins of serum were identified, showing an apparent molecular weight of 77,000 and 51,000, respectively. If serum samples preincubated with the tritiated endotoxin form have undergone isoelectric focusing under non-denaturing conditions one radioactive peak appears which coincides with the precipitate obtained by immunoelectrophoresis against anti-human Gc-globulin and anti-human transferrin. Radioimmunoprecipitation experiments of serum showed that tritiated endotoxin of E. coli 0 111: B 4 was only found in the precipitate obtained with anti-Gc-globulin, antitransferrin, and polyvalent antiserum against human serum. By isoelectric focusing of purified proteins 3H-lipopolysaccharide of E. coli 0 111: B 4 was only found associated with human Gc-globulin and transferrin.     

Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 X 10(4)/cell), followed by monocytes (7.3 X 10(4)/cell) and lymphocytes (4.0 X 10(4)/cell). Compared with conventional analysis of binding of 125I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a "docking" type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias.     

A novel, internally competitive polymerase chain reaction for quantification of human cytomegalovirus DNA in human leukocytes

The rapid, low cost estimation of Human Cytomegalovirus (HCMV) viral load in the blood of immunosupressed individuals is crucial for the timely diagnosis of transplant recipients with clinically significant HCMV infections requiring treatment. To this end we have developed a novel polymerase chain reaction (PCR) assay with internal controls for competitive quantification of cytomegalovirus DNA in human leukocytes. The reaction simultaneously amplifies, under reduced stringency conditions, a 136 bp fragment of the HCMV LA gene together with a single anonymous 200 bp fragment of human DNA using a single-primer pair. The primers used are specific for the HCMV gene sequence but contain a single mismatch that permits the amplification of the competing human DNA fragment. Quantification is achieved by comparison of the amount of the two products. The assay quantifies between 10(3)and 10(6)HCMV genomes in 10(6)leukocytes, a range that permits low-level clinically unimportant HCMV infections to be distinguished from those likely to cause serious disease. The advantages of the method are that no external controls are needed, quantification is achieved from a single reaction and no separate measurement of host DNA concentration is required.     

Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.     

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components.

We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the resulting fluorescent nuclei was used to define the relationship between cytoplasmic release of nucleic acids and gene expression. Endothelial cells were stained after i.v. administration, and epithelial cells were stained after i.t. administration. The delivery of nucleic acids was also more homogeneous with i.v. administration of lipoplex than with i.t. administration. After i.t. administration, it was notable that high concentrations of fluorescent nuclei correlated with low GFP expression. This suggested that toxicity was associated with high local concentrations of cationic lipoplexes. The ratio of GFP-expressing cells to fluorescent nuclei indicated that capillary endothelial cells were more efficient in gene expression per delivery event than were pulmonary epithelial cells. Thus, the greater gene expression efficiency of i.v. administered lipoplexes was due not only to the initial distribution but also to the greater efficiency of the vascular endothelial cells to appropriately traffic and express the foreign gene.     

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal,osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."     

Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes.

The possible participation of proteases in superoxide (O2-) production by human polymorphonuclear leukocytes (PMN) and monocytes was explores using various protease inhibitors and substrates. Protease inhibitors of serine proteases and synthetic inhibitors that modify the active site of serine proteases. Substrates used were synthetic substrates of the chymotrypsin type as well as trypsin type of protease. All these inhibitors and substrates inhibited O2- oroduction by human PMN and monocytes induced by cytochalasin E and concanavalin A, though PMN were more sensitive to these inhibitors and substrates than monocytes. Inhibition appeared rapidly even when the inhibitors were added at the same time as the stimulants, during the "induction time of O2-production" or at the time of maximum O2- production, whereas much greater inhibition was observed when the cells were preincubated with the inhibitors. These observations suggest that enzymatically active serine proteases are essential for these phagocytic cells to initiate and maintain the O2- production in response to the stimuli. The inhibitory effect of the inhibitor and substrate for chymotrypsin type protease was greater than that of those substances for trypsin-type protease. Macromolecular inhibitors also inhibited the O2- production. These findings suggest that the serine proteases involved in the O2- production by human PMN and monocytes are similar to chymotrypsin rather than trypsin, and are possibly located at the cell surface membrane.     

Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface.

Superoxide anion (O-2-) generation by human peripheral blood polymorphonuclear leukocytes is enhanced when these cells encounter appropriate soluble or particulate stimuli. O-2- generation requires intact, viable cells and proceeds independently of phagocytosis. To investigate the possibility that the O-2--generating system is associated with the outer surface of the polymorphonuclear leukocyte plasma membrane, we have examined the effects upon O-2- production of p-diazobenzenesulfonic acid, a reagent which can react predominantly with proteins of the external cell membrane. When normal human polymorphonuclear leukocytes were preincubated with cytochalasin B (to minimize endocytosis) and then exposed to the surface-active lectin, concanavalin A, the cells were stimulated to generate O-2- in a concentration- and time-dependent fashion and selectively to discharge the granule-associated enzyme, lysozyme, into the surrounding medium. These responses, as well as cellular binding of [H] concanavalin A, could be blocked by alpha-methyl-D-mannoside. Brief treatment (less than 5 min at 4 degrees C) of the cells with p-diazobenzenesulfonic acid (1.0-5.0 mM) significantly interfered with concanavalin A-mediated O-2- generation but had no influence upon lysozyme release or upon binding of [3H] concanavalin A. The diazonium salt did not alter cell viability or the specific activity of the cytoplasmic enzyme, lactate dehydrogenase (inhibitable under conditions which allowed entry of this reagent into the cytosol). p-Diazobenzenesulfonic acid, therefore, very likely exerted its effects at the cell surface of the intact polymorphonuclear leukocyte, selectively inhibiting O-2- production (either directly or indirectly) without influencing another response to lectin-cell contact: release of lysozyme. These results support the possibility that a polymorphonuclear leukocyte ectoenzyme is responsible for O-2- production.     

