Purinergic receptor agonists modulate phagocytosis and clearance of apoptotic cells in macrophages

Phagocytosis plays an important role in controlling inflammation and antigen cross-presentation through the uptake of apoptotic bodies from dying cells. As dying cells are known to release nucleotides and other “danger signals”, we investigated whether extracellular nucleotides may affect phagocytosis through binding to P2 purinergic receptors on phagocytic cells. We here show that the purinergic receptor agonists, ATP, ADP, α,β-methylene ATP (α,β-meATP), 3′-O-(4-benzoyl)benzoyl ATP, UTP and UDP, increased phagocytosis of latex beads, and some of them increased endocytosis and/or macropinocytosis of dextran by macrophages. The enhanced phagocytosis could be inhibited by pre-treatment with the P2X and P2Y antagonists, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid and suramin, and the P2Y₁-selective antagonist, MRS2179. The nucleotides induced upregulation in macrophages of the β2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (α_vβ3, CD51/CD61), both of which are involved in recognition and internalization of apoptotic cells. In addition, ATP and α,β-meATP increased adhesion of apoptotic cells to macrophages, both in vitro and in vivo, and α,β-meATP had a small effect on adhesion of necrotic cells. The nucleotides had no effect on adhesion of viable cells. We propose that engagement of the P2 receptors (P2X₁, or P2X₃) by extracellular nucleotides released from dying cells increases the ability of macrophages to bind apoptotic bodies, thus enhancing their ability to internalize and present antigens from the dying cells.     

Nucleotide responses of human neutrophils.

Since human polymorphonuclear neutrophils (PMN) exposed to ATP or its poorly hydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), respond with increases in intracellular calcium and enhanced O2- responses to the chemotactic peptide N'-formyl-Met-Leu-Phe (fMLP), we systematically evaluated responses of PMN to various nucleotides. The P2X and P2Y receptor agonists, 2-methylthioadenosine triphosphate and beta, gamma-methyleneadenosine triphosphate, failed to induce increases in intracellular calcium and did not desensitize PMN to increases in intracellular calcium induced by ATP gamma S. Since it has been suggested that P2Z receptor occupancy with the ATP4- species caused nonselective increases in cell permeability, the ability of ATP to induce increases in intracellular calcium was evaluated in the presence and absence of extracellular Ca2+ and Mg2+. In the presence of these cations, 5-fold greater concentrations of ATP were required. The effects of ATP4- were not associated with changes in cell membrane permeability. This suggests that ATP4- is the active species but that its effect on PMN is not linked to a nonselective increase in permeability of the cell membrane. With respect to responses of PMN to purine and pyrimidine nucleotides as defined by increases in intracellular calcium, the rank order of potency for the nucleotides was ATP = UTP greater than ATP gamma S greater than or equal to ITP greater than GTP greater than or equal to CTP. These responses were blocked by pretreatment of PMN with pertussis toxin. Prior exposure of PMN to ATP gamma S blocked cellular responses (calcium increases) to these nucleotides but not to fMLP. Likewise, exposure of PMN to any nucleotides blocked subsequent cellular responses to ATP gamma S but not to fMLP. These data support the concept that nucleotide responses of PMN utilize either a common receptor or a common signal transduction pathway involving a guanine nucleotide binding protein in events leading to elevations in intracellular calcium. Nucleotide interaction with PMN does not follow the established pattern of responses associated with P2X or P2Y purinergic receptor occupancy.     

Chemotactic and Phagocytic Activity of Blood Neutrophils in Allergic Asthma

Allergic asthma is a chronic inflammatory airway disease, and has been considered a T helper-2-biased response. Studies suggest that neutrophils may be associated with exacerbation and asthma severity. We sought to evaluate the chemotactic activity and phagocytic capacity by peripheral blood neutrophils from individuals with controlled and uncontrolled allergic asthma, and compare the results with non-asthmatic controls groups. Blood neutrophils were isolated from 95 patients: 24 with controlled asthma, 24 uncontrolled asthma, 24 healthy subjects and 23 patients with IgE-mediated allergies other than asthma. The neutrophil chemotaxis, stimulated with LPS, autologous serum or homologous serum, was determined using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by NBT test. The phagocytic digestion phase and chemotaxis by neutrophils from asthmatic patients was higher than in non-asthmatic controls (p??<?0.05). Autologous serum-induced neutrophil chemotaxis in patients with uncontrolled asthma was greater (p??<?0.05) than in other study groups. The ingestion phase of phagocytosis showed similar values in asthmatics and non-asthmatics. We conclude that the blood neutrophil from controlled and uncontrolled asthmatic patients exhibit activation markers, particularly phagocytic digestion and chemotactic activities.     

