Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons.     

The macrophage-derived lectin, MNCF, activates neutrophil migration through a pertussis toxin-sensitive pathway.

The macrophage-derived neutrophil chemotactic factor (MNCF) is a D-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, D-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by D-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.     

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily

Summary Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8, suggested gene symbol IL8) is a cytokine that chemoattracts and activates neutrophils. Using a panel of human-rodent cell hybrids that preferentially segregate human chromosomes and in situ hybridization, the MDNCF/IL-8 gene was placed on the human gene map at position 4q12-q21. This is the same location where at least three other members (platelet factor 4, melanoma growth stimulatory activity, and interferon- induced factor) of the platelet factor 4 gene superfamily reside. In addition, a restriction fragment length polymorphism was identified using MDNCF as a probe in screening genomic DNA digested with HindIII from unrelated individuals.On leave from Genetics Section, Instituto Nacional do Cancer (RJ)/ Department of Genetics, Universidade Federal do Rio de Janeiro, Brazil     

Actin filaments are involved in the regulation of trafficking of two closely related chemokine receptors, CXCR1 and CXCR2

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37 degrees C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.     

Down-regulation of high affinity interleukin 2 receptors in a human tumor T cell line. Interleukin 2 increases the rate of surface receptor decay

We have described a human tumor T cell line, IARC 301, which constitutively expresses high affinity interleukin 2 (IL2) receptors, and showed that after binding to its receptors, IL2 is endocytosed and degraded. Here we present evidence that IL2 down-regulates its own high affinity receptors. Within 1 h, IL2 induces a 60% decrease in surface receptor expression. In order to maintain this down-regulation, IL2 concentration must be high enough for the receptors to be saturated throughout the incubation. The effect of IL2 on the kinetics of receptor internalization was investigated with two approaches. First, the initial rate of IL2 internalization was measured, and no difference could be detected whether the receptors were saturated with IL2 or only partially occupied. Second, the initial rate of surface receptor decay was followed and found to be significantly decreased in the presence of IL2. Although the half-life of IL2 receptors is very short in the absence of IL2, t 1/2 approximately 65 min, suggesting that these receptors are constantly endocytosed, it can still be reduced to t 1/2 approximately 25 min when the receptors are saturated with ligand. This suggests that occupied receptors are internalized faster than and independently from free receptors. The difference in internalization rates can explain the observed receptor down-regulation.     

Rapid constitutive internalization and externalization of epidermal growth factor receptors in isolated rat hepatocytes. Monensin inhibits receptor externalization and reduces the capacity for continued endocytosis of epidermal growth factor

It was previously demonstrated that freshly isolated rat hepatocytes can internalize severalfold more epidermal growth factor (EGF) molecules than the number of surface EGF receptors, suggesting extensive reutilization of receptors during endocytosis (Gladhaug, I. P. & Christoffersen, T. (1987) Eur. J. Biochem. 164, 267-275). The present report attempts to explore the pathways involved in the externalization of EGF receptors. Incubation of hepatocytes at 37 degrees C in the absence of ligand increased the surface receptor pool by 50-100% within 45 min. Pretreatment with monensin inhibited the turnover of the surface EGF receptor pool by 50-60% within 10 min and blocked the temperature-dependent externalization of receptors. Cycloheximide caused a slower attenuation of the surface receptor pool, whereas tunicamycin and chloroquine did not significantly affect the exchange of receptor pools. Monensin reduced the surface receptor pool and the endocytic uptake in corresponding proportions, without affecting the internalization of prebound EGF. Endocytic uptake was unaffected by chloroquine and slightly reduced by cycloheximide. The internalization of unoccupied receptors and the endocytosis of prebound EGF followed similar kinetics (t1/2 approximately 5 min), suggesting that unoccupied receptors are internalized at a rate comparable to that of occupied receptors. The results suggest that there is a rapid turnover of the surface pool of EGF receptors with constitutive internalization of unoccupied surface receptors and externalization of internal receptors. This is consistent with, but does not prove, a true recycling of the EGF receptors in the hepatocytes. The monensin-sensitive externalization pathway determines the capacity for continued endocytosis of EGF.     

The hepatic interleukin-6 receptor. Down-regulation of the interleukin-6 binding subunit (gp80) by its ligand.

