Adhesion Of Activated Neutrophils Research Articles

Different glycoforms of alpha-1-acid glycoprotein contribute to its functional alterations in platelets and neutrophils.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.

Transfusion-Related Acute Lung Injury: An Update

Transfusion-associated acute lung injury (TRALI) has emerged as a leading cause of transfusion-related morbidity and mortality. TRALI is characterized by acute non-cardiogenic pulmonary edema and respiratory compromise in the setting of transfusion. The study of TRALI has been hampered by inadequate case definitions and an incomplete understanding of the pathologic mechanisms. Recent consensus conferences took an important first step by providing a framework for case definition. Recent advances in the understanding of the pathogenesis of TRALI have also occurred. TRALI has been primarily attributed to donor leukocyte antibodies that are thought to interact with recipient neutrophils, resulting in activation and aggregation in pulmonary capillaries, release of local biologic response modifiers causing capillary leak, and lung injury. An alternate mechanism termed the "two hit" or "neutrophil priming" hypothesis postulates that a pathway to neutrophil activation and aggregation can occur without leukocyte antibodies. A first event such as sepsis or trauma can induce pulmonary endothelial activation, release of cytokines, and priming of neutrophils. A subsequent second event such as exposure to lipids, cytokines or antibodies in a blood component would then cause activation of adherent neutrophils and a release of bioreactive molecules leading to lung injury. There are limited clinical and animal studies to support the "two hit" model. These proposed mechanisms are not mutually exclusive in that donor leukocyte antibody can be pathogenic in both models and have implications for new strategies to prevent TRALI.

Neutrophil Activation in Anti‐Proteinase 3‐Positive Vasculitis – a Prospective Study

In vitro studies have demonstrated that antineutrophil cytoplasmic antibodies (ANCA) have the capacity to activate neutrophils. Whether circulating neutrophils in patients with vasculitis are activated is under debate. Eight consecutive patients with antiproteinase 3 (PR3) positive acute vasculitis were included in this prospective study. Neutrophil expression of adhesion molecules, Fc-receptors and the ANCA-antigen PR3 was analysed and clinical characteristics were documented at inclusion and after 1, 3, 6 and 9 months in the same individuals. As additional markers of inflammation and endothelial activation interleukin-8 and soluble vascular cell adhesion molecule-1 in serum were analysed at the same time points. The expression of adhesion molecules on circulating neutrophils, CD62L and CD11b after in vitro N-formyl-methionyl-leucyl-phenylalanine stimulation was significantly decreased at diagnosis and after 1 month but returned to normal levels after 3-9 months. The neutrophil expression of Fc-receptor IIIb (CD 16) was decreased at diagnosis but normalized after 1-9 months. The main finding was an activated neutrophil adhesion phenotype at diagnosis and after 1 month, with normalized expression of adhesion molecules at 3-9 months. A pathological regulation of adhesion molecules may have implications on the endothelial damage seen in vasculitis.

The effect of fever‐like temperatures on neutrophil signaling

The effect of fever on neutrophils has not been explored. We tested the hypothesis that fever-like temperature spikes affect neutrophil signaling and function. Prior 60 min, 42 degrees C heat exposure inhibited p38 MAPK, ERK, PI3-Kinase/Akt, and NF-kappaB activation in TNF-alpha-challenged suspended neutrophils. Using pharmacological inhibitors and an inhibitory peptide transduced into neutrophils by a HIV-TAT sequence, we found that p38 MAPK and NF-kappaB mediate TNF-alpha-mediated delayed apoptosis in suspended neutrophils. Heat exposure (39-42 degrees C) did not affect constitutive apoptosis but abrogated TNF-alpha-delayed apoptosis in these suspended cells. In contrast, adhesion-dependent functions were not inhibited. Furthermore, we found that heat exposure neither blocked p38 MAPK, ERK, and NF-kappaB activation in neutrophils on fibronectin nor prevented delayed apoptosis by TNF-alpha when cells interacted with fibronectin. Above and beyond apoptosis, TNF-alpha initiated NF-kappaB-dependent gene transcription. Heat exposure blocked this effect in suspended neutrophils but not in neutrophils on fibronectin. Finally, we show that beta2-integrins, which are not necessary for TNF-alpha-induced NF-kappaB activation at 37 degrees C, transduce costimulatory signals allowing NF-kappaB activation after heat exposure. The effect could protect circulating neutrophils from TNF-alpha activation, while not interfering with activation of adherent neutrophils. Fever could make neutrophils more parsimonious.

