Adhesion Of PMNs Research Articles

Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion.

The binding of neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial cells (ECs) presents special requirements in the regulation of intercellular adhesion. ECs that are stimulated by certain agonists, including thrombin and cytokines (tumor necrosis factor alpha, interleukin-1), generate molecular signals that induce the adhesion of PMNs (endothelial cell-dependent neutrophil adhesion). Our experiments demonstrate that the mechanism of binding induced by thrombin is distinct from that induced by the cytokines based on the time courses, the requirement for protein synthesis, and differential binding of HL60 promyelocytic leukemia cells to ECs activated by the two classes of agonists. The rapid EC-dependent PMN adhesion (initiated in minutes) that occurs when the ECs are stimulated by thrombin is temporally coupled with the accumulation of platelet-activating factor, a biologically active phosphoglyceride that remains associated with ECs and that activates PMNs by binding to a cell surface receptor. A portion of the newly synthesized platelet-activating factor (PAF) is on the EC surface, as demonstrated by experiments in which the rate of hydrolysis of PAF synthesized by activated ECs was accelerated by extracellular PAF acetylhydrolase. When ECs were treated with exogenous PAF they became adhesive for PMNs; the PMN binding was prevented by incubating the ECs with PAF acetylhydrolase or by treating the PMNs with competitive PAF receptor antagonists. Thus PAF associated with the EC plasma membrane induces PMN binding, an observation supported by experiments in which PAF in model membranes (liposomes) stimulated rapid PMN adhesion to ECs and to cell-free surfaces. In addition, competitive antagonists of the PAF receptor inhibited the binding of PMNs to ECs activated by thrombin and other rapidly acting agonists, but not to ECs activated by tumor necrosis factor alpha, indicating that PAF that is endogenously synthesized by ECs can mediate neutrophil adhesion. These experiments demonstrate a novel mechanism by which a cell-associated phospholipid, PAF, can serve as a signal for an intercellular adhesive event.

The effect of cytomegalovirus infection on the adherence of polymorphonuclear leucocytes to endothelial cells

Adherence of polymorphonuclear leucocytes (PMN) to the endothelial lining of blood vessels is an essential component of the inflammatory response. In this study the effect of cytomegalovirus (CMV) infection on the adherence of PMNs has been examined using an in vitro model system. Human umbilical venous endothelial cells (HUVEC) were grown on fibronectin-coated plastics. CMV infection of HUVEC resulted in the appearance of viral antigens in a small percentage of the cells. At 24 h post-infection when no virus-induced cytopathic effect could be observed in the cell monolayers, the adherence of PMNs was significantly increased. The virus-induced adherence effect was cell bound and could not be induced by soluble components in the medium of the virus-infected cells. The augmentation of the PMN adherence to CMV-infected endothelium was sensitive to tunicamycin suggesting that the virus infection induces the expression of glycoproteins on the HUVEC membranes which are responsible for the PMN adherence. Thus CMV infection of the endothelium results in an increased adherence of PMNs. In the in vivo situation systemic viral infection can potentially lead to infection of blood vessel endothelium and thus can induce a damage of endothelium. This phenomenon could play a role in the atherogenesis process.

Effect of tumor necrosis factor on granule release and LTB4 production in adherent human polymorphonuclear leukocytes

Tumor necrosis factor (rTNF) has previously been shown to induce PMN chemotaxis, stimulate PMN adhesion to vascular endothelium and stimulate hydrogen peroxide secretion from PMNs adhered to biological surfaces. We investigated the activity of both rTNF alpha and rTNF beta on adherent and suspension cultures of human PMNs. rTNF alpha selectively stimulated the release of the specific granule in a dose dependent manner. Exocytosis of the specific granule was measured with an enzyme-immunoassay for lactoferrin and a radioassay for vitamin B12-binding protein. Adherent PMNs released up to 60% of the total lactoferrin content of the cells with no increase in myeloperoxidase (MPO) secretion when stimulated with 0.1-10 nM rTNF alpha. The PMNs in suspension cultures also selectively released the specific granule, although total release was reduced suggesting that adherence of PMNs increased their ability to respond to physiological stimuli. When PMNs in suspension cultures or adherent cells were stimulated with rTNF alpha, no LTB4 production was detectable, yet the cells retained the ability to synthesize LTB4 when stimulated with calcium ionophore A23187. Neither rTNF alpha or rTNF beta stimulated the release of the azurophilic granule, measured by the secretion of MPO and neutrophil elastase activity. These results suggest that a function of rTNF alpha and rTNF beta on PMNs is the release of the contents within the specific granule.

