CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.     

Anti-inflammatory actions of lipoxin A(4) stable analogs are demonstrable in human whole blood: modulation of leukocyte adhesion molecules and inhibition of neutrophil-endothelial interactions

We have examined in whole blood the actions of 2 lipoxin A(4) (LXA(4)) stable analogs, 15-R/S-methyl-LXA(4) and 16-phenoxy-LXA(4), for their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA(4) analogs in nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA(4) analogs also attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor (PAF), interleukin-8, or C-reactive protein-derived peptide 201-206 with IC(50) values of 0.2 to 1.9 micromol/L, whereas they did not affect lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-stimulated expression of E-selectin and intercellular adhesion molecule-1 on HCAEC. These LXA(4) analogs markedly diminished adhesion of neutrophils to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. Combining LXA(4) analogs with dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with dexamethasone, but not with LXA(4) analogs, also decreased neutrophil attachment. Together, these results indicate that LXA(4) stable analogs modulate expression of both L-selectin and CD11/CD18 on resting and immunostimulated leukocytes and inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18 expression. These actions are additive with those of glucocorticoids and may represent a novel and potent regulatory mechanism by which LXA(4) and aspirin-triggered 15-epi-LXA(4) modulate leukocyte trafficking.     

Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis

We have investigated the adhesive capacity of neutrophils after spontaneous apoptosis, which occurs during in vitro culture. Apoptotic neutrophils show reduced adhesion to E selectin and the CD18 integrin ligand fibrinogen. Neutrophil apoptosis is associated with changes in the levels of surface expression of key receptors that mediate neutrophil adhesion events. Notably, apoptotic neutrophils show reduced expression of L-selectin/selectin ligand. In contrast, CD11b/CD18 and CD11c/CD18 integrins are expressed at increased levels. The reduced capacity for adhesion of apoptotic neutrophils may be achieved by very different mechanisms. Regulation of the levels of surface expression of receptors/ligand may control selectin-mediated adhesion, possibly as a result of protease/sialidase activity. In contrast, modulation of integrin-mediated adhesion may involve functional uncoupling of receptors present on the surface of the apoptotic cell without alteration in levels of surface expression. The altered adhesive potential of the apoptotic neutrophil may serve to limit release of its histotoxic contents and reduce inappropriate tissue injury.     

老年急性心肌梗死患者中性粒细胞黏附分子的表达和溶栓治疗的影响

目的观察老年急性心肌梗死患者中性粒细胞表面黏附分子L-selectin和CD11b的表达以及溶栓治疗对其影响. 方法用免疫荧光技术和流式细胞术测定30例正常对照组和32例老年急性心肌梗死患者溶栓前后中性粒细胞表面黏附分子L-selectin和CD11b的表达. 结果与对照组相比,急性心肌梗死患者中性粒细胞L-selectin的表达下降47.3%（P＜0.01）,而CD11b的表达升高145.5%（P＜0.01）.溶栓治疗前后中性粒细胞L-selectin和CD11b的表达无明显变化. 结论老年急性心肌梗死患者中性粒细胞L-selectin表达下调,CD11b表达上调,提示中性粒细胞被激活.溶栓治疗对中性粒细胞表面黏附分子的表达无影响.     

The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a CD49d-dependent manner

The acute release of neutrophils from the bone marrow is a critical step in their trafficking to sites of inflammation. This process is stimulated by systemically acting inflammatory mediators, such as the CXC chemokines. In this study we have used a novel in situ perfusion system of the rat femoral bone marrow to directly investigate the role of specific adhesion molecules in chemokine-stimulated neutrophil mobilization. We show here that neutrophils mobilized in response to rat macrophage inflammatory protein-2 (MIP-2) shed L-selectin and expressed significantly higher levels of CD11b and CD49d. However, inhibition of L-selectin sheddase activity with KD-IX-73-4 had no effect on the number of neutrophils mobilized in response to rat MIP-2. Blockade of CD18, using a neutralizing monoclonal antibody (mAb), did not inhibit neutrophil mobilization but unexpectedly increased the rate and number of neutrophils released from the bone marrow in response to chemokine, suggesting that CD18 could play a role in neutrophil retention within the bone marrow. Blockade of CD49d using either a selective mAb or a specific antagonist resulted in a dramatic inhibition (> 75%) of the chemokine-stimulated neutrophil mobilization from the bone marrow. These data reveal contrasting roles for CD18 and CD49d in the retention and release of neutrophils from the bone marrow.     

CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation.

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, "enkephalinase"). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu-phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.     