Functional Changes En Thp-1 Human Monocytic cells after Stimulation with Lipopolysaccharide of oral Microorganisms and Granulocyte Macrophage colony Stimulating Factor

Abstract

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobaclerium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using FITC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10–11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2–3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nuclealum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.     

Functional Changes En Thp-1 Human Monocytic cells after Stimulation with Lipopolysaccharide of oral Microorganisms and Granulocyte Macrophage colony Stimulating Factor

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobaclerium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using FITC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10-11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2-3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nuclealum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.     

Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll,Ficoll and Spontaneous Sedimentation Methods

Studies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.     

Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo

Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate that these molecules mediate LPS-induced neutrophil migration in vitro and in vivo. Apyrase, a nucleotide scavenger, reduced the ability of LPS-stimulated monocytes to recruit neutrophils, as assayed using a modified Boyden chamber. This effect resulted from the inhibition of IL-8 release from monocytes. Furthermore, LPS-induced IL-8 release by monocytes was attenuated significantly by P2Y6 receptor antagonists, RB-2 and MRS2578. Reciprocally, UDP, the selective P2Y6 agonist, induced IL-8 release by monocytes. As for LPS, the media of UDP-stimulated monocytes were chemotactic for neutrophils; IL-8 accounted for approximately 50% of neutrophil migration induced by the media of LPS- or UDP-treated monocytes in transendothelial migration assays. It is important that in the murine air-pouch model, extracellular nucleotides were instrumental in LPS-induced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity.     

P2 receptor mRNA expression profiles in human lymphocytes,monocytes and CD34+ stem and progenitor cells

Background  

Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34⁺ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.     

Coupling of airway ciliary activity and mucin secretion to mechanical stresses by purinergic signaling

The mucociliary clearance system is comprised of three components, ion transport activities controlling the height of airway surface liquid (ASL), mucin secretion, and ciliary activity. These activities in humans are controlled principally by local agonists, extracellular nucleotides and nucleosides released from the epithelium. Importantly, mechanical stresses stimulate goblet cell mucin secretion, ciliary beating, and Cl⁻ and fluid secretion through mechanically induced nucleotide release. Emerging evidence also implicates co-secretion of nucleotides and mucin from goblet cells as a source of extracellular agonist. At rest, ATP is released onto airway surfaces at 370 fmol/min cm², but only 3% of released ATP is recovered in ASL. Secreted UTP meets with a similar fate. A wide variety of hydrolytic and transphosphorylating ecto-enzymes convert the triphosphate nucleotides into ADP, AMP, and adenosine, UDP, UMP, and uridine. Of these, ATP, adenosine, UTP, and UDP act as agonists at apical P2Y₂ (ATP, UTP), P2Y₆ (UDP), and A_2B (adenosine) receptors on ciliated and/or goblet cells to regulate mucociliary clearance.     

Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes

The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.     

Nandrolone decanoate pre-treatment attenuates unweighting-induced functional changes in rat soleus muscle