微小循環における血管内皮細胞の役割 血管透過性こう進と多形核白血球血管外遊走

After intravenous injection of colloidal carbon, subcutaneous injection of bradykinin (BK, 5-5000 ng/50μl) into the scrotum of rats induced carbon deposit along the venular trees of the cremaster muscle, since BK induced the gap formation between endothelial cells of venules. Endothelial cells of the collecting venules were most sensitive to BK and kept the gaps open longer than 20 min, whereas venules upstream with a diameter of about 10μm were less sensitive and closed the gaps within 5 min. Thus, individual endothelial cells show different sensitivity along the venular trees within a distance of 400 to 500 μm. Vital microscopy of microcirculation of hamster cheek pouch disclosed that polymorphonuclear leukocytes (PMNs) cross the endothelial layer to the interstitial space by four steps; adhesion to endothelial cells, penetration between adjacent endothelial cells, stay beneath the pericyte basement membrane (BM) and cross the pericyte BM. The experiments revealed that the adhesion of PMNs by LTB4 was attributable to change in the PMN membrane and not to that in the vascular endothelial cells. Thus, the endothelial cells seem to take a passive role to the PMN penetration. Rapid and magnificent development of the techniques in culturing endothelial cells must be supplemented with the knowledge, which was aquired by in vivo observation and precise analyses of the nature of the endothelial cells in living animals under the microscope.

The Effects of Oral or Intravenous Citrate on the Activation of Neutrophils In Vitro

Topical citrate has been shown to significantly reduce the severity and incidence of corneal ulceration in alkali-burned rabbit eyes. Subcutaneous injection of citrate significantly reduced the severity of ulceration. This study was performed to determine the plasma citrate and ionized calcium levels after systemic administration of citrate. Repeated ingestion of citric acid capsules showed a small increase in the plasma citrate level, but no significant change in the level of ionized serum calcium 30 min after the last ingestion. A single dose of Polycitra-LC (6.672 g of citrate) more than doubled the baseline citrate concentration but only induced a 5% decrease in the level of ionized serum calcium 30 min after ingestion. Neither regimen of orally administered citrate produced any significant effect on the respiratory burst or adherence of in vitro polymorphonuclear leukocytes (PMNs) resuspended in their respective plasma. In comparison studies, higher levels of plasma citrate and lower levels of ionized serum calcium were achieved through plateletpheresis. The respiratory burst and adherence of PMNs were not statistically different from baseline PMNs. However, there was an inverse correlation between the micromolar increase in citrate or the micromolar decrease in calcium and the respiratory burst. These results suggest that the use of oral citrate alone may not be practical, but does not preclude the use of oral citrate as a supplement to topical citrate.

Neutrophil adherence to human endothelium in vitro occurs by CDw18 (Mo1, MAC-1/LFA-1/GP 150,95) glycoprotein-dependent and -independent mechanisms.

Components of the CDw18 leukocyte surface glycoprotein complex (Mo1/LFA-1/GP 150,95 or MAC-1, LFA-1 family) are required for some adhesion-related functions of human neutrophils (PMNs). We evaluated the ability of monoclonal antibodies (MoAb) directed against specific determinants on the CDw18 glycoproteins to inhibit neutrophil adherence to cultured human endothelial cells (EC) stimulated by a variety of agonists, including thrombin and leukotriene C4, which induce the EC-dependent adhesion of PMNs. MoAb 60.3, an antibody that binds to an epitope common to the 3 heterodimer subunits of the neutrophil CDw18 complex, potently inhibited (90-100%) the rapid (5-30 minute) adherence response stimulated by N-formyl-methyionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating factor, phorbol myristate acetate, Ionophore A23187, and tumor necrosis factor. MoAbs directed against epitopes on the alpha polypeptide of the CD11b (Mol, MAC-1) heterodimer also inhibited PMN adherence to EC and to cell-free surfaces induced by these agonists. In contrast, the anti-CDw18 MoAbs had a trivial effect on maximal EC-dependent neutrophil adherence stimulated by thrombin and leukotriene C4, and incompletely inhibited PMN adherence induced by these agonists under submaximal conditions. These findings indicate that there is an alternative mechanism for neutrophil adherence, presumably resulting from molecular alterations of the EC surface, that does not require the PMN CDw18 glycoproteins. They also suggest that the inability to adhere to endothelium may not completely account for the defect in chemotaxis that is observed in vivo in neutrophils that are deficient in the CDw18 complex.

