Cardiopulmonary effects of a brief exposure to cigarette smoke in the guinea pig

To investigate whether the acute inflammatory response produced by acute cigarette smoke exposure is associated with alterations in pulmonary and cardiovascular function, using a respirator we exposed 8 guinea pigs to 350 ml of cigarette smoke diluted in 2,000 ml room air for 10 min. Lung volumes, pressure volume curves and flow volume curves were performed. Measurements of pulmonary artery pressure, systemic pressure, heart rate and cardiac output were taken at baseline, immediately after the smoke exposure, and 1.5 h after smoke exposure. Aliquots of peripheral blood for total white cell count and differential count were obtained. After animal sacrifice, the right lung was lavaged and white cell count and differential count performed. We found that acute exposure to cigarette smoke produced a peripheral blood neutrophilia which rose progressively from a baseline of 36.3 +/- 8% to a maximum of 72.2 +/- 7.7% at 1.5 h after exposure. There was pulmonary neutrophilia with 24.6 +/- 6.3% neutrophils in the lavage fluid of the smoke-exposed animals compared to 8.4 +/- 4.3% in the control animals. Immediately after smoke exposure, there was acute airflow obstruction with a decrease in the peak flow and forced expiratory flow between 25-75% vital capacity. This was associated with airtrapping, as shown by a transient increase in residual volume. There was a slight decrease in systemic blood pressure in the smoke-exposed animals associated with a nonsignificant decrease in the heart rate, and the cardiac output remained stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of smoke exposure on the cellular and cytokine profile in isolated perfused lungs

The aim of this study was to assess the acute effects of cigarette smoke exposure on cellular and cytokine profile in BAL fluids in an isolated perfused rabbit assay. The experimental animals were categorized into four groups: (1) unexposed controls and (2) cigarette smoke-exposed animals perfused with autologous whole blood; (3) unexposed controls and cigarette smoke-exposed; (4) cigarette smoke-exposed animals perfused with Krebs' Ringer solution containing 5% bovine serum albumin and glucose. Cigarette smoke induced an increase in total cell numbers (mainly alveolar macrophages in BAL fluids) and an increase in the permeability index of BAL. Levels of interleukin 8 were also significantly decreased in BAL fluids due to acute effects of cigarette smoke exposure. The most likely explanation for cigarette smoke-induced increase of inflammatory cells in BAL in lungs is because of the release of pre-existing cells from reservoirs within the lungs. The acute effects of cigarette smoke-induced increase of pulmonary epithelial permeability may also play an important role in the cellular recruitment into airspaces from the lung reservoirs.     

Reduced expression of flavocytochrome b558, a component of the NADPH oxidase complex,in neutrophils from patients with myelodysplasia

OBJECTIVE: Patients with myelodysplasia (MDS) show a disturbed production of ROS in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils. Because generation of ROS is mediated by the NADPH oxidase complex, a component of which is flavocytochrome b558, we investigated whether the expression of flavocytochrome b558 in neutrophils from MDS patients is affected. MATERIAL AND METHODS: Neutrophils were stimulated with fMLP and GM-CSF, and plasma membrane expression of flavocytochrome b558 and specific granule markers were assessed by fluorescence-activated cell sorting analysis. Protein levels of the flavocytochrome b558 subunits gp91phox and p22phox in whole neutrophil lysates were detected by Western blotting. RESULTS: Stimulation of neutrophils with GM-CSF and fMLP increased the flavocytochrome b558 plasma membrane expression. The fMLP-induced translocation of flavocytochrome b558 was reduced in neutrophils from MDS patients (140%+/-9% vs 180%+/-13%, p<0.05). Analysis of cell surface expression of markers of flavocytochrome b558 containing granules (CD35 and CD66b) indicated that exocytosis of these granules in response to fMLP stimulation was not affected in MDS patients. Western blot analysis demonstrated a decreased protein expression level of the flavocytochrome b558 subunits gp91phox and p22phox in neutrophils from MDS patients. CONCLUSION: Our results indicate both a lower basal protein level and a disturbed fMLP-induced increase in plasma membrane expression of flavocytochrome b558 in neutrophils from MDS patients, which together might play a role in decreased ROS production.     