不稳定型心绞痛患者白细胞CD11b/CD18表达的变化

目的：观察不稳定型心绞痛患者中性粒细胞和单核细胞膜CD11b/CD18表达的变化。方法：选择经冠状动脉造影确诊的不稳定型心绞痛32例和正常人20例，用流式细胞仪直接免疫荧光法检测中性粒细胞和单核细胞膜CD11b/CD18表达。结果：不稳定型心绞痛患者中性粒细胞和单核细胞膜CD11b/CD18表达较正常对照组均显著增加（P＜0.01）；且CD11b／CD18表达的增加与心绞痛的发作有关，而与冠状动脉狭窄程度和病变支数无关。结论：不稳定型心绞痛患者中性粒细胞和单核细胞膜CD11b/CD18表达明显增加，其增加程度与心绞痛的发作有关。     

Neutrophil adhesion to endothelial cells and factors affecting adhesion in patients with Beh?et's disease.

OBJECTIVES: To study the in vitro adhesion of polymorphonuclear leucocytes (PMNLs) to endothelial cells in patients with Behçet's disease (BD), and the humoral and cellular factors which may contribute to adhesion. METHODS: A total of 118 patients with BD and 60 healthy controls were studied. In vitro adhesion of chromium-51 labelled normal neutrophils to human umbilical vascular endothelial cell (HUVEC) monolayers were studied in the presence of normal serum or serum obtained from patients with BD. Adhesion of neutrophils from patients with BD to HUVEC stimulated with tumour necrosis factor (TNF), interleukin-1 (IL-1), and lipopolysaccharide (LPS) and adhesion molecule (CD11a, CD11b, CD18 and L-selectin) expression on the patient's neutrophils and lymphocytes were determined, and the serum concentration of IL-8 was measured. RESULTS: Sera from patients with BD were found to enhance the adherence of normal PMNLs to HUVEC monolayers in vitro. Patients' sera induced an increase in surface expression of CD11a and CD18 on normal neutrophils and intercellular adhesion molecule-1 (ICAM-1) expression on HUVECs. The number of CD11a positive neutrophils was greater in the blood of patients with BD than in that of healthy controls (89.4% v 71%; p < 0.001). Pretreatment of HUVECs with IL-1 alpha, TNF alpha or LPS resulted in an increased adhesion of patients' PMNLs greater than that observed for normal PMNLs. Monoclonal antibodies to CD11a, CD11b, CD18, and ICAM-1 caused varying degrees of inhibition of neutrophil adhesion. The concentration of IL-8 was also found to be significantly increased in sera of patients with BD (490 (SD 470) pg/ml) compared with normal controls (97.5 (56.3) pg/ml). CONCLUSION: Abnormalities of neutrophils, endothelial cells, or both, have been suggested to be responsible for many of the clinical manifestations of BD. Our findings may explain the underlying mechanism of neutrophil accumulation in Behçet's lesions.     

Altered surface expression of effector cell molecules on neutrophils in myelodysplastic syndromes

The surface expression of effector cell molecules on neutrophils was examined in 18 patients with myelodysplastic syndromes (MDS) and 20 healthy control subjects. The MDS patients were further classified as low clinical risk (L-MDS, n  = 7) and high clinical risk (H-MDS, n  = 11). The expression of Fc receptors for IgG (FcR), complement receptors (CR) and cellular adhesion molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. The effect of granulocyte colony-stimulating factor (G-CSF) and tumour necrosis factor-alpha (TNF) on L-selectin shedding and CR up-regulation on neutrophils was also examined. The percentage of FcRI-positive neutrophils and CD11b/CR3 expression on neutrophils were significantly increased in the H-MDS patients when compared to the controls. In contrast, the expression of FcRII, FcRIII, L-selectin, LFA-1 and CD18 on neutrophils was significantly reduced in the H-MDS patients compared with the controls. The L-MDS neutrophils exhibited lower expressions of CR1, L-selectin, LFA-1 and CD18 than those of the controls. Neutrophils from some H-MDS patients showed impaired L-selectin shedding and CR up-regulation after stimulation with G-CSF or TNF, although these were not significantly different when assessed in the whole H-MDS group. These findings suggest that an altered surface expression of effector cell molecules and an impaired modulation of cellular adhesion molecules on neutrophils may contribute to the increased susceptibility to bacterial infections in MDS patients.     