Thrombin stimulates neutrophil adherence by an endothelial cell-dependent mechanism: characterization of the response and relationship to platelet-activating factor synthesis.

Thrombin, a serine coagulation protease that is generated at sites of tissue injury and inflammation, stimulates the adherence of PMNs and neutrophils to EC. We found that thrombin enhanced the adhesion of neutrophils to primary monolayers of human umbilical vein EC when assayed by the binding of 111Indium-labeled PMNs to the EC, the recovery of unlabeled PMNs after incubation with thrombin-treated EC, and by phase contrast and scanning electron microscopy (SEM). SEM demonstrated that thrombin caused PMNs to intimately adhere to the EC plasma membrane and under some conditions to become polarized. The thrombin-stimulated adherence was a rapid, time-dependent response with an onset within 1 minute of addition of thrombin, a peak at 5-10 minutes, and a decline thereafter. The response was concentration-dependent over the range 0.01-2 U/ml thrombin, and required active thrombin. Prothrombin, factor Xa, and fibrinogen were not effective. Thrombin-stimulated PMN adherence was dependent on the EC, because thrombin did not significantly stimulate neutrophils to adhere to albumin-coated petri dishes, subendothelial matrices, or primary cultures of smooth muscle cells. Human EC, when treated with thrombin, also produce platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The concentration-response relationships and time courses for thrombin-stimulated PMN adherence and PAF production were tightly correlated. Furthermore, PAF itself stimulated the adherence of PMNs to EC, and pretreatment of PMNs with PAF selectively inhibited their adherence response to thrombin. These findings demonstrate two novel biologic activities of thrombin, the stimulation of EC-dependent adherence of PMNs and the production of PAF by EC, and suggest that they are functionally related. In addition, they suggest that thrombin may act as a plasma-derived humoral mediator of inflammation under some conditions.

Leukotrienes C4 and D4 stimulate human endothelial cells to synthesize platelet-activating factor and bind neutrophils.

Human endothelial cells in culture produced platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with leukotriene C4 or D4 (LTC4 or LTD4). Other arachidonate metabolites did not induce the synthesis of PAF. Accumulation of PAF was a prolonged response with maximal accumulation after 10-20 min of stimulation. Half of this amount remained after 90 min of stimulation. The PAF synthesized by endothelial cells remained associated with these cells. LTC4 and LTD4 also induced the adherence of human neutrophils (polymorphonuclear leukocytes; PMNs) to the endothelial cell monolayer. Adhesion was an endothelial cell-mediated process because PMNs adhered to monolayers that had been stimulated and washed prior to PMN addition, and neither LTC4 nor LTD4 stimulated PMNs in the absence of endothelial cells. The time course of PMN adhesion paralleled that of PAF accumulation by endothelial cells, and exogenously added PAF induced adherence. PMNs specifically desensitized to PAF showed only 39% of the agonist-stimulated adherence of control PMNs. We conclude that the PAF synthesized and retained by LTC4- or LTD4-stimulated endothelial cells may induce the adherence of previously unstimulated PMNs. This process may be relevant to inflammation, thrombosis, and mechanisms of vascular injury, including atherosclerosis.

Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.

Highly purified human thrombin stimulates the adherence of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC). When Indium-labeled PMNs were incubated with primary monolayers of cultured human umbilical vein EC, the basal adherence was 10 +/- 1% of the PMNs at 5 min. Addition of thrombin (2 U/ml) increased the mean adherence to 42 +/- 15%. Enhanced neutrophil adherence in response to thrombin was confirmed by experiments with unlabeled leukocytes, examined by phase contrast and scanning electron microscopy. The action of thrombin was on the EC, since it did not directly stimulate PMN adhesiveness when measured by aggregation or by adherence to nylon fiber columns. Furthermore, enhanced neutrophil adherence occurred when endothelial monolayers were treated with thrombin and washed before adding 111Indium (111In)-labeled PMNs. Thrombin that had been inactivated with antithrombin III and heparin did not enhance neutrophil adherence. Prothrombin, Factor Xa, and fibrinogen were also ineffective. The stimulated adherence of PMNs was maximal 5 min after incubation of the EC with thrombin, and decreased thereafter. The response was dose-dependent, with half-maximal stimulation at 0.2-0.25 U thrombin/ml. The enhanced PMN adherence caused by thrombin may result in part from the production of platelet-activating factor (PAF) by the stimulated EC since thrombin-stimulated EC synthesize PAF with a time course and concentration dependence that are similar to the time and concentration relationships for thrombin-stimulated PMN adherence, PAF itself promoted neutrophil adherence to the EC monolayers, and pretreatment of PMNs with PAF decreased the adherence stimulated by thrombin and PAF, but not adherence stimulated by N-formylmethionyl-leucyl-phenylalanine and C5a fragments, which indicates specific desensitization of PAF-mediated adherence. These studies demonstrate the endothelial cell-dependent stimulation of PMN adherence by thrombin, a novel mechanism of enhanced leukocyte adherence that may be important in interactions between the coagulation and inflammatory systems.

