Effect of cytokines and chemokines on sickle neutrophil adhesion to fibronectin

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37 degrees C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 micro) was determined (60 min, 37 degrees C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10-100 pg/ml), GCSF (1- 10 ng/ml) or IL-8 (1-100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1-100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.     

CD157 is an important mediator of neutrophil adhesion and migration

CD157, a glycosylphosphatidylinositol (GPI)-anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca(2+) concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]-ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)-differentiated (CD157(+)/CD38(-)) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and beta(2) integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.     

Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation- independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin- binding domain of FN by a novel divalent cation-independent mechanism.     

Prednisolone interferes with neutrophil adhesion and neutrophil mediated endothelial injury

Adhesion of leukocytes to vascular endothelium is a crucial step in inflammation. This interaction may result in damage of the endothelial cells (EC). We evaluated the effects of prednisolone on adhesive interactions between human polymorphonuclear neutrophil granulocytes (PMN) and human umbilical vein endothelial cells (HUVEC) as well as PMN mediated cytotoxicity to HUVEC (as release of 51chromium), mediated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), lipoxin A4 (LXA4), and the calcium ionophore A23187 in vitro. Prednisolone dose-dependently interfered with adhesion and cytotoxicity induced by fMLP. Prednisolone (at 10 microM) led to a 39% reduction of adhesion and an almost complete inhibition of cytotoxicity, mainly by effects on the PMN. Prednisolone also interfered with cytotoxicity induced by LXA4 by effects on PMN as well as on HUVEC. Adhesion and cytotoxicity induced by the calcium ionophore A23187 was not affected in any way by prednisolone. Thus, in these in vitro models of vasculitis, prednisolone interferes with adhesive and cytotoxic interactions induced by receptor-dependent agonists. These protective effects of prednisolone might explain some of the beneficial effects of glucocorticoids in the treatment of vasculitis.     

Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease

Background and aims  

The etiology of pouchitis after coloproctomucosectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis is still unknown. Beside changes in luminal antigens, the immunological predisposition is assumed to be responsible. In previous electrophysiological studies, we showed that mucosal barrier and transport function in pouchitis is markedly reduced. Thus, the aim of the present study was to analyze barrier function on the molecular level.     

Secretory leucocyte proteinase inhibitor: inhibition of fibronectin degradation by neutrophil elastase.

Degradation of surface-bound fibronectin of the upper respiratory tract by human leucocyte elastase (HLE) was shown to favour colonization of these mucosal surfaces by Gram-negative bacteria. We investigated the degradation of fibronectin by purified HLE and by enzymes released from stimulated human polymorphonuclear leucocytes (PMNs), in the presence of recombinant secretory leucocyte proteinase inhibitor (rSLPI) and alpha 1-proteinase inhibitor (alpha 1-PI), the two main inhibitors of HLE within the airways. Our results show that HLE degraded fibronectin at concentrations as low as 0.2 nM. To inhibit the degradation of fibronectin by pure HLE in an experimental system in which the enzyme was premixed with inhibitor, a twofold molar excess of rSLPI and an equimolar concentration of alpha 1-PI were required. On the other hand, a fivefold molar excess of rSLPI was necessary to inhibit degradation of fibronectin by enzymes released from stimulated neutrophils. In order to estimate the role of oxidants generated by stimulated PMNs in the activation of the inhibitory capacity of rSLPI by stimulated PMNs, we preincubated PMNs with antioxidants such as superoxide dismutase, methionine, catalase or Na-azide prior to stimulation of the cells. Under these conditions, a threefold molar excess of rSLPI over released HLE was required to inhibit the degradation of fibronectin, raising the possibility that either exogenous or endogenous antioxidants in the lung could be important in improving the efficacy of this therapeutic antiprotease. We conclude that a molar excess of rSLPI to HLE is always necessary to inhibit fibronectin degradation by HLE, and that addition of antioxidants partly prevents the inactivation of rSLPI by oxidants released from stimulated PMNs.     

Laminin and fibronectin in cell adhesion: enhanced adhesion of cells from regenerating liver to laminin.

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5--6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.     