Extracellular nucleotides have been shown to induce vasodilatation of conductance arteries by release of the endothelium-derived hyperpolarizing factor (EDHF). As small resistance arteries are of greater importance for blood pressure regulation, a whole rat mesenteric arterial bed preparation was used in the present study when evaluating the physiological relevance for EDHF in mediating nucleotide dilatation. Dilatory responses were examined after pre-contraction with noradrenaline in the presence of 10 mM indomethacin. Adenosine-5'-O-thiodiphosphate (ADPbetaS), adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced vasodilatation (pEC50=6.5-7 and E(max)=40-70%), while uridine diphosphate (UDP) was ineffective. Endothelium-derived hyperpolarizing factor was studied in the presence of 0.5 mM Nvarpi-nitro-L-arginine (L-NOARG). ADPbetaS and UTP induced strong and potent EDHF-dilatations, while ATP only had a weak effect (E(max)=25%). After P2X1 receptor desensitization with 10 microM alphabeta-methylene-adenosine triphosphate, the ATP response was significantly increased (E(max)=65%). Putatively, this could be because of simultaneous activation of both endothelial P2Y receptors and P2X1 receptors on smooth muscle cells, which resulted in the release of EDHF and subsequent hyperpolarization, and depolarization, respectively. Nitric oxide (NO) was studied in the presence of 60 mM K+. ADPbetaS, ATP and UTP induced weak NO dilatations, suggesting a minor role for NO as compared with EDHF. In conclusion, extracellular nucleotides stimulate dilatation by activating P2Y(1) and P2Y(2)/P2Y(4) receptors, but not P2Y(6) receptors. The dilatory responses are mediated primarily by EDHF in the peripheral vascular bed.     

Effects of L-carnitine on neutrophil functions in aged rats

Several age-related alterations occur at the cellular level in the immune system leading to a decrease in the immune response. The present study was designed to determine the effect of L-carnitine on impaired neutrophil functions of aged rats. For this reason, superoxide anion radical production, chemotaxis and phagocytic activity were studied in the neutrophils obtained from the peripheral blood of young and old rats. We orally gavaged L-carnitine (50 mg/kg b.w. per day) or control vehicle into young (2 months) and aged (24 months) rats for 30 consecutive days. The neutrophils of aged rats exhibited an increase in superoxide anion production and decline in phagocytosis and chemotaxis when compared with that in young rat neutrophils. Superoxide anion production in aged rats was significantly decreased by L-carnitine treatment which was accompanied with a significant enhancement of chemotactic and phagocytic activity being restored to control levels. These findings demonstrated that L-carnitine is capable of restoring the age-related changes of neutrophil functions.     

Peptides and their constituent amino acids influence the immune response and phagocytosis in different ways.

To elucidate the role of immunoactive amino acids (which are capable of stimulating the immune response) in the peptide regulation of antibody production and phagocytic processes we have studied the influence of some fragments of natural peptides and the amino acids included in them on the thymus-dependent immune response to sheep red blood cells (SRBC) and on the phagocytosis of Staphylococcus by murine peritoneal neutrophils. It has been found that the effects of amino acids, as well as of peptides that they form, on the immune response and on phagocytosis were diverse. Glutamic and aspartic acids, threonine and valine stimulated both the immune response and phagocytosis. Glycine and isoleucine influenced neither the immune response nor phagocytosis, whereas lysine, proline, tyrosine and leucine did not influence the immune response, but enhanced the phagocytic activity of neutrophils. Arginine inhibited the immune response but stimulated phagocytosis. Peptide TTKD section (the fragment 77-80 of murine Thy-1-antigen) contained in C- and N-terminus amino acids (T and D) which stimulated the antibody production and phagocytosis, and lysine (K) which stimulated phagocytosis only, enhanced both processes. Peptides LGIP and PYIK (the fragments 49-53 and 66-69 of murine Thy-1 antigen) which contained both immunologically inert amino acids (L, G, I, P, Y, K) and phagocytosis stimulating amino acids (L, Y, P, K) influenced neither the immune response nor the phagocytic activity of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil Functions in Immunodeficiency Due to DOCK8 Deficiency