Effect of fibronectin on adherence of neutrophils from newborn infants.

Neutrophils (polymorphonuclear leukocytes [PMNs]) from infants and adults increased in adherence over a 20-min incubation as measured in a morphological assay. At each time point, PMNs from healthy infants had significantly diminished adherence when compared with those of adults; PMNs from sick newborn infants showed intermediate adherence values. Moreover, PMNs from healthy infants had significantly diminished membrane fluorescence for fibronectin when compared with those of adults. Preincubation with exogenous fibronectin did not augment the adherence of PMNs from infants or adults. Deficient adherence of PMNs from newborns may be related to decreased membrane fibronectin.

Adherence of Human Polymorphonuclear Leukocytes to Endothelial Monolayers: Effects of Temperature, Divalent Cations, and Chemotactic Factors on the Strength of Adherence Measured With a New Centrifugation Assay

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.

Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl- phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.

THE ROLE OF FIBRONECTIN IN NEUTROPHIL ADHERENCE IN NEWBORN INFANTS

Deficiencies of neutrophil (PMN) function, including adherence, may contribute to the increased susceptibility of newborn infants to bacterial infection. The basis for the decreased adherence is currently unknown; however, fibronectin (FN) has an important role in adherence of phagocytes and other cells. The purpose of this study was 1) to compare PMN adherence FOR newborn infants and adults, 2) to investigate the effects of exogenous FN on newborn PMN adherence and, 3) to determine if there are quantitative differences in membrane bound FN between PMNs from infants and adults. PMNs were obtained from cord blood samples of healthy term neonates and adults, and separated with Dextran and Ficoll-Hypaque. 1 × 106 PMNs were tumbled in 250 ug of FN or phosphate buffered saline (control) at 37°C for 30 min, washed, and adhered to 15 mm glass coverslps (CS) for 5,10,20 and 30 mins. CS were washed, stained, and the number of adherent PMNs calculated. For determination of membrane bound FN, PMNs (5 × 105) were incubated with a 1:2 dilution of fluorescein conjugated F(ab1)2 goat anti-human FN for 30 min at 4°C, washed, and adhered to CS. PMNs from newborn infants (n=25) demonstrated diminished adherence when compared to adults (n=30) at 5,10,20 and 30 min (p<.005). Following preincubation with FN, there was no increase in the adherence of PMNs from newborn infants, whereas, adult PMNs demonstrated enhanced adherence at 10 min(p<.025). PMNs from newborn infants demonstrated significantly diminished membrane fluorescence for FN when compared to adults: 75.2 ± 5.4% (fluorescent positive cells) vs. 88.7 ± 7.3%, p=.05. Thus, deficient newborn PMN adherence may be related to decreased membrane FN.

Effects of a chemotactic factor, N-formylmethionyl peptide, on adherence, superoxide anion generation, phagocytosis, and microtubule assembly of human polymorphonuclear leukocytes.

FMLP promoted microtubule assembly in PMNs at concentrations which were chemotactic for the cells. At higher concentrations than those required for chemotaxis, FMLP enhanced the adherence of PMNs to nylon glass fibers. With colchicine, PMN adherence was inhibited, but upon exposure to FMLP, PMN adherence could be restored. The effect of FMLP on PMN adherence was transitory and was no longer evident by 5 min. At concentrations similar to those employed in the adherence studies. FMLP induced the cells to briefly generate O-2, since ferricytochrome C reduction was no longer evident by 5 min. Pretreatment of the PMNs with cytochalasin B enhanced the release of O-2 by PMNs exposed to FMLP. On the other hand, there was no effect of FMLP on phagocytosis of C3-coated particles. These results suggest that FMLP induces responsive cells to develop a hyperadherent plasma membrane which is largely independent on microtubule control. Since oligopeptides similar of AMLP are formed in bacteria, it is likely that the action of N-formylated peptides is important in regulating the inflammatory response.