冠心病患者中性粒细胞和单核细胞CD11b/CD18表达的研究

目的 :探讨不同类型冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达的变化。方法 :选择经冠状动脉造影确诊的 49例心绞痛患者 ,30例急性心肌梗死患者和 2 0例正常人 ,用流式细胞仪直接免疫荧光法检测中性粒细胞和单核细胞膜 CD11b/CD18表达。结果 :冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达较正常对照组均显著增加 （P<0 .0 1） ;不稳定性心绞痛和急性心肌梗死患者中性粒细胞和单核细胞膜 CD11b/CD18表达显著高于稳定性心绞痛患者 （P<0 .0 1）。与正常对照组比较 ,心绞痛患者组中性粒细胞和单核细胞计数无变化 （P>0 .0 5 ） ,而急性心肌梗死组明显增加 （P<0 .0 1）。急性心肌梗死患者中性粒细胞和单核细胞膜 CD11b/CD18表达与梗死范围无关。结论 :冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达明显增加 ,其增加程度与心肌缺血的类型有关。     

Expression of L-Selectin,CD43, and CD44 in Synovial Fluid Neutrophils from Patients with Inflammatory Joint Diseases

Objective. To study the expression of L-selectin, CD43, and CD44 on peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with inflammatory joint diseases, and to investigate the presence of soluble L-selectin in both SF and plasma from patients with acute and chronic arthritis. Methods. PB and SF neutrophils were isolated from 13 patients with rheumatoid arthritis (RA) and 17 patients with various inflammatory joint diseases other than RA. Expression of L-selectin, CD43, CD44, CD11a, and CD11b was determined in both unstimulated and in vitro—activated cells by immunofluorescence flow cytometry. Soluble L-selectin levels were estimated in SF and plasma by a semiquantitative radioimmunoassay. Results. Neutrophils from SF showed diminished expression of L-selectin compared with PB neutrophils; CD43 expression and CD44 expression were decreased in SF neutrophils from most patients. In contrast, SF neutrophils exhibited significantly increased expression of CD11b, to an extent similar to that seen with in vitro—activated PB neutrophils. Soluble L-selectin was detected at similar levels in SF and PB. Conclusion. The phenotypic profile of SF neutrophils (low levels of L-selectin, CD43, and CD44, and high levels of CD11b) from most patients with RA or other inflammatory joint conditions resembles that observed in in vitro—activated neutrophils. Our results suggest that SF neutrophils are activated to a similar degree in inflammatory joint diseases with different pathogenic mechanisms.     

Beta 2-integrin and L-selectin are obligatory receptors in neutrophil aggregation [see comments]

We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2- integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates on the basis of size and color, as well as antibody binding to these two adhesive molecules. Formyl peptide-stimulated aggregate formation was measured for individual populations fluorescently labeled red (LDS-751) or green (CD44-FITC), and interpopulation red-green cell conjugates. Blocking either the beta 2-integrin or L-selectin adhesive epitope with monoclonal antibody on individual cell populations resulted in an approximately 50% reduction in two-color aggregation as compared with that in unblocked samples. Shedding the L-selectin on a cell population by preincubation with complexes of lipopolysaccharide and its plasma membrane binding protein also decreased aggregation to a control population by approximately 50%. We examined the aggregation of neutrophils from patients genetically deficient in beta 2-integrin and clinically leukocyte adhesion deficient (LAD). LAD adhesion to normal neutrophils was dependent on the expression of L-selectin on LAD cells and beta 2-integrin on normal cells. Thus, the minimum requirement for adhesion between two mixed populations of neutrophils was that one population expressed the beta 2-integrin and the other expressed the L- selectin adhesive epitope.     

Protective effects of 3,4-dihydroxyphenyl lactic acid on lipopolysaccharide-induced cerebral microcirculatory disturbance in mice

This study intended to examine the effect of 3,4-dihydroxyphenyl lactic acid (DLA), a major ingredient of Salvia miltiorrhiza, on lipopolysaccahride (LPS) -induced mouse cerebral cortical microcirculatory disturbance. Velocity of red blood cells in, and albumin leakage from venules, and the numbers of leukocytes rolling on, and adherent to the venular wall were determined by an up-right microscope after LPS (5 mg/kg/h) infusion with or without administration of DLA (5 mg/kg/h). Expression of adhesion molecules CD11b/CD18 and L-selectin on neutrophils, plasma concentration of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were evaluated by flow cytometry. Concentration of TNF-α in supernatants of LPS-stimulated mononuclear cells was evaluated in vitro by flow cytometry as well. LPS exposure significantly increased the number of rolling and adherent leukocytes as well as albumin leakage, and decreased the velocity of red blood cells in venules. In addition, LPS stimulation apparently increased the expression of CD11b/CD18 on neutrophils, the concentration of plasma TNF-α, and the production of TNF-α from mononuclear cells. Treatment with DLA significantly ameliorated LPS-induced insults in mice, including cerebral microcirculatory disturbance, the expression of CD11b/CD18 on neutrophils, and the increased concentration of plasma TNF-α and the production of TNF-α from mononuclear cells.     

Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.     

Expression of adhesion molecules during apoptosis of circulating neutrophils in COPD

STUDY OBJECTIVES: Neutrophil accumulation occurs in the lungs of patients with COPD. This can be due to increased recruitment and/or delayed tissue clearance. Previous studies have described alterations in circulating neutrophils in these patients that can facilitate the former. Dysregulation of neutrophil apoptosis may contribute to the latter. This study investigated the potential abnormalities of the apoptotic process in COPD patients. DESIGN: Prospective study. SETTINGS: Outpatient clinic in a urban, tertiary hospital. PATIENTS: Fourteen stable patients with COPD, 8 smokers with normal lung function, and 8 healthy nonsmoking subjects. MEASUREMENTS AND RESULTS: We cultured circulating neutrophils that had been harvested from the study subjects at 2, 6, and 24 h. Apoptosis was assessed using flow cytometry by annexin binding and CD16 expression. The surface expression of the adhesion molecules Mac-1 (CD11b) and L-selectin (CD62L) also was determined by flow cytometry. The percentage of apoptotic neutrophils increased with time similarly in all groups. However, the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) was lower, during apoptosis in the neutrophils of patients with COPD. CONCLUSIONS: These results show that, quantitatively, in vitro neutrophil apoptosis in COPD patients occurred at a rate similar to that found in healthy individuals and smokers with normal lung function. Qualitatively, however, the increased surface expression of Mac-1 (CD11b) and the decreased surface expression of L-selectin (CD62L) observed in the apoptotic neutrophils of COPD patients indicate increased activation during the apoptotic process. This may be relevant for the pathogenesis of COPD.     

Relationship between interleukin-8 and neutrophil adhesion molecules in rheumatoid arthritis

To evaluate the role of interleukin-8 (IL-8) on the activation of neutrophils in rheumatoid arthritis (RA), we measured IL-8 and the adhesion molecules, L-selectin (CD62L), CD11b and CD18, on neutrophils in paired peripheral blood and synovial fluid of RA patients. Synovial fluid IL-8 levels were significantly increased compared to peripheral blood. L-selectin was split off and CD 11b and CD18 were upregulated on neutrophils in the synovial fluid. A positive correlation occurred between the IL-8 concentration and CD18 or CD11b densities on neutrophils in the synovial fluid (r=0.75,P <0.005 andr=0.60,P < 0.05, respectively). Peripheral blood neutrophils of the patients were desensitised with IL-8 in vivo, as shown by the significantly lower L-selectin shedding after in vitro IL-8 stimulation: 1.6 times decrease for patients vs 3.2 for controls (P < 0.05). In conclusion, these results add further evidence for the role of IL-8 in the activation of neutrophils in RA.     

Neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats

RATIONALE: Neutrophils accumulate in pulmonary capillaries during acute inflammation. Initial events in injury recognition and sequestration do not occur through selectin-mediated rolling. Cytoskeletal rearrangements, as assessed by submembrane F-actin rims, result in poorly deformable neutrophils that may not pass through capillaries. OBJECTIVE: To test the hypothesis that neutrophils sequestering during pneumonia contain F-actin rims and to determine the roles of CD11/CD18, L-selectin expression, and neutrophil-platelet adhesion in neutrophil sequestration. METHODS: Neutrophils were compared in blood obtained simultaneously from venous and arterial sites before and 4 h after instillation of Streptococcus pneumoniae or Escherichia coli in rats. MEASUREMENTS AND MAIN RESULTS: At 4 h of pneumonia, the number of neutrophils was greater in the venous blood entering the lungs than in the arterial blood leaving the lungs, indicating that neutrophil sequestration was occurring. More neutrophils entering the lungs contained F-actin rims than did neutrophils exiting, and the venous-arterial difference in F-actin-rimmed neutrophil counts completely accounted for sequestration. In E. coli pneumonia, in which neutrophil adhesion is mediated by CD11/CD18, CD18 blockade 15 min before blood samples were obtained did not prevent this sequestration of F-actin-rimmed neutrophils. Neutrophils expressing high or low levels of L-selectin or of neutrophils that bound platelets while circulating did not preferentially sequester. CONCLUSIONS: Neutrophils with cytoskeletal rearrangements preferentially sequester within the lungs during pneumonia, and this sequestration is not due to CD11/CD18-mediated adhesion, L-selectin expression, or platelet adhesion to neutrophils, suggesting that cytoskeletal rearrangements result in sequestration of neutrophils.     