Neutrophils activation can be diminished by apolipoprotein A-I

High density lipoprotein (HDL) has anti-inflammatory function. To investigate the effects of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, on activated neutrophils, we stimulated neutrophils in vitro with fMLP and PMA, as a receptor-binding and a nonreceptor-binding stimuli, respectively, and incubated ApoA-I with those neutrophils. Three conditions were utilized: 1) resting neutrophils + ApoA-I (0, 2.5 ,5, 10 μg/mL respectively), 2) fMLP(10 −7 mol/L)-activated neutrophils + ApoA-I (0, 2.5, 5, 10 μg/mL respectively), and 3) PMA(10 −7 mol/L)-activated neutrophils + ApoA-I (0, 2.5 ,5, 10 μg/mL respectively). After incubation, we measured neutrophils adhesion to fibronectin, oxidative bust (O 2 − and H 2O 2 production), degranulation (release of MPO and elastase), and L929 cell mortality which were attacked by release-out of cytokines in activated neutrophils (using MTT). Our results showed that in vitro ApoA-I inhibits fMLP- and PMA- activated neutrophil adhesion, oxidative burst, degranulation and L929 cell mortality. These inhibition effects of ApoA-I on fMLP-activated neutrophils are more powerful than that on PMA-activated neutrophils. ApoA-I has no effect on resting neutrophils. We concluded that ApoA-I could diminish the function of activated neutrophils.

Stimulation of the inflammatory system by reamed and unreamed nailing of femoral fractures

It has been suggested that reamed intramedullary nailing of the femur should be avoided in some patients with multiple injuries. We have studied prospectively the effect of femoral reaming on the inflammatory process as implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiple-organ failure (MOF). We studied changes in the levels of serum interleukin-6 (IL-6) (proinflammatory cytokine), neutrophil CD11b (C3) receptor expression (activated neutrophil adhesion molecule), serum soluble intracellular adhesion molecule (s-ICAM-1), serum soluble E-selectin (the soluble products of endothelial adhesion molecules) and plasma elastase (neutrophil protease) in a series of patients with femoral fractures treated by nailing. We have also compared reamed nailing with unreamed nailing. We found that the levels of serum IL-6 and elastase rose significantly during the nailing procedure indicating a measurable ‘second hit’. There was no clear response in leukocyte activation and no difference in the release of endothelial adhesion molecule markers. There was no significant difference between groups treated by reamed and unreamed nailing. Although clinically unremarkable, the one patient who died from ARDS was shown to be hyperstimulated after injury and again after nailing, suggesting the importance of an excessive inflammatory reaction in the pathogenesis of these serious problems. Our findings have shown that there is a second hit associated with femoral nailing and suggest that the degree of the inflammatory reaction may be important in the pathogenesis of ARDS and MOF.

Actin Polymerisation Regulates Integrin-Mediated Adhesion as Well as Rigidity of Neutrophils

Activation of adherent neutrophils causes them to convert from selectin-mediated rolling to integrin-mediated immobilisation and migration. Migration is known to depend on formation and redistribution of filamentous (F) actin, but although immobilisation in seconds parallels early cortical actin polymerisation, no link has been proven. We tested the effect of the actin-polymerising agent jasplakinolide (10μM) on adhesive and mechanical properties of neutrophils. Pretreated cells were able to adhere and roll on immobilised platelets in a flow-based adhesion assay, but whereas untreated rolling cells became immobilised in seconds when chemotactic formyl peptide (fMLP, 0.1μM) was superfused over them, the cells treated with jasplakinolide continued rolling. Pretreatment with jasplakinolide also blockedde novoexpression of integrin CD11b and shape change which otherwise occurred in minutes after treatment with fMLP. Jasplakinolide directly caused actin polymerisation within neutrophils, evidenced by a marked increase in rigidity (resistance to aspiration into a 5μm micropipette) and increase in association of actin with the Triton-insoluble cytoskeleton. These results indicate that rearrangement of the actin cytoskeleton regulates integrin-mediated adhesion of activated neutrophils, as well as their migration and mechanical properties.

