Adhesive Properties Of Neutrophils Research Articles

Effect of taxifolin, a conjugate of taxifolin with glyoxylic acid, and naringenin on the functional activity of neutrophils

The aim of the present work was to study the effects of taxifolin, a conjugate of taxifolin with glyoxylic acid, and naringenin on phagocytosis of latex beads by neutrophils and adhesive properties of these cells. It was shown that taxifolin had no effect on the phagocytic activity and adhesive properties of neutrophils. A conjugate of taxifolin with glyoxylic acid strongly inhibited the phagocytic activity of neutrophils. At the same time, the conjugate significantly enhanced adhesion of these cells. Naringenin decreased phagocytic activity of neutrophils, though to a lesser extent than the conjugate did. Naringenin also inhibited adhesion of neutrophils. Thus, the data collected during our study indicate that polyphenols may influence the functional activity of neutrophils that can play an important role in the modulation of inflammation, especially when prolonged activation of neutrophils leads to tissue damage.

Beneficial Effects of Soluble Guanylyl Cyclase Stimulation and Activation in Sickle Cell Disease Are Amplified by Hydroxyurea: In Vitro and In Vivo Studies.

The complex pathophysiology of sickle cell anemia (SCA) involves intravascular hemolytic processes and recurrent vaso-occlusion, driven by chronic vascular inflammation, which result in the disease’s severe clinical complications, including recurrent painful vaso-occlusive episodes. Hydroxyurea, the only drug frequently used for SCA therapy, is a cytostatic agent, although it appears to exert nitric oxide/soluble guanylyl cyclase (sGC) modulating activity. As new drugs that can complement or replace the use of hydroxyurea are sought to further reduce vaso-occlusive episode frequency in SCA, we investigated the effects of the sGC agonists BAY 60-2770 (sGC activator) and BAY 41-2272 (sGC stimulator) in the presence or absence of hydroxyurea on SCA vaso-occlusive mechanisms and cell recruitment both ex vivo and in vivo. These agents significantly reduced stimulated human SCA neutrophil adhesive properties ex vivo in association with the inhibition of surface β2-integrin activation. A single administration of BAY 60-2770 or BAY 41-2272 decreased tumor necrosis factor cytokine–induced leukocyte recruitment in a mouse model of SCA vaso-occlusion. Importantly, the in vivo actions of both agonists were significantly potentiated by the coadministration of hydroxyurea. Erythroid cell fetal hemoglobin (HbF) elevation is also a major goal for SCA therapy. BAY 41-2272 but not BAY 60-2770 at the concentrations employed significantly induced γ-globin gene transcription in association with HbF production in cultured erythroleukemic cells. In conclusion, sGC agonist drugs could represent a promising approach as therapy for SCA, for use either as stand-alone treatments or in combination with hydroxyurea.SIGNIFICANCE STATEMENTThis preclinical study demonstrates that stimulators and activators of sGC are potent inhibitors of the adhesion and recruitment of leukocytes from humans and in mice with sickle cell anemia (SCA) and may represent a promising approach for diminishing vaso-occlusive episode frequency in SCA. Hydroxyurea, a drug already frequently used for treating SCA, was found to potentiate the beneficial effects of sGC agonists in in vivo studies, implying that these classes of compounds could be used alone or in combination therapy.

Heme Induces Significant Neutrophil Adhesion in Vitro Via an Nfκb-Dependent Pathway