Neutrophil chemotactic defects have been reported previously in patients with hyper-IgE syndrome. Bi-allelic mutations in dedicator of cytokinesis 8 (DOCK8) gene usually cause an autosomal recessive hyper-IgE syndrome phenotype. Data are lacking about expression of DOCK8 protein in neutrophils or the possible role of DOCK8 in neutrophil function. We sought to determine if DOCK8 protein is expressed in neutrophils and if DOCK8 plays a role in neutrophil function. The expression of DOCK8 protein was assessed in neutrophils from healthy volunteers with and without activators. Neutrophil chemotaxis, phagocytosis and superoxide generation were studied in neutrophils from DOCK8-deficient patients compared to neutrophils from healthy controls before and after stimulation with activators: phorbol 12-myristate 13-acetate (PMA) or N-Formylmethionyl-leucyl-phenylalanine (fMLP). DOCK8 protein is expressed in resting neutrophils from healthy controls, with a significant increase in DOCK8 expression after stimulation. Neutrophil functions were assessed in 6 DOCK8-deficient patients. All patients had the same non-sense mutation (c.C5134A, p.S1711X). Normal chemotaxis was recorded in 4/6 patients while a mild to moderate chemotaxis defect was recorded in 2/6. Superoxide generation was mainly normal in neutrophils from all six patients and phagocytosis was normal in five patients tested. We conclude that DOCK8 protein is expressed in resting human neutrophils and DOCK8 expression is increased after stimulation with either PMA or fMLP. Most patients with a disease-causing mutation in DOCK8 have normal neutrophil functions, while a minority showed a mild to moderate chemotactic defect.     

Pyrimidine Nucleotide-Evoked Inhibition of Cyclic AMP Accumulation in Equine Epithelial Cells

Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotides. ATP evoked cAMP accumulation in control cells and this response was unaffected by pertussis toxin. There is, therefore, no indication that ATP activates the pertussis toxin-sensitive inhibitory pathway. The UTP-evoked inhibition of cAMP accumulation was abolished by isobutylmethylxanthine (IBMX, 5 mM) and so the negative control over cAMP levels appears to be mediated by receptors that are selectively activated by pyrimidine nucleotides and permit control over phosphodiesterase activity.     

Platelet enhancement of O2-. responses in stimulated human neutrophils. Identification of platelet factor as adenine nucleotide

Human neutrophils stimulated with immune complexes in the presence of platelets show enhanced superoxide anion (O2-.) responses that are proportional to the amount of agonist present and the number of platelets added. Platelet related enhancement of O2-. responses also occurs with the neutrophil agonists phorbol ester, formyl chemotactic peptide and zymosan particles. Pretreatment of platelets with cycloheximide does not alter their ability to enhance O2-. responses of neutrophils. In parallel with platelet-related enhancement of O2-. responses of immune complex-stimulated neutrophils, secretory release of myeloperoxidase is also increased. The platelet effects on O2-. responses can be reproduced with platelet lysates or with supernatant fluids which have been obtained from thrombin or immune complex-stimulated platelets and are rich in ATP and ADP content. Solutions containing ATP and ADP in amounts similar to those found in supernatant fluids of activated platelets reproduce the enhancement of O2-. responses in N-formyl methionyl leucyl phenylalanine (FMLP) or immune complex-activated neutrophils. The platelet factor responsible for the effects of neutrophils is heat-stable, elutes in gel sieving chromatography near the position of phenol red and does not, in the absence of a neutrophil agonist, trigger an O2-. response. With formyl peptide-stimulated neutrophils, ATP and ADP enhance O2-. responses while the responses are depressed by addition of AMP or adenosine. In immune complex-stimulated neutrophils, adenosine and all adenine nucleotides enhance the O2-. responses. Taking advantage of this information, treatment of ATP or of supernatant fluids from thrombin-stimulated platelets with alkaline phosphatase (resulting in formation of adenosine) converts the O2-. enhancing activity for formyl peptide-activated neutrophils into an inhibitory activity. In contrast, using immune complex-activated neutrophils, similar manipulations of ATP or supernatant fluids from stimulated platelets result only in enhanced O2-. responses. These data support the conclusion that the platelet-derived factor responsible for enhanced O2-. responses in neutrophils is ATP/ADP. In FMLP stimulated neutrophils, the presence of ATP or ADP leads to enhanced increases in intracellular levels of Ca++ as determined by the fura-2 probe, while the presence of AMP or adenosine results in inhibition of the increases in FMLP induced elevations in cytosolic Ca++. These data demonstrate a direct relationship between effects of adenine compounds on FMLP induced changes in cytosolic Ca++ and the associated O2-. responses.     