Inhibition of phospholipase D blocks activation of fibrinogen-adherent neutrophils by tumor necrosis factor.

Tumor necrosis factor stimulates fibrinogen-adherent neutrophils to produce a dramatic oxidative burst; the resulting superoxide and other products may contribute to tissue damage in severe infection. Inhibitors of the phospholipase D pathway blocked this activation without affecting cell adherence or nonspecific activation by phorbol myristate acetate. The phospholipase D pathway appears to be involved in the activation of adherent neutrophils by tumor necrosis factor, and this pathway may be a target for modulation of tumor necrosis factor's effects.

Neutrophil but not Monocyte Activation Inhibits P-Selectin-Mediated Platelet Adhesion

Selectins are Ca(2+)-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of Activated Neutrophil Adhesion to Endothelium by Soluble Adhesion Protein GMP140

Neutrophils and monocytes, but not lymphocytes, adhered strongly to plastic surfaces coated with GMP140, a protein of endothelial cells and platelets. This adhesion of neutrophils was mediated by GMP140 and not by the CD18 integrin complex. By contrast, GMP140 in solution inhibited the CD18-dependent adhesion of tumor necrosis factor-alpha-activated neutrophils to plastic surfaces and resting endothelium, but not of resting neutrophils to tumor necrosis factor-alpha-activated endothelium. Thus, the binding of a soluble form of an adhesion protein selectively inhibited another set of adhesive events. Soluble GMP140 may be important in maintaining the nonadhesiveness of neutrophils in the circulation and may serve to limit inflammatory reactions.

Lymphotoxin Induces Secretion of Granule Proteins From Adherent Neutrophils: Possible Role of Intracellular Free Calcium

Lymphotoxin (LT) can activate human neutrophils. Using a hemolytic plaque assay to detect secretion of lactoferrin and myeloperoxidase (MPO) from single adherent neutrophils, we showed that LT induced secretion from both primary and secondary granules. Incubation of cells with cytochalasin B was required for MPO secretion, and it enhanced lactoferrin secretion. Pertussis toxin, which blocks a G-protein in the plasma membrane, inhibited LT-induced exocytosis of MPO, but not of lactoferrin. Incubation with LT did not induce any detectable changes of the cytoplasmic free [Ca2+] in neutrophils. On the other hand, secretion of granule proteins from adherent neutrophils in response to LT was blocked by loading neutrophils with quin-2 in order to increase the intracellular calcium buffering capacity. This was achieved at a concentration of quin-2, at which the secretion induced by the phorbol ester PMA and the chemotactic peptide FMLP was unaffected. Trifluoroperazine (TFP), a dual protein kinase C and calmodulin inhibitor, significantly inhibited the LT-mediated secretion of lactoferrin from adherent granulocytes. The PMA effect was unaltered by TFP under these conditions, suggesting that the inhibitory effect was on a calcium-calmodulin dependent step. The secretion induced by TNF and GM-CSF was also blocked by buffering changes in the intracellular [Ca2+] and inhibited to a similar extent by TFP. Our results suggest that calmodulin and minute changes in the cytoplasmic free [Ca2+] may be involved in a common signal transduction pathway engaged in activation of adherent neutrophils by several cytokines.

“Autoregulation” of Human Neutrophil Activation In Vitro: Regulation of Phorbol Myristate Acetate-Induced Neutrophil Activation by Cell Density

Phorbol myristate acetate (PMA, 10(-7) M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This "autoregulatory" phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2'7'-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2'7'-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response.