Background: Intravascular hemolysis, a major complication of sickle cell anemia and malaria among other diseases, incurs the release of excessive quantities of hemoglobin and heme from red blood cells. If not adequately sequestered by hemoglobin- and heme-binding proteins, these molecules may incur oxidative stress, thrombosis and cellular activation. Heme is now recognized as a red cell DAMP (damage-associated molecular pattern) that stimulates inflammasome formation in macrophages and can induce neutrophil extracellular trap release under certain circumstances. Aim: This study evaluated the in vitro effects of heme on the adhesive properties of human neutrophils. Methods: Neutrophils were separated from the peripheral blood of healthy individuals and their adhesion in the presence/absence of heme was compared by static adhesion assays using myeloperoxidase, for quantification of cell adhesion (30 min, 37oC, 5% CO2). Results: Heme (50 µM) significantly increased the adhesion of neutrophils to fibronectin (FN) and to recombinant ICAM-1 (an endothelial ligand), when compared to non-treated neutrophils (43.7±4.6%; 10.9±21.4 %, respectively, n=5, P<0.001 for FN and 38.1±3.6%; 6.9±0.8%, respectively, n=6, P<0.001 for ICAM-1). Interestingly, heme induced neutrophil adhesion even more efficiently than the potent pro-inflammatory cytokine, TNF-α (200 ng/ml) (35.5±5.2% adhesion to FN, n=6, P<0.05). Furthermore, inhibition of the NFκB transcription factor with the pharmacological inhibitor, BAY 11-7082 (20 μM), abolished heme-stimulated neutrophils adhesion to FN (reduced from 41.8±6.7% to 7.4±0.5%. n=6; P<0.001). Flow cytometry demonstrated that while TNF-α significantly increases the expression of the Mac-1 integrin subunit, CD11b (data not shown, P<0.05), but not the LFA-1 integrin subunit CD11a (data not shown, P>0.05), on the surface of neutrophils, heme did not augment CD11b or CD11a expression (P>0.05). In contrast, heme significantly augmented the active conformations of these two β2 integrin subunits, as demonstrated by epitope-specific antibodies (CD11b; heme: 520.5±51.8; basal 123.7±16.7 MFI, n=10, P<0.001 and CD11a; heme: 69.8±3.9; basal: 43.8±2.0 MFI, n=6, P>0.001). Inhibition of NFκB translocation with BAY 11-7082 (20 μM) significantly decreased the activity of the LFA-1 integrin on the surface of neutrophils after heme stimulation (reduced to 47.6±4.2 MIF, n=6; P<0.05). To assess whether heme-induced neutrophil adhesive properties are mediated by cytoskeletal rearrangements, we evaluated the effects of cytochalasin D (0.5 μg/ml), an inhibitor of actin polymerization. While cytochalasin D inhibited TNF-α-induced neutrophils adhesion (data not shown, P<0.05), this compound did not significantly alter the adhesive properties of heme-stimulated neutrophils to FN (heme: 22.8±2.3%, p<0.001; cytochalasin D: 22.5±2.0%, p=0.4; n=6). Pre-incubation of heme-stimulated neutrophils with the antioxidants, ascorbic acid (120 µM; 3.5 hours) and α-tocopherol (1mM; 15 min), reduced their adhesion to FN by 14.4±9.2% and 46.5±5.5% respect. n=6, p<0.05). Conclusion: We therefore demonstrate, herein, that heme is a potent activator of neutrophil adhesive properties, increasing the ligand affinity of the β2 integrins via a mechanism that is apparently mediated by an NFkB-dependent pathway. The mechanism of neutrophil activation appears to differ from that stimulated by TNF-α and may involve, in part, the generation of reactive oxygen species. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in the vaso-occlusive process in sickle cell disease and other vascular inflammatory processes, our findings further support the idea that cell-free heme represents a major therapeutic target in the hemolytic diseases. DisclosuresNo relevant conflicts of interest to declare.

Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity

Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions. Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry. In the presence of a TNF-α inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-α, and this increased expression was also inhibited by simvastatin. TNF-α also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression. Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.

Increased Neutrophil Adhesive Properties In Patients with Venous Thromboembolism and Residual Vein Thrombosis and High D-Dimer Levels