Lipopolysaccharide modulates chemotactic peptide-induced actin polymerization in neutrophils

To study the effect of endotoxin (LPS) on the basal and chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP)-induced alterations in neutrophil cytoskeleton, we purified (greater than 98%) LPS-free neutrophils (LPS- less than 10 pg/ml LPS), compared their cytoskeletal organization to that of circulating neutrophils, and examined the effect of LPS exposure on the basal and fMLP-induced change in the cytoskeleton as reflected by F-actin content and distribution. Shape, F-actin content and distribution were monitored by FACS analysis and fluorescence microscopy of NBDphallicidin-stained cells. The F-actin content of basal and fMLP-activated, purified LPS- cells is similar to that of circulating neutrophils (defined as cells drawn in LPS- buffers at 37 degrees C and analyzed after less than 10 seconds of ex vivo manipulation). LPS- cells are round with a diffuse F-actin distribution. Exposure of LPS- cells to LPS causes cell polarization and F-actin redistribution without net gain in F-actin content. Peptide activation of the LPS- cell causes actin polymerization, which is preceded by a brief lag time. Exposure of LPS- cells to LPS (LPS+) enhances fMLP-induced actin polymerization by: 1) increasing the maximal extent of polymerization; 2) shortening the lag time preceding polymerization and increasing the rate of polymerization; and 3) lowering fMLP dose required for half maximal F-actin response. The enhancement depends on LPS dose, duration of exposure, and temperature. To examine the mechanism whereby LPS enhances fMLP-induced actin polymerization, we determined the predominant end for filament growth in LPS- and LPS+ cells, the number of actin nuclei generated in LPS- and LPS+ by fMLP activation, and the number and affinity of fMLP receptors on LPS- and LPS+ cells by 3[H]fMLP binding. Actin polymerization in both LPS- and LPS+ occurs predominantly by monomer addition to the barbed ends of nuclei, and the number of actin nuclei in basal and fMLP-activated LPS- and LPS+ cells is similar. LPS+ cells express three times more fMLP receptors than LPS- cells. The results show that LPS- cells are similar in cytoskeletal organization to circulating neutrophils, LPS causes shape change without change in F-actin content, and LPS enhances fMLP-induced actin polymerization response in neutrophils. The results suggest that LPS enhancement of actin polymerization response is associated with an increase in the number of fMLP receptors expressed on the cell surface.     

Chronic treatment with P2-purinergic receptor agonists induces phenotypic modulation of the HL-60 and U937 human myelogenous leukemia cell lines.

In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil/monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATP gamma S (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear/cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATP gamma S, HL-60 cells showed no obvious changes in morphology, viability, or the levels of beta-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a greater than 90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.     

Inhibition of human neutrophil receptor-mediated uptake of N-formyl-met-leu-phe by platelet factor 4 (59-70).

Human platelet factor 4 (PF4) and a substituent dodecapeptide designated PF4(59-70) elicited human neutrophil and monocyte chemotaxis with a similar concentration-dependence and maximal responses equal to that attained by chemotactic fragments of C5 (C5fr). At maximally chemotactic concentrations, PF4(59-70) stimulated the secretion by neutrophils of approximately 40% and 60% of the respective quantities of beta-glucuronidase and beta-glucosaminidase released by 10(-6) M N-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to the deactivation of chemotaxis achieved by preincubation of neutrophils with other chemotactic factors, prior exposure to 10(-6)M PF4(59-70) for 2 min, or 20 min at 37 degrees, enhanced by 1.5- to 2-fold the chemotactic responses of neutrophils evoked by optimal concentrations of fMLP, C5fr, leukotriene B4, and PF4(59-70). Concentrations of PF4(59-70) which enhanced neutrophil chemotaxis inhibited the rate of receptor-mediated internalization of [3H]fMLP at 37 degrees and 18 degrees, but at 0 degrees failed to alter the binding affinity or the number of receptors for [3H]fMLP. Preincubation of neutrophils at 37 degrees with concentrations of PF4(59-70) which enhanced neutrophil chemotaxis also did not affect the subsequent binding of [3H]fMLP at 0 degrees. The inhibition by PF4(59-70) of the receptor-mediated internalization of [3H]fMLP was not mimicked by other positively charged compounds. The specific inhibition of receptor-mediated internalization of fMLP may explain the enhanced chemotactic responsiveness of neutrophils preincubated with PF4(59-70).