Agonist-induced beta-adrenergic receptor internalization on intact human mononuclear leukocytes: effect of temperature of mononuclear leukocyte separation

The hydrophilic ligand 3H-CGP 12177 was used to measure beta-adrenergic receptors on intact human mononuclear leukocytes (MNLs). A single homogeneous class of receptor sites was found, with KD value of 0.71 +/- 0.04 nmol/L and Bmax of 3.0 +/- 0.4 fmol/10(6) cells (mean +/- SEM; n = 12). The receptor affinity (KD) and density (Bmax) were similar when measured on MNLs, purified lymphocytes, and a T-lymphocyte-enriched population from the same individual. Preincubation of intact MNLs with 1 mumol/L isoproterenol at 37 degrees C for 20 minutes reduced the number of surface receptors, measured by 3H-CGP 12177 binding at 4 degrees C for 20 hours, by approximately 70% (receptor internalization) without affecting KD. This effect was reversible, and surface receptors completely reappeared when binding was investigated at 37 degrees C for 40 minutes. Receptor internalization was similar when either isolated MNLs or whole blood was incubated with isoproterenol. Agonist-induced receptor internalization was stable during MNL isolation from whole blood at 4 degrees C but was partially or completely lost from MNLs prepared at 20 degrees C.     

Accumulation of a newly developed fluoroquinolone, OPC-17116, by human polymorphonuclear leukocytes.

The accumulation of OPC-17116 by human polymorphonuclear leukocytes was studied by comparison with three other new quinolones, ofloxacin, levofloxacin, and DR-3354. The intracellular/extracellular concentration ratio was the highest for OPC-17116, being 66.2, followed by 9.8 for levofloxacin, 7.6 for ofloxacin, and 5.8 for DR-3354. Furthermore, it was suggested that the accumulation of these new quinolones was partially related to their active transport system. The elution of OPC-17116 and ofloxacin from cells was rapid, but the elution of OPC-17116 decreased at a high extracellular pH and increased at a low extracellular pH. The accumulation of OPC-17116 as well as that of the other new quinolones was satisfactorily high, thus indicating that they are expected to be useful for the treatment of various kinds of infections, particularly infections caused by cytozoic bacteria.     

Evidence for reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. A new microbicidal pathway for polymorphonuclear leukocytes.

In anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), the nitric oxide synthase inhibitor N omega-monomethyl-L-arginine (NMMA) produced a modest decrease in uptake of staphylococci from supernatants (P less than 0.02, n = 7), and a marked decrease in the killing of cytoplast-associated bacteria (P less than 0.001, n = 7). After 60 min of incubation with bacteria, NMMA-treated cytoplasts had a mean of over 3.5 times as many live, CKP-associated staphylococci as did controls (51% of the inocula versus 14%), despite having taken up fewer. Effects on both uptake and killing were reversible by L-arginine but not by D-arginine. Results were the same with other granule-poor cytoplasts (U-cytoplasts, U-CYT), which, unlike CKP, retain activatable oxidase activity. Killing by intact PMN, including those from a patient with chronic granulomatous disease, was not inhibited by NMMA. Thus, the ability to discern effects of NMMA correlated with the paucity of granules, without regard to the presence or absence of activatable oxidase. We propose that the generation of reactive nitrogen intermediates serves as an additional microbial killing pathway in PMN, and that cytoplasts can be used to help delineate the spectrum of susceptible targets.     

Evidence for receptor-mediated hepatic uptake of pullulan in rats

Fluorescein-labeled pullulan (FP-60; MW 58,200) was prepared by reaction with FITC according to the method of de Belder and Granath. The hepatic distribution of FP-60 was examined using a specific high-performance size-exclusion chromatography. Intravenously administered FP-60 was rapidly eliminated from the blood circulation followed by an appreciable distribution to the liver. A marked dose-dependency was seen in the hepatic uptake of FP-60 which was markedly reduced by the coadministration of both asialofetuin and arabinogalactan. Measurement of the hepatocellular localization demonstrated the overwhelming distribution of FP-60 in the parenchymal liver cell fraction. Furthermore, microscopic examination revealed that FP-60 was effectively endocytosed by the parenchymal liver cells. Radiolabeled pullulan ([¹²⁵I]P-60) was prepared by ¹²⁵I-labeling the tyramine derivative of pullulan which was synthesized by the cyano-transfer method. [¹²⁵I]P-60 was predominantly accumulated in sliced rat liver tissue at 37°C, which was drastically inhibited by the addition of both asialofetuin and arabinogalactan. The kinetic parameters of the specific binding of [¹²⁵I]P-60 to monolayered hepatocytes at 0°C were almost identical to those for asialofetuin. The binding of [¹²⁵I]P-60 to isolated parenchymal cells was significantly inhibited by arabinogalactan and asialofetuin, however dextran, the same glucan as pullulan, did not affect the binding of [¹²⁵I]P-60. It was found that pullulan, which is bound to the asialoglycoprotein receptor with high affinity, is subsequently internalized to the hepatocyte via receptor-mediated endocytosis.