Abstract 2301 Introduction:Venous Thromboembolism (VTE) is a multifactorial disease that affects 1:1000 individuals worldwide, with a recurrence rate of about 25% in 10 years. Although many risk factors for VTE are well defined, first presentation and recurrence depend, at least in part, on as yet unknown etiologic factors. Studies in animal models show a tight relation between inflammation and hemostasis, as well as the infiltration of neutrophils in the venous wall after the induction of venous thrombosis. Neutrophils also participate in different stages in the formation and resolution of venous thrombosis. Methods: In this study, we investigated the adhesive properties of neutrophils in VTE patients. We hypothesized that increased adhesive properties of these cells, either as an individual baseline characteristic or as an acquired alteration after a previous VTE episode, could be associated with the thrombotic process. The patient population consisted of 22 VTE patients (14F:8M; median age: 46.1 years) that had completed at least 6 months of oral anticoagulation. Twenty-two healthy volunteers matched to VTE patients by age, gender and ethnic background were used as controls. Neutrophil adhesion was measured by a static adhesion assay in triplicate. Peripheral blood was collected with heparin and neutrophils were separated on Histopaque® (Sigma-Aldrich, St. Louis, MO, USA). Isolated neutrophils (2.2×106 cells/ml) were allowed to adhere to fibronectin (FN)-coated 96-well plates (30 min, 37°C, 5%CO2). Non-adherent cells were then removed by washing and adherent cells calculated as the percentage of cells adhered, compared to a standard curve of known cell concentrations and using a colorimetric enzyme assay. Results are expressed as means ± standard error of mean (SEM) and were compared using the Mann-Whitney test. Results: Overall, adhesion of neutrophils from VTE patients (25.40% ±2.35) was not increased when compared to the control group (21.25%±1.20 p=0.2). However when only patients at a higher risk of recurrence (n=13) - here defined as the presence of elevated D-dimer (higher than 0.5mg/L) and residual vein thrombosis - were analyzed, a statistically significant increase in cell adhesion compared to matched controls was observed (26.70%±2.08 and 21.36%±1.26, respectively, p = 0.04). When these patients (higher recurrence risk; n=13) were compared to the remaining VTE patients (standard recurrence risk, n=9), a non significant increase in neutrophil adhesion was observed (26.70%±2.08 vs 23.51%±5.03 respectively, p=0.1). Conclusions: We demonstrate that neutrophil adhesion is increased in patients with VTE with characteristics associated with increased recurrence risk. In addition, we also observed a non-significant difference in neutrophil adhesion in these patients compared to other VTE patients. Our results suggest that the increased adhesive properties of neutrophils in VTE patients could play a role in the exacerbation of inflammation, and in the pathophysiology of VTE. Further studies are warranted to study whether neutrophil adhesiveness could be used as a biomarker of VTE recurrence. Disclosures:No relevant conflicts of interest to declare.

Inhibition of phosphodiesterase 9A reduces cytokine-stimulated in vitro adhesion of neutrophils from sickle cell anemia individuals

Leukocyte adhesion to vessel walls may initiate vaso-occlusion in sickle cell anemia (SCA); however, the extent to which inflammation participates in this mechanism is not understood. This in vitro study investigated whether inflammatory molecules, commonly augmented in SCA, can affect neutrophil adhesive properties and whether cyclic guanosine monophosphate (cGMP)-elevating agents can inhibit such adhesion. Effects of Interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines, BAY 73-6691 [phosphodiesterase (PDE)-9A-inhibitor], and BAY 41-2271 (guanylate-cylase stimulator) on the adhesive properties of neutrophils from healthy control (CON) and steady-state SCA individuals were determined using static-adhesion assays. SCA neutrophils demonstrated increased adhesive properties, compared to CON neutrophils; IL-8, TNF-α and GM-CSF increased CON neutrophil adhesion and further increased SCA neutrophil adhesion to fibronectin (FN). The PDE9A inhibitor, BAY-73-6691, significantly reduced basal CON neutrophil and SCA neutrophil adhesion; this was accompanied by decreased SCA neutrophil surface expressions of the L-selectin and CD11b adhesion molecules. BAY-73-6691 also significantly reduced cytokine-stimulated CON neutrophil and SCA neutrophil adhesion to FN; however, this was not accompanied by alterations in adhesion-molecule presentation. The chronic inflammatory nature of SCA may contribute to leukocyte adhesive functions in SCA. Furthermore, elevation of leukocyte cGMP may be an interesting approach for inhibition of leukocyte adhesion to the vessel wall, even in the presence of inflammatory stimuli.