Effects of cardiopulmonary bypass on the expression of adhesive molecules in children undergoing cardiac surgery due to congenital heart disorders

BACKGROUND: Infants and small children who undergo cardiac surgery due to congenital heart disorders, are at risk of developing inflammatory complications and multi-organ failure. AIM: To assess the changes in the expression of adhesive molecules on neutrophils and in peripheral blood plasma in children undergoing cardiac surgery with or without the use of cardiopulmonary bypass (CPB). METHODS: In 18 children who underwent surgery with the use of CPB and in 7 children who underwent surgery without CPB, the expression of CD11b, CD11c and CD62L molecules on neutrophils and soluble adhesive molecules - L-selectin and ICAM-1, was assessed before, during and after cardiac surgery. RESULTS: A significant increase in the expression of CD11b on neutrophils at the time of the initiation of CPB was observed. The most pronounced decrease in the expression of CD11c was detected at the end of surgical procedure. There was also a negative correlation between L-selectin concentration measured at the end of CPB and CPB duration as well as between ICAM-1 concentration and duration of hypothermia. CONCLUSIONS: Cardiac surgery influences the expression of adhesive molecules on neutrophils and in blood plasma. These changes are more pronounced in children who undergo CPB, and depend on CPB duration as well as the use of hypothermia.     

Down-regulation of L-selectin expression in neutrophils by nonsteroidal anti-inflammatory drugs: role of intracellular ATP concentration

L-selectin is an adhesion molecule that plays an essential role in the early events of the inflammatory response. Our group has recently described that several nonsteroidal anti-inflammatory drugs (NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In this work, we have studied potential mechanisms involved in the shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did not involve any detectable intracellular calcium flux. Pretreatment of neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of protein kinase C (PKC), or with inhibitors of protein tyrosine kinases such as tyrphostin A25 or herbimycin A did not prevent the NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an inhibitor of L-selectin proteolysis was able to block the effect of NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration that highly correlated with the differential ability of NSAIDs to trigger L-selectin shedding (r = 0.8, P <.01). In agreement with this finding, azide plus 2-deoxy-D-glucose, 2 metabolic blockers, also induced a rapid L-selectin shedding (65% +/- 8%) without affecting the neutrophil viability, activation, or expression level of other surface molecules with soluble isoforms such as CD16 and CD59. These data indicate that the maintenance of L-selectin on the neutrophil surface requires energy consumption, which suggests that L-selectin is shed in neutrophils by default. Interestingly, NSAIDs seem to cause the shedding of L-selectin, at least in part, through the reduction of the intracellular ATP concentration.     

Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets

In an in vitro flow model, unstimulated neutrophils rolled steadily over a surface coated with platelets, until superfusion of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) caused a dose-dependent (10(-11) to 10(-7) mol/L) transition from rolling to stationary attachment in seconds, followed more slowly by neutrophil shape change and spreading on the surface, However, at low concentrations of Ca2+ and Mg2+ (0.1 mmol/L and 0.05 mmol/L, respectively, rather than physiologic 1 mmol/L and 0.5 mmol/L), neutrophils first halted but then started to roll again and to detach from the surface over 5 to 10 minutes. At the low cation concentration, stopping was largely inhibited by antibodies to the neutrophil integrins CD18 or CD11b, but not CD11a. When neutrophils were pretreated with antibodies to CD11b or CD18 in 1 mmol/L Ca2+ 0.5 mmol/L Mg2+, stopping was not prevented but delayed. However, if antibodies were also included with the superfused fMLP, stopping was inhibited, and detachment followed. This indicates that CD11b/CD18 was newly expressed during shape change and mediated the second phase of neutrophil immobilization and spreading in a cation-dependent manner. Prestimulated neutrophils also bound to platelets and spread, but immobilization was blocked if they were perfused with antibody to CD18 or CD11b or with low Ca2+ and Mg2+. Examining the cation-dependence further, it was evident that the presence of Mg2+ was essential for integrin-mediated adhesion and that the Mg2+ concentration determined whether immobilization could be maintained or was transient. Continuous superfusion of fMLP was also essential for maintenance of stable adhesion and spreading. Thus, activation of constitutive CD11b/CD18 rapidly and reversibly converted rolling to stationary attachment, whereas maintenance of adhesion and neutrophil spreading required continual expression of additional CD11b/CD18 that was only functional at physiologic Mg2+. Continual activation and deactivation of CD11b/CD18 during de novo expression could mediate immobilization and onward migration of neutrophils in vivo, and activated platelets appear capable of supporting this process as well as endothelial cells.