TREM-1 ligand expression on platelets enhances neutrophil activation

The triggering receptor expressed on myeloid cells 1 (TREM-1) plays an important role in the innate immune response related to severe infections and sepsis. Modulation of TREM-1-associated activation improves the outcome in rodent models for pneumonia and sepsis. However, the identity and occurrence of the natural TREM-1 ligands are so far unknown, impairing the further understanding of the biology of this receptor. Here, we report the presence of a ligand for TREM-1 on human platelets. Using a recombinant TREM-1 fusion protein, we demonstrate specific binding of TREM-1 to platelets. TREM-1-specific signals are required for the platelet-induced augmentation of polymorphonuclear leukocyte (PMN) effector functions (provoked by LPS). However, TREM-1 interaction with its ligand is not required for platelet/PMN complex formation, which is dependent on integrins and selectins. Taken together, the results indicate that the TREM-1 ligand is expressed by platelets, and the TREM-1/ligand interaction contributes to the amplification of LPS-induced PMN activation. Our results shed new light on our understanding of TREM-1 and its role in the innate inflammatory response in infections and might contribute to the development of future concepts to treat sepsis.     

Fibronectin in an extracellular matrix of cultured endothelial cells supports platelet adhesion via its ninth type III repeat : A comparison with platelet adhesion to isolated fibronectin

We investigated the involvement of different domains of fibronectin in mediating platelet adhesion to fibronectin in the extracellular matrix (ECM) of cultured endothelial cells under flow conditions. Polyclonal anti-fibronectin antibodies were absorbed with Sepharose to which no protein, intact fibronectin, or different fibronectin fragments had been coupled to obtain supernatants (Sups) (Sup(0), Sup(FN), and Sup(name of the fragment), respectively) from which a specific part of the antibodies had been removed. Treatment of the ECM before perfusion with Sup(0) resulted in a 36% decrease in platelet coverage, whereas treatment with Sup(FN) resulted in maximal adhesion. Treatment of the ECM with supernatants from which antibodies directed against the gelatin- or heparin-binding domain had been removed showed the same inhibition as treatment with Sup(0). Removal of antibodies directed to the 120-kDa cell-binding domain resulted in a level of adhesion equal to the level found when the ECM was treated with Sup(FN). Further analysis of this central region showed that only treatment with supernatants from which antibodies directed to the ninth type III repeat (III-9) of fibronectin had been removed resulted in a significantly higher adhesion than treatment with Sup(0). Studies of adhesion to the fragments themselves showed that only fragments containing III-10 were able to support adhesion. Mutation of the Arg-Gly-Asp (RGD) sequence into Arg-Gly-Glu (RGE) in one of those fragments resulted in a complete loss of adhesive capacity. These data suggest that platelet adhesion to fibronectin in the ECM depends on III-9, whereas III-10 does not seem to be required. For platelet adhesion to isolated fibronectin, an intact RGD sequence seems to be crucial.     

The role of plasma fibronectin in platelet adhesion to collagen

Human washed platelets were eluted from columns of Sepharose 4B linked to different preparations of collagen in order to evaluate cell adhesion. Collagen preparations characterized by low and high affinity toward platelets were identified. In our experiments, fibronectin purified from human plasma modified platelet adhesiveness, though not dramatically. When washed platelets, resuspended in a buffer containing fibronectin, were filtered on a low-affinity collagen-Sepharose, a significant increase in their adhesion occurred. A similar modification could be observed when platelets were allowed to adhere to the same collagen-Sepharose preconditioned with fibronectin. The effect of fibronectin was otherwise negligible when the high-affinity collagen was used for the experiments.     

E-selectin-dependent neutrophil adhesion to Rickettsia rickettsii- infected endothelial cells

Increased neutrophil or HL60 cell adhesion to Rickettsia rickettsii- infected endothelial cells (ECs) was observed at 6 to 8 hours after the initiation of infection, diminishing by 24 hours. Similar increases were observed using formaldehyde-fixed neutrophils. Cellular association and likely the intracellular presence of rickettsiae was required for enhanced neutrophil adhesion, because culture medium conditioned by infected cells or rickettsiae rendered noninfective by pretreatment with tetracycline were ineffective at inducing neutrophil adhesion. Increases in neutrophil adhesion caused by infection were blocked by pretreatment of ECs with cycloheximide, suggesting the involvement of new protein synthesis in the cells' response. Flow cytometric analysis of infected cells showed increases in cell surface expression of E-selectin compared with uninfected control cells. Furthermore, incubation of 6- to 8-hour infected cells with a blocking monoclonal antibody against E-selectin (BB11) inhibited neutrophil adhesion an average of 61%. These results suggest the involvement of E- selectin in neutrophil adhesion to infected ECs occurring early in the course of the infection process. EC-initiated recruitment of neutrophil adhesion during rickettsiae infection could contribute to the pathologic changes associated with Rocky Mountain Spotted Fever.     