Interleukin-6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6-binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp130). We studied the role of gp80 in binding, internalization and down-regulation of the hepatic IL6-receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp80 with parental cells indicate that gp80 is responsible for low affinity binding (Kd = 500 pM) of IL6. Furthermore, gp80 is rate-limiting in internalization and degradation of IL6. Internalization resulted in a rapid down-regulation (t1/2 approximately 15-30 min) of IL6-binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6-binding sites at the cell surface required greater than 8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down-regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.     

Differential modes of regulation of cxc chemokine-induced internalization and recycling of human CXCR1 and CXCR2

Studies of human neutrophil IL-8 receptors, CXCR1 and CXCR2, have shown that the two receptors are differentially regulated by ELR⁺-CXC chemokines, that they differ functionally and may have diverse roles in mediating the inflammatory process. To elucidate the role of CXCR1 and CXCR2 in inflammation and to delineate the basis for the divergent regulation of these receptors by IL-8 and NAP-2, we characterized the IL-8- and NAP-2-induced mechanisms regulating the expression of each receptor, focusing on receptor internalization and recycling. Using HEK 293 cell transfectants, IL-8 was shown to induce significantly higher levels of CXCR2 internalization than NAP-2. Moreover, although CXCR2 bound IL-8 and NAP-2 with similarly high affinity, IL-8 functionally competed with and displaced NAP-2, and prompted high levels of internalization, similar to those induced by IL-8 alone. In a system providing an identical cellular milieu for reliable comparisons between CXCR1 and CXCR2, we have shown that the mechanisms controlling the internalization of CXCR1 diverge from those regulating CXCR2 internalization. Whereas IL-8-induced internalization of CXCR1 was profoundly dependent on a region of the carboxyl terminus expressing six phosphorylation sites, internalization of CXCR2 was primarily regulated by a membrane proximal domain of the carboxyl terminus that does not express phosphorylation sites. Analysis of receptor re-expression on the plasma membrane indicated that at early time points following removal of free ligand and incubation of the cells at 37°C, receptor recycling accounted for recovery of CXCR1 and CXCR2 expression, whereas at later time points other processes may be involved in receptor re-expression. Phosphorylation-independent mechanisms were shown to direct both receptors to the recycling pathway. The differential control of CXCR1 vs CXCR2 internalization by IL-8 and NAP-2, as well as by phosphorylation-mediated mechanisms, suggests that a chemokine- and receptor-specific mode of regulation of internalization may contribute to the divergent activities of these receptors.     

Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes.

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.     

Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling

The kinetics of receptor internalization and recycling was directly determined in adipocytes by measuring 125I-insulin binding to total, intracellular, and cell-surface insulin receptors. In the absence of insulin 90% of all receptors were on the cell-surface and 10% were intracellular. Insulin (100 ng/ml) rapidly altered this distribution by translocating surface receptors to the cell-interior through a temperature and energy dependent process. Surface-derived receptors were seen within cells as early as 30 s and accumulated intracellularly at the rate of approximately 20,000/min (t 1/2 = 2.7 min). After 6 min the size of the intracellular receptor pool plateaued (for up to 2 h), with 30% of surface receptors residing within the cell. This plateau was due to the attainment of an equilibrium between receptor uptake and recycling, since removal of insulin (to stop receptor uptake) was followed by both a rapid depletion of intracellular receptors and a a concomitant and stoichiometric reappearance of receptors on the cell-surface. Receptors were efficiently recycled, with little or no net loss observed even after 4 h of insulin treatment; however, recycling could be partially inhibited (approximately 10%) by several agents (e.g. chloroquine and Tris). Tris treatment of adipocytes in the presence of insulin led to 50% loss of surface and total receptors at 2 and 4 h, respectively. Since chloroquine prevented the decrease in total receptors, but not the loss of surface receptors, it appears that Tris impairs recycling by diverting a portion of incoming receptors to a chloroquine-inhibitable degradative site. From these results we conclude that: 1) insulin triggers endocytotic uptake of insulin-receptor complexes; 2) internalized receptors are then rapidly reinserted into the plasma membrane, and the receptors can traverse this recycling pathway within 6 min; 3) prolonged recycling does not normally result in measurable receptor loss, but when receptors are prevented from recycling, they become trapped intracellularly and are shunted to a chloroquine-sensitive degradative pathway; and 4) chloroquine and Tris are only partially effective inhibitors of receptor recycling.     

Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils

Actinonin and amastatin are low-molecular-weight inhibitors of aminopeptidases associated with cell surfaces. The purpose of this study was to determine their effects on human neutrophil functions such as chemotaxis and phagocytosis. Both actinonin and amastatin enhanced chemotaxis to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. On the other hand, the effects of both agents on neutrophil phagocytosis were varied. Bacterial attachment to neutrophils was slightly affected by these agents. However, actinonin enhanced the internalization of bacteria by neutrophils. Neutrophil leucine aminopeptidase activity was also determined and was found to be weakly inhibited by these agents.     

The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. We show that internalization of asialoglycoprotein by HepG2 hepatoma cells is accompanied by a rapid (t1/2 = 0.5-1 min) depletion of surface asialoglycoprotein receptors. This is followed by a rapid (t1/2 = 2-4 min) reappearance of surface receptors; most of these originate from endocytosed cell-surface receptors. The loss and reappearance of asialoglycoprotein receptors is specific, and depends on prebinding of ligand to its receptor. HepG2 cells also contain abundant receptors for both insulin and transferrin. Endocytosis of asialoglycoprotein and its receptor has no effect on the number of surface binding sites for transferrin or insulin. We conclude that binding of asialoglycoprotein to its surface receptor triggers a rapid and specific endocytosis of the receptor-ligand complex, probably due to a clustering in clathrin-coated pits or vesicles.     

Phorbol esters and calcium ionophores inhibit internalization and accelerate recycling of receptors in macrophages

Exposure of macrophages to phorbol esters or the calcium ionophore A23187 increases the number of several surface receptors due to recruitment of receptors from internal pools (Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38, 569-576). We have examined the mechanism by which these agents increase surface receptor number. Cells which were preloaded with either fluid phase or receptor-mediated ligands did not lose ligand following exposure to ionophore or phorbol ester. The rate of movement of ligands to the lysosome was also unaffected. These results suggest that A23187 does not induce the fusion of ligand-containing compartments with the cell surface. Ionophore treatment did, however, produce a severalfold increase in the rate at which unoccupied receptors reappear on the cell surface. These results suggest that the compartment of receptors affected by the ionophore formed subsequent to the dissociation of ligand from receptor. The altered rate of receptor reappearance was transitory (90 s), and the increase in receptor number was subsequently maintained by a decrease in the rate of internalization. Changes in the rate of receptor internalization did not correlate with changes in the rate of fluid phase pinocytosis, suggesting that the effect on receptor internalization was selective.     

Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling

We have used an iodinated, photoreactive analog of insulin, 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 degrees C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 degrees C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 degrees C compared to 37 degrees C and essentially negligible at 12 degrees C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.     

DNA‐based probes for flow cytometry analysis of endocytosis and recycling

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane‐bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo‐TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor‐antibody complex behaves differently to the receptor‐ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.      

Role of the proteasome in modulating native G-CSFR expression

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.     

Binding and cross-linking of recombinant mouse interferon-gamma to receptors in mouse leukemic L1210 cells; interferon-gamma internalization and receptor down-regulation

Recombinant E. coli-derived murine IFN-gamma (Mu-rIFN-gamma; 5 X 10(7) U/mg) was radiolabeled with 125I by the chloramine-T method without loss of its antiviral activity. The 125I-Mu-rIFN-gamma showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 X 10(-10)M. Binding of 125I-Mu-rIFN-gamma to cells was inhibited by both natural (glycosylated) and rIFN-gamma, but not by IFN-alpha/beta. Receptor-bound 125I-Mu-rIFN-gamma was rapidly internalized when incubation temperature was raised from 4 degrees C to 37 degrees C. On internalization, almost no IFN-gamma degradation was observed during 16 hr incubation. 125I-Mu-rIFN-gamma binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-gamma, but not with IFN-alpha/beta. This receptor down-regulation was dose-dependent: 90% reduction of 125I-Mu-rIFN-gamma binding was observed after preincubation with 100 U/ml. After removal of IFN-gamma from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of 125I-Mu-rIFN-gamma to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000. The formation of such a complex appeared highly specific on the basis of the following criteria: it could be inhibited by the addition of Mu-rIFN-gamma but not by Mu-rIFN-alpha/beta, it was not obtained in cells pretreated with IFN-gamma to induce down-regulation of IFN-gamma receptors, and it was also identified in the IFN-alpha/beta-resistant L1210R cell line, known to be sensitive to IFN-gamma and which we have recently shown to express IFN-gamma receptors.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Acceleration of human neutrophil apoptosis by TRAIL

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-alpha also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-kappaB, suggesting TRAIL does not activate NF-kappaB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.     

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.