Characterization of Siglec-5 (CD170) expression and functional activity of anti-Siglec-5 antibodies on human phagocytes

OBJECTIVE: The Siglec family of proteins consists of at least 10 members with immunoglobulin and lectin domains and with similar sialic acid-binding properties. Many Siglec family members are expressed on hematopoietic cells and are involved in cell/cell interactions. Some family members are suspected of regulating cellular processes through specific signaling pathways. Monoclonal antibodies were generated against specific epitopes of Siglec-5 (CD170) and were used to determine expression of Siglec-5 on normal blood and marrow cells and cell lines. The antibodies also were used to elucidate functional activity for Siglec-5 on blood neutrophils. METHODS: Flow cytometry and ELISA were used to determine the specificity of the monoclonal antibodies for Siglec-5 and to determine expression patterns. Chemiluminescence assays were employed to measure the oxidative burst activity of whole blood or purified neutrophils following treatment with the anti-Siglec-5 antibodies. RESULTS: Cell surface expression analysis demonstrated that the protein was expressed on gated human neutrophil and monocyte populations, both in the blood and bone marrow. Expression on neutrophils was enhanced by one-hour treatment with fMLP or TNF-alpha. Epitope-specific anti-Siglec-5 monoclonal antibodies did not directly activate human neutrophils; however, antibody binding augmented neutrophil oxidative burst activity as determined by fMLP-induced luminol-dependent chemiluminescence. CONCLUSION: Data demonstrating expression of Siglec-5 on cells of the myelomonocytic lineage and alteration of its expression by inflammatory stimuli suggest a role for this protein in cell/cell interactions following microbial exposure.     

Decreased expression of the common acute lymphoblastic leukaemia antigen (CALLA/CD10) on neutrophils from patients with thermal injury

Summary. The common acute lymphoblastic leukaemia antigen (CALLA/CD10) is a normal component of the circulating neutrophil cell surface membrane. In order to examine the potential functional significance of CALLA/CD10 we analysed the expression of this molecule on neutrophils isolated from thermal injury patients, since these patients have a well-documented constellation of neutrophil defects affecting their microbicidal functions. Expression of neutrophil CALLA/CD10 was monitored by indirect immunofluorescence and flow cytometry. We observed that CALLA/CD10 expression was quantitatively reduced on burn patient neutrophils, compared to healthy donors ( P < 0.001). In contrast, burn patient neutrophils expressed normal levels of class I HLA molecules and the C3bi receptor. Reduced expression of CALLA/CD10 was not associated with neutrophil activation or exposure to plasma 'factor(s)' in vivo . Analysis of normal bone marrow neutrophils by cell sorting indicated that expression of CALLA/CD10 occurs late in neutrophil maturation, since 25% of polymorphonucleated bone marrow neutrophils did not express cell surface CALLA/CD10. Attempts to examine the chemotactic responses of CALLA/CD10 positive and negative neutrophils from burn patients were hampered by previous exposure of these cells to chemoattractants in vivo . Collectively, our findings suggest that burn patient peripheral blood neutrophils may be deficient in CALLA/CD10 due to insufficient maturation time in the bone marrow following thermal injury.     

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.     

Time course of cigarette smoke-induced pulmonary inflammation in mice.

Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease. In the present study a murine model of tobacco smoke-induced emphysema was used to investigate the time course of airway and pulmonary inflammatory response, with a special emphasis on pulmonary dendritic cell (DC) populations. Groups of mice were exposed to either cigarette smoke or to control air for up to 24 weeks. In response to cigarette smoke, inflammatory cells (i.e. neutrophils, macrophages and lymphocytes) progressively accumulated both in the airways and lung parenchyma of mice. Furthermore, a clear infiltration of DCs was observed in airways (10-fold increase) and lung parenchyma (1.5-fold increase) of cigarette-exposed mice at 24 weeks. Flow cytometric analysis of bronchoalveolar lavage (BAL) DCs of smoke-exposed mice showed upregulation of major histocompatability complex II molecules and costimulatory molecules CD40 and CD86, compared with BAL DCs of air-exposed mice. Morphometric analysis of lung histology demonstrated a significant increase in mean linear intercept and alveolar wall destruction after 24 weeks of smoke exposure. In conclusion, the time course of the changes in inflammatory and dendritic cells in both bronchoalveolar lavage and the pulmonary compartment of cigarette smoke-exposed mice was carefully characterised.     

Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM-CSF and role of the Bcr gene

Although devoid of proliferative capacity, polymorphonuclear neutrophils (PMN) express receptors for haemopoietic growth factors and need growth factors for survival and functional stimulation. This study showed that in vitro treatment of human PMN with GM-CSF for up to 48 h increases cell surface expression of the β2-integrin molecules CD11b/CD18 and CD11c/CD18 and of the receptor for the chemotactic peptide fMLP. Such modifications are usually expression of PMN activation. PMN treated with GM-CSF also displayed increased phagocytosis of latex particles and enhanced oxidative burst and superoxide anion release. Since integrins mediate PMN adhesion to endothelium, homotypic adhesion, chemotaxis/phagocytosis and the triggering of respiratory burst, our results suggested that functional stimulation of PMN persisted following prolonged exposure of PMN to growth factors and that it was not a temporary phenomenon which lasted only for the first 12–24 h of treatment. We also used oligonucleotides antisense to the Bcr gene mRNA to inhibit expression of the gene and evaluate its function in PMN, following the recent observation that PMN from Bcr-null mutant mice produced increased amounts of reactive oxygen metabolites upon activation. The antisense oligonucleotides had no effect on the parameters investigated. This may indicate that increased production of O⁻₂ by neutrophils in which the Bcr gene is not expressed requires either that gene expression is absent in the earlier stages of myeloid differentiation/maturation, so that when inhibition occurs in the terminally differentiated neutrophils their functional status is no longer influenced, or that the residual low-level expression of the gene which may be present in the antisense-treated cells is sufficient to provide a normal response to stimulation.     

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.     

Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation

Human neutrophils are primed in the presence of complexes of lipopolysaccharide (LPS) with its serum binding protein (LBP) in a manner dependent on CD14. Cellular consequences of priming include increased responsiveness, the upregulation of surface proteins including the adhesive integrin CD11b/CD18 (Mac-1), the increased binding of certain ligands to CD11b/CD18, and the concurrent shedding of the L-selectin homing receptor. Because expression of both CD11b/CD18 and L-selectin is obligatory for formyl peptide-stimulated neutrophil aggregation in vitro (Simon et al, Blood 82:1097, 1993), we have examined the consequences of bacterial endotoxin on the expression of neutrophil adhesive molecules. We observed that the exposure of neutrophils to LPS/LBP, while enhancing the surface numbers and adhesive function of CD11b/CD18 for latex particles, did not induce aggregation. In contrast, as the LPS/LBP concentration increased (ED50 = 30 ng/mL LPS/LBP), the ability of neutrophils to aggregate decreased in parallel with the shedding of L-selectin. Moreover, when L-selectin adhesive activity was blocked by treatment with Fab fragments of Dreg- 200, aggregation was inhibited to an extent roughly proportional to the available L-selection. Blocking of LPS/LBP with CD14-specific monoclonal antibodies suppressed L-selectin shedding and preserved formyl peptide-stimulated aggregation. Taken together, the data suggest that inhibition of neutrophil aggregation by LPS/LBP is related to the expression of L-selectin via CD14 rather than LPS inhibition of CD11b/CD18 function during cellular stimulation.     