Mechanisms underlying neutrophil adhesion to apical epithelial membranes.

Crypt abscesses allow prolonged apposition of activated neutrophils to the epithelial surface of the colon. Adhesion of neutrophils to both the vascular endothelium and basolateral epithelial membrane share common effector molecules but are distinct processes. This study aimed to define the mechanisms that effect adhesion, independent of transmigration, to the apical epithelium. HT29 (cl 19A) cells were grown to confluency and incubated with neutrophils under conditions of: (i) neutrophil stimulation with phorbol-myristate-acetate; (ii) monolayer stimulation with interferon gamma, tumour necrosis factor alpha (IFN gamma, TNF alpha); and (iii) recent epithelial cell trypsinisation. These experiments were carried out in the presence of neutralising antibodies to CD18, CD11b, LFA-1, E-selectin, P-selectin, intracellular adhesion molecule 1 (ICAM-1), and ICAM-2; a novel CD11b/CD18 antagonist, neutrophil inhibitory factor (rNIF); adenosine receptor agonists (5'N-ethycarboxamido adenosine/N6-cylopentyladenosine (NECA/CPA)) and a platelet activating factor (PAF) receptor antagonist lexipafant. Adhesion of stimulated neutrophils to resting monolayers was Mac-1, CD18 dependent and ICAM-1, ICAM-2, E-selectin, P-selectin, PAF independent. Cytokine activated monolayers exhibited higher binding of neutrophils which was inhibited by rNIF and aCD18. Recently trypsinised monolayers bound neutrophils in a CD11b/CD18 and CD18 independent manner. Adenosine agonists failed to influence neutrophil adhesion under any condition. This study shows neutrophil adhesion to apical epithelial membranes is similar to that at the epithelial basolateral membrane, though different to that seen at the vascular endothelium. These results highlight regional differences in neutrophil adhesion molecule usage.     

Src family kinases mediate neutrophil adhesion to adherent platelets

Polymorphonuclear leukocyte (PMN)-platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of "outside-in" signaling by Src-family tyrosine kinases (SFKs) in the regulation of alphaMbeta2-integrin-dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in beta2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from alphaMbeta2(-/-), hck(-/-)fgr(-/-), and hck(-/-)fgr(-/-)lyn(-/-) mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of alphaMbeta2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for alphaMbeta2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin-PSGL-1-mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.     

纤粘连蛋白介导的平滑肌细胞粘附迁移与粘着斑激酶的磷酸化

目的　探讨纤粘连蛋白介导的血管平滑肌细胞粘附和迁移与粘着斑激酶 (focaladhesionkinase ,FAK)的磷酸化的关系。方法　不同浓度的纤粘连蛋白 (fibronectin ,FN)刺激培养的血管平滑肌细胞 (smoothmusclecells,SMCs) ,观察细胞粘附反应 ,统计铺展比率。免疫沉淀和Wsternblot分别检测FAK及FAK磷酸化的表达量。利用改良的BoydenChamber测SMCs迁移。结果　FN有效地促进了SMC粘附 ,其铺展比率、迁移细胞数均显著高于对照 (P <0 0 5 ) ,且随FN浓度递增而增加。其中 2 0、40、6 0 μg ml组分别为 (75 6± 6 5 ) %、(80 9± 5 4) %和 (82 4± 7 9) % ,无组间差异 ,但均高于 5 μg ml的 (2 0 8± 3 2 ) %和 10 μg ml组的 (32 8± 4 7) % ,各组迁移细胞数也从 16 8± 3 6 HFP 2 0 0×增加到48 9± 6 1 HFP 2 0 0×。不同浓度FN作用后均有FAK的表达 ,FN10 μg ml即可致FAK磷酸化。表明FN介导SMCs粘附和迁移时伴有显著的FAK活化。结论　FN诱导平滑肌细胞粘附和迁移可能是通过FAK介导的 ,对其活性进行调控将有助于抑制血管损伤后内膜平滑肌细胞的迁移。     

Intercellular adhesion molecule-1-dependent neutrophil adhesion to endothelial cells induces caveolae-mediated pulmonary vascular hyperpermeability