Role for cAMP‐protein kinase A signalling in augmented neutrophil adhesion and chemotaxis in sickle cell disease

The significance of the leukocyte in sickle cell disease (SCD) pathophysiology is becoming increasingly recognised; we sought to examine whether the chemotactic properties of neutrophils of SCD individuals may be altered and, further, to better understand the signalling events that mediate altered SCD neutrophil function. Adhesion to immobilised fibronectin (FN) and chemotaxis of control and SCD neutrophils were assessed using in vitro static adhesion assays and 96-well chemotaxis chamber assays. Adhesion assays confirmed a significantly higher basal adhesion of SCD neutrophils to FN, compared with control neutrophils. Chemotaxis assays established, for the first time, that SCD neutrophils demonstrate greater spontaneous migration and, also, augmented migration in response to IL-8, when compared with control neutrophils. Co-incubation of SCD neutrophils with KT5720 (an inhibitor of PKA) abrogated increased basal SCD neutrophil adhesion, spontaneous chemotaxis and IL-8-stimulated chemotaxis. Stimulation of SCD neutrophils with IL-8 also significantly augmented SCD neutrophil adhesion to FN with a concomitant increase in cAMP levels and this increase in adhesion was abolished by KT5720. Interestingly, the adhesive properties of neutrophils from SCD individuals on hydroxyurea therapy were not significantly altered and results indicate that a reduction in intracellular cAMP may contribute to lower the adhesive properties of these cells. Data indicate that up-regulated cAMP signalling plays a significant role in the altered adhesive and migratory properties in SCD neutrophils. Such alterations may have important implications for the pathophysiology of the disease and the cAMP-PKA pathway may represent a therapeutic target for the abrogation of altered leukocyte function.

Hydroxyurea Reverses Increased Adhesive Properties of Neutrophils in Sickle Cell Disease.

Neutrophils play an important role in sickle cell disease (SCD) and the vaso-occlusion that characterizes the disorder; having a central and, possibly, initiating role in the vaso-occlusive (VO) process due to their adhesion to both the vascular endothelium and red cells. Hydroxyurea (HU) therapy significantly decreases the neutrophil count in SCD, however, to our knowledge, the effects of HU therapy upon the augmented adhesive properties of these cells has not been investigated. As such, the effects of HU therapy on the adhesion of SCD neutrophils to the endothelial cell adhesion molecule, ICAM-1, were investigated utilizing an in vitro static adhesion assay. Neutrophils were isolated from peripheral blood of healthy controls, SCD individuals in steady state and SCD patients on HU therapy (SCDHU; 20–30 mg/kg/day, 3-month minimum duration) by separation over a ficoll-paque gradient. Separated neutrophils were resuspended in RPMI medium and allowed to adhere to recombinant ICAM-1-coated 96-well plates (5x106cells/ml, 30 min, 37°C, 5% CO2). Adhesion to ICAM-1 was calculated, using a standard curve, as the percentage of the original cell suspension adhered. SCD neutrophil adhesion to ICAM-1 was approx. doubled (17.09±2.99 %; n=10) compared to control neutrophils (8.89±1.10 %; n=9; P<0.05); in contrast, adhesion of neutrophils from SCDHU patients was similar to that of control cells (8.12±2.79 %; n=6; P<0.05 compared to SCD). Nitric oxide (NO) bioavailability is decreased in SCD, and HU may be a NO donor. Thus, we measured levels of the NO second messenger, cyclic guanosine monophosphate (cGMP), in subjects' neutrophils, since cGMP is an inhibitor of cell adhesion in some cell types. Cyclic adenosine monophospate (cAMP) may also be important for activating cellular adhesion, thus neutrophil cAMP was also evaluated. Whilst intracellular cGMP levels were not significantly different in SCD neutrophils compared to controls (0.110±0.022 pMol/107 cells and 0.142±0.036 pMol/107 cells, n≥11, respectively, P>0.05), cGMP levels in SCDHU neutrophils were significantly higher (0.241±0.023 pMol/107 cells, n=9, P<0.01 compared to SCD). In contrast, whilst cAMP levels are significantly increased in SCD neutrophils (4.55 ±0.38 pMol/106 cells and 2.15±0.38 pMol/106 cells, n≥14, SCD and control, respectively, P<0.001), cAMP levels were significantly lower in SCDHU neutrophils than in SCD neutrophils (3.26 ±0.46 pMol/106 cells, n=14, P<0.05). Flow cytometry demonstrated, however, that the surface expressions of the major neutrophil adhesion molecules were unaltered on SCDHU neutrophils compared to SCD neutrophils; mean expression levels of the CD11a, CD11b, CD18, CD49d and CD29 integrin subunits were unaltered (P>0.05, data not shown). Results indicate that the HU-dependent inhibition of neutrophil adhesion does not appear to be due to any change in surface integrin expression, but may be the consequence of alterations in adhesion molecule activity (affinity or avidity). Thus, we demonstrate that HU therapy significantly reduces the ability of neutrophils of SCD neutrophils to adhere to the endothelial adhesion molecule, ICAM-1. Such an inhibition of the augmented adhesive properties of SCD neutrophils may be important for the prevention of VO processes. A concomitant increase in neutrophil cGMP levels was seen following HU therapy, whilst a decrease in neutrophil cAMP levels was observed, indicating that alterations in the intracellular cyclic nucleotide (modulators of cell adhesion) balance may constitute one of the beneficial effects of HU therapy on neutrophil function.