We investigated the role of caveolae in the mechanism of increased pulmonary vascular permeability and edema formation induced by the activation of polymorphonuclear neutrophils (PMNs). We observed that the increase in lung vascular permeability induced by the activation of PMNs required caveolin-1, the caveolae scaffold protein. The permeability increase induced by PMN activation was blocked in caveolin-1 knockout mice and by suppressing caveolin-1 expression in rats. The response was also dependent on Src phosphorylation of caveolin-1 known to activate caveolae-mediated endocytosis in endothelial cells. To address the role of PMN interaction with endothelial cells, we used an intercellular adhesion molecule (ICAM)-1 blocking monoclonal antibody. Preventing the ICAM-1-mediated PMN binding to endothelial cells abrogated Src phosphorylation of caveolin-1, as well as the increase in endothelial permeability. Direct ICAM-1 activation by crosslinking recapitulated these responses, suggesting that ICAM-1 activates caveolin-1 signaling responsible for caveolae-mediated endothelial hyperpermeability. Our results provide support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of PMNs adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury.     

Mammalian reticulocytes lose adhesion to fibronectin during maturation to erythrocytes.

We describe three situations in which a large fraction of circulating red blood cells attach tightly and specifically to fibronectin: (i) rabbits made anemic by repeated bleeding, (ii) patients with hemolytic anemia and functional asplenia and splenectomized normal humans, and (iii) splenectomized mice. Upon induction of anemia in rabbits, the proportion of circulating red blood cells capable of specifically attaching to fibronectin-coated plastic increased in parallel with the number of reticulocytes. Fibronectin-adherent red cells were barely detectable when the rabbit had recovered from the anemia. Attachment of reticulocytes to fibronectin was specific; cells did not attach to dishes coated with albumin, laminin, or collagen. None of these proteins promoted the attachment of normal erythrocytes. About 75% of the erythrocytes from splenectomized mice (but not control mice) also attached specifically to fibronectin 40 days after surgery. The effect of splenectomy was incomplete and transient; adherent cells were not detectable 8 weeks after splenectomy. As judged by labeling studies with [35S]methionine, newly emergent reticulocytes preferentially attached to fibronectin. We suggest that about half of the reticulocytes in erythropoietically unstressed mice lose their ability to attach to fibronectin, possibly due to loss of fibronectin-adhesive components, during passage through the spleen. The others lose their ability to interact with fibronectin before release, in the bone marrow, or in some extrasplenic site.     

Abnormal neutrophil adhesion in sickle cell anaemia and crisis: relationship to blood rheology

Defects in neutrophil adhesion and migration may contribute to the susceptibility to infection seen in sickle cell anaemia (SCA). These dynamic defects may be influenced by abnormalities in blood rheology found in this disorder. A whole blood model was used to study neutrophil adhesion in SCA patients: neutrophil adhesion to protein coated glass was quantitated by measuring the rate of disappearance of neutrophils from heparinized whole blood circulating through a perfusion chamber. Twenty-three adult patients (Hb SS) were studied in asymptomatic steady state, of whom nine were also studied during pain crisis, both before and 4-7 d after conventional therapy. Red cell and granulocyte filterability and whole blood and plasma viscosity were also measured. The half-time for disappearance from the perfusion system (t1/2) of neutrophils from patients in the steady-state was 93.5 +/- 8.4 min, compared to 49.1 +/- 2.8 min in normal age-matched controls (P = 0.001). In crisis t1/2 was further prolonged to 170.0 +/- 16.1 min (P = 0.01 v. steady state). After therapy, t1/2 decreased to 57.0 +/- 4.5 min (P = 0.001 v. pre-therapy state and P = 0.009 v. steady state) and was comparable to Hb AA controls. These findings reveal a neutrophil adhesion defect in SCA which worsens in crisis but is corrected following supportive therapy. Red cell filterability (expressed as average resistance to flow and pore-clogging particles) and white cell filterability (expressed as pore-clogging particles) were also abnormal in SCA and were found to correlate with neutrophil adhesion. Plasma viscosity also correlated with adhesion t1/2. The defect appears to be related to abnormal blood flow properties in SCA but the rheological factors cannot fully explain either the steady-state defect or the marked changes in neutrophil adhesion during crisis.     

High insulin enhances neutrophil transendothelial migration through increasing surface expression of platelet endothelial cell adhesion molecule-1 via activation of mitogen activated protein kinase

AIMS/HYPOTHESIS: There is increasing evidence that hyperinsulinaemia is linked with the development of atherosclerosis in patients with diabetes. However, the mechanisms by which hyperinsulinaemia causes accelerated atherosclerosis, especially with respect to leukocytes transendothelial migration, are poorly understood. We examined whether hyperinsulinaemia directly affects neutrophil transendothelial migration and surface expression of related endothelial adhesion molecules. METHODS: Experiments on the transmigration of neutrophils from healthy volunteers and from patients with Type II (non-insulin-dependent) diabetes mellitus across human umbilical vein endothelial cells cultured in insulin-rich medium using cell-culture inserts were carried out. Migrated neutrophils were quantified by measuring their myeloperoxidase activities, and the surface expression of endothelial adhesion molecules was examined using an enzyme immunoassay. RESULTS: High insulin (over 50 microU/ml for 24 h) enhanced neutrophil transendothelial migration in a dose-dependent manner. This was associated with increased expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of intercellular adhesion molecule-1 (ICAM-1), P-selectin or E-selectin. Both phenomena were attenuated by pretreatment with a tyrosine kinase inhibitor, especially a mitogen-activated protein kinase inhibitor, but not by inhibitors of other second messengers. In addition, a mitogen-activated protein kinase activator, anisomycin, by itself enhanced both neutrophil transendothelial migration and PECAM-1 expression within 3 h in a dose-dependent manner. Pretreatment with nitric oxide synthase inhibitors had no effect on these events. CONCLUSION/INTERPRETATION: These results suggest that hyperinsulinaemia could accelerate atherosclerosis by directly enhancing neutrophil transendothelial migration through increasing endothelial PECAM-1 expression via mitogen-activated protein kinase activation.     

A reassessment of the energy requirements for neutrophil migration: adenosine triphosphate depletion enhances chemotaxis

In view of previous studies demonstrating a significant correlation between adenosine triphosphate (ATP) depletion and impairment of chemotaxis (CTX) during granulocyte (PMN) storage, we sought to quantitate the relationship between CTX and PMN energy metabolism. We incubated PMNs at 37 degrees C with 2-deoxyglucose (2-dg) in the presence of 5 mmol/L glucose. As expected, ATP inhibition by 2-dg was time-dependent (T 1/2, 18 minutes) and dose-dependent, with half- maximal inhibition of ATP (ID50) with 1.3 +/- .3 mmol/L 2-dg. Similar concentrations of 2-dg inhibited lactate generation, phagocytosis, superoxide anion generation, and degranulation. The random migration of PMNs was inhibited by somewhat higher concentrations of 2-dg (ID50, 12 mmol/L). In contrast, up to 40 mmol/L 2-dg did not inhibit CTX toward synthetic peptides or activated serum. In fact, 2-dg consistently increased the CTX of PMNs toward 10(-8) mol/L f-Met-Leu-Phe (fMLP), to a maximum of 450% of control CTX using 15 mmol/L 2-dg. Half-maximal stimulation (ED50) of CTX occurred at 6.3 +/- 1.0 mmol/L 2-dg. Although maximal CTX toward optimal concentrations of fMLP was consistently increased with 2-dg, the ED50 of CTX to fMLP was unchanged (ED50 with glucose, 2.0 +/- 0.6 nmol/L fMLP; ED50 with 2-dg 2.2 +/- 0.7 nmol/L fMLP), and 2-dg did not increase fMLP receptors. In the absence of glucose, 2-dg exerted similar effects on ATP and CTX, but at doses 30- to 50-fold lower than in the presence of glucose. Other glycolytic inhibitors (iodoacetamide and sodium fluoride) exerted similar effects. Additional studies indicated that CTX enhancement by 2-dg (a) required Mg++ but not Ca++, (b) occurred with PMNs from a patient with chronic granulomatous disease, (c) was unaltered in the presence of inhibitors of proteolysis, (d) was not due to generation of a soluble agent, (e) was not due to alterations in PMN adherence, and (f) was not due to inhibition of glycosylation. We conclude that the chemotaxis, but not the random migration, of PMNs is surprisingly resistant to inhibition of energy metabolism and depletion of ATP, since concentrations of 2-dg that decreased ATP and other cell functions by more than 50% not only did not inhibit, but actually stimulated, CTX. These studies also indicate that the previously reported correlation between ATP depletion and CTX impairment observed in stored PMNs are not causally related.