Inhibition of Increased Sickle Neutrophil Adhesion to Fibronectin and ICAM-1 by a Nitric Oxide Donor.

Leukocytes play an important role in sickle cell disease (SCD); increased numbers of leukocytes are associated with increased SCD morbidity and mortality and leukocytes participate significantly in the vaso-occlusive process. Nitric oxide (NO), a signaling gas involved in vaso-relaxation and platelet aggregation regulation, has recently been implicated as important in SCD pathophysiology. NO plasma bioavailability is thought to be decreased in SCD, and NO therapy has been proposed for SCD and vaso-occlusive crises treatment. The possible consequences of decreased NO bioavailability in SCD are innumerous and have yet to be fully explored. As NO is an important inflammatory mediator and may be important for the inhibition of leukocyte adhesion and migration mechanisms, we compared nitric oxide metabolites and cGMP levels (second messenger for NO) in neutrophils from normal subjects and SCD patients (SCD neutrophils) and then compared to the adhesive properties of these cells and observed the effect of NO donating agents on this adhesion. Neutrophils were isolated from whole blood by separating on a Ficoll gradient. NO metabolites/cGMP level were measured in neutrophil extracts using specific assays. Levels of nitric oxide metabolites in SCD neutrophils did not differ signicantly from those in normal neutrophils (17.34 ± 7.02%, n=10; 16.30 ± 7.00%, n=8; respectively). Furthermore, levels of cGMP (0.11 ± 0.02 pMol/1x107 neutrophils, n=9) in SCD neutrophils were not significantly different to those observed in normal neutrophils (0.142 ± 0.036 pMol/1x107 neutrophils, n=7; p>0.05). Cell adhesion to fibronectin and ICAM-1 was compared using static adhesion assays. Neutrophils from SCD patients demonstrated a significantly greater adhesion to FN-coated plates (FN= 20mg/ml) than normal neutrophils (18.73 ± 6.24%, n=18; 13.52 ± 5.15%, respectively, n=15; p=0.016). Similarly, neutrophils from SCD patients demonstrated significantly higher adhesion to ICAM-1 (10mg/ml) when compared to normal neutrophils (19.51 ± 9.02%, n=13; 10.98 ± 3.95%, respectively n=9; p=0.025). Co-incubation of SCD neutrophils with a nitric oxide donor, sodium nitroprusside (SNP,10mM) abolished their increased adhesion to both FN and ICAM-1 to levels similar to those of normal neutrophil adhesion (16.53 ± 2.27 %, reduced to 12.61 ± 1.98%, n=8; p= 0.002 for fibronectin and 19.12 ± 3.00% reduced to 16.49 ± 2.60, n=8; p=0.009 for ICAM-1). In contrast, SNP did not affect normal neutrophil adhesion to either FN (11.59 ± 1.02% and 12.67 ± 1.35%, following incubation n=11; p>0.05) or ICAM-1 (12.33 ± 0.25% to 11.95 ± 0.62%, n= 5; p> 0.05) significantly. Although NO bioavailability may be decreased in the plasma of SCD patients due to NO sequestration by cell-free hemoglobin, our data indicate that NO dynamics are not significantly altered in the leukocytes of patients in SCD, as indicated by levels of NO metabolites and the major second messenger for NO, cGMP, are not significantly different in sickle cell neutrophils. We speculate that increased neutrophil adhesive properties in SCD may not be the consequence of decreased NO bioavailability, rather, other factors such as altered cytokine levels may mediate increased cell adhesion. However, whilst increased adhesion appears not to be mediated by NO-dependent mechanisms, an NO donor significantly reduced increased SCD neutrophil adhesion to both FN and ICAM-1, indicating that NO and NO donating drugs may also benefit SCD patients by reducing increased leukocyte adhesion, a mechanism important for the vaso-occlusive process.

Systemic inflammation during paediatric cardiopulmonary bypass: changes in neutrophil adhesive properties

Systemic inflammation during paediatric cardiopulmonary bypass: changes in neutrophil adhesive properties

Down‐regulation by tumor necrosis factor‐α of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism

Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties.

The chemotactic receptor for human C5a anaphylatoxin

Host defence and inflammatory responses are controlled and amplified by receptor-mediated events often initiated by a chemotactic factor that directs the approach of phagocytic cells. Complement receptors CR1 and CR3 are responsible for the phagocytic and adhesive properties of neutrophils, whereas the C5a receptor mediates the pro-inflammatory and chemotactic actions of the complement anaphylatoxin C5a. In addition to stimulating chemotaxis, granule enzyme release and superoxide anion production, this receptor stimulates upregulation of expression and activity of the adhesion molecule MAC-1, and of CR1, and a decrease in cell-surface glycoprotein 100MEL-14 on neutrophils. In vivo, the C5a receptor may participate in anaphylactoid and septic shock. The human C5a receptor was cloned from U937 and HL-60 cells and identified by high affinity binding when expressed in COS-7 cells. The deduced amino-acid sequence of the receptor reveals the expected motifs befitting its interaction with cellular GTP-binding proteins.

Stimulation of human neutrophil adhesive properties by adenine nucleotides.

Inasmuch as adenine nucleotides may be secreted by platelets during inflammation, we sought to determine whether ATP and related compounds could serve as stimuli of neutrophil (polymorphonuclear cells, PMN) activation as manifested by an increase in their adhesive properties. Exposure of isolated human PMN to ATP or its nonhydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate) did indeed stimulate an increase in cellular adhesive function as assessed by an increase in the surface expression of the leukocyte adhesion-promoting glycoprotein, Mo1 (CD11b/CD18), the initiation of PMN aggregation, and (in the case of ATP) the attachment of increased numbers of albumin-coated polystyrene latex beads. However, this increase in PMN adhesive function was not accompanied by the generation of products of the respiratory burst. These in vitro data suggest the possible influence of secreted adenine nucleotides in promoting neutrophil adhesion-dependent interactions at inflammatory sites in vivo.

Neutrophil FC-receptor expression in cells of the circulating and marginated pools.

A method for rosetting neutrophils was developed and optimal rosetting conditions determined. The adhesive properties of neutrophils and their ability to form rosettes were studied for cells in the circulating pool, and following exercise when the circulating pool had been enriched by neutrophils from the marginated pool. We found the marginated pool to be the same as the circulating pool in terms of rosetting subpopulations and the in vitro adhesive properties of these subpopulations, indicating that the two pools can be considered kinetically as one. We also showed that non-rosetting neutrophils can develop the ability to form rosettes following adhesion to glass in the presence of autologous serum.