Aminobenzoic acid hydrazide, a myeloperoxidase inhibitor, alters the adhesive properties of neutrophils isolated from acute myocardial infarction patients

Acute myocardial infarction (AMI) is associated with vascular inflammation, including activation and adherence of neutrophils to vascular endothelial cells via CD11b/CD18 intercellular adhesion molecule interactions. Myeloperoxidase (MPO) induces CD11b surface expression in polymorphonuclear neutrophils (PMNs); however, its role in regulating adhesion in AMI is not well characterized. This study investigates the effects of aminobenzoic acid hydrazide (ABAH), an inhibitor of MPO, antibodies specific for CD11b, on the adhesion of PMNs isolated from AMI patients to endothelial cells. Human neutrophils were isolated from the peripheral blood of 20 patients with AMI or 20 healthy participants as control using Percoll density gradient centrifugation. The major biochemical indicators were detected with different biochemical analyses. The effects of ABAH and anti-CD11b antibodies on neutrophil adhesion to endothelial cell were measured using adhesion assays in vitro. The adhesion rate was significantly higher for neutrophils isolated from AMI patients than healthy individuals (P < 0.001). ABAH significantly inhibited MPO activity in PMNs isolated from AMI patients. Neutrophil adhesion was significantly reduced upon treatment with 10 and 20 μM ABAH in a dose-dependent manner. Treatment with anti-CD11b antibodies also significantly reduced neutrophil adhesion in comparison with the untreated control group (P < 0.001). Thus, both ABAH and anti-CD11b antibodies reduced PMN adhesion. Further studies are necessary to determine whether MPO enhances neutrophil adhesion to endothelial cells in AMI patients through the upregulation of CD11b expression on the surface of neutrophils, which is abrogated by ABAH.     

The role of complement in cigarette smoke-induced chemotactic activity of lung fluids

To help explain increased recruitment of leukocytes to smokers' lungs, we examined the possibility that the smoke-induced chemotactic activity in lung fluids may be partly complement-dependent. Acute inhalation exposure of rats to smoke from 2R1 Kentucky Reference cigarettes caused an increase in the leukocyte chemotactic activity of their lung secretions (collected by bronchoalveolar lavage 1 h after exposure). Prior depletion of circulating complement, by intravenous administration of purified cobra venom factor 24 h beforehand, prevented the smoke-induced rise in lavage fluid chemotactic activity. Crossed immunoelectrophoretic analysis of lavage C3 from smoke-exposed animals demonstrated a modified form of the molecule with increased electrophoretic (anodal) mobility. When 2 strains of mice, C5-deficient DBA/2 and C5-sufficient BALB/c, were acutely exposed to cigarette smoke, only the C5-sufficient animals (BALB/c) had increased neutrophil chemotactic activity in their lung fluids 1 h after exposure. These results show that the increased chemotactic activity in lung fluids of laboratory animals immediately after acute cigarette smoke exposure is complement-dependent.     

Subcellular distribution and mobilization of MAC-1 (CD11b/CD18) in neonatal neutrophils

The CD11b/CD18 (Mac-1) heterodimeric surface glycoprotein contributes to a broad range of adherence-dependent neutrophil inflammatory functions. Previous investigations have indicated that diminished expression or regulation of Mac-1 may underlie abnormalities of stimulated adhesion and chemotaxis of neonatal neutrophils in vitro and inflammatory deficits in human neonates. To define the pathogenic mechanisms contributing to these findings, we compared the distribution and translocation of Mac-1 in subcellular fractions of neonatal and adult neutrophils before and after chemotactic stimulation. The total cell content of Mac-1 and the proportions of Mac-1 in beta fractions (vitamin B12 binding protein-rich granules), pre-gamma fractions (gelatinase-rich granules), or gamma fractions (plasma membrane) of neonatal neutrophils were comparable with those of adult neutrophils. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 nmol/L, 37 degrees C, 15 minutes), neonatal neutrophils demonstrated (1) diminished translocation of Mac-1 from pre-gamma fractions (P less than .05), and (2) diminished surface expression of Mac-1 (P less than .05), as compared with healthy adult neutrophils. As shown in enzymatic and immunochemical assays, neonatal cells contained significantly (P less than .01) diminished levels of neutrophil gelatinase. In response to FMLP (0.1 to 10 nmol/L, 37 degrees C, 15 minutes), neonatal suspensions also released significantly (P less than .001) less gelatinase, as compared with adult neutrophil suspensions. These observations demonstrate that diminished mobilization of Mac-1 from gelatinase-rich granular pools in neonatal neutrophils is associated with abnormal surface expression of this glycoprotein after chemotactic stimulation. This abnormality may contribute, in part, to abnormal migratory properties of neonatal neutrophils in response to inflammatory stimuli.     

Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) causes upregulation of neutrophil surface CD11b/CD18 expression, and enhances the adhesion of neutrophils to cultured human endothelial cells in vitro. Systemic administration of GM-CSF results in a rapid, transient decrease in circulating phagocyte numbers. Using a nonhuman primate model (Cynomolgus), we provide histologic evidence that this transient leukopenia is associated with the margination of neutrophils in the pulmonary microcirculation. In four animals receiving 2 to 15 micrograms/kg recombinant human GM-CSF (rhGM-CSF), light microscopic sections of lung contained 36 +/- 8, 17 +/- 7, 21 +/- 6, and 15 +/- 8 (mean +/- SD, n = 20) neutrophils within a graticule grid, as compared with two control animals receiving saline injections whose lung sections contained 2.1 +/- 1.6 and 3.1 +/- 2.1 (mean +/- SD, n = 20) neutrophils within the same grid. Scanning electron microscopy shows activated leukocytes adherent to pulmonary vascular endothelium, but no morphologic evidence of endothelial damage, and no migration of cells into the extravascular space. Margination is associated with an increase in surface expression of CD11b/CD18 on circulating phagocytes, which could contribute to the adhesion to capillary endothelial cells, but CD11b/CD18 levels remain elevated even when demargination is complete. In vitro, monoclonal antibodies (MoAbs) to CD18 and CD11b were able to inhibit neutrophil aggregation and adhesion to endothelium. FMLP-induced neutrophil aggregation was inhibited by 39.8% +/- 11.5% and 44.8% +/- 12.3%, respectively, by MoAbs to CD18 and CD11b (P less than .0005, n = 4 for both); a similar effect was demonstrated on TPA-induced aggregation. MoAb CD18 reduced the adhesion of unstimulated neutrophils to endothelium by 44% (P less than .01, n = 7), and inhibited the amount of GM-CSF-stimulated adhesion by 74% (P less than .001, n = 7), while MoAb to CD11b produced a reduction of unstimulated neutrophil adhesion by 30%, and of GM-CSF-stimulated adhesion by 40% (P less than .01, n = 5, for both). However, when administered in vivo, MoAb CD18 produced only a small, albeit significant, amelioration of GM-CSF-induced margination in vivo, while MoAb CD11b was without effect. These results show that GM-CSF-induced transient leukopenia is associated with enhanced neutrophil adherence to pulmonary vascular endothelium, but suggest that the beta 2 leukocyte integrins CD11/CD18 play only a minor role in this process.     

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.     

Cigarette smoking: Effects on P selectin expression and subsequent inflammatory responses in ethanol-induced gastric mucosal damage

Abstract Experimental evidence has shown that cigarette smoking potentiates ulcer formation and delays ulcer healing. The exact mechanisms are still not completely defined. We investigated the effects of cigarette smoking on P selectin expression and its actions on neutrophil infiltration and nitric oxide formation in ethanol-induced gastric mucosal damage. In the present study, rats were exposed to cigarette smoke for three 1 h periods. Cigarette smoke exposure significantly up-regulated P selectin expression in the gastric mucosa. Cigarette smoke exposure following ethanol challenge substantially potentiated the mucosal damage, which was accompanied with an increase in myeloperoxidase and inducible nitric oxide synthase activities. Anti-neutrophil serum completely abolished these changes, whereas aminoguanidine produced similar effects without affecting myeloperoxidase activity. Taken together, cigarette smoke exposure can up-regulate P selectin expression, which facilitates neutrophil rolling, margination and infiltration when challenged with ethanol. These actions activate inducible nitric oxide synthase activity, which may contribute to the potentiating effect of cigarette smoking on ethanol-induced gastric ulceration in rats.     

Cigarette smoke-induced acute airway impairment]

Cigarette smoking has been implicated in many pulmonary disorders, including chronic bronchitis and chronic obstructive lung disease. Cigarette smoking is associated with increased airway responsiveness. Acute exposure to cigarette smoke increases airway responsiveness in a dose-dependent manner. A superoxide is involved in airway hyper-responsiveness induced by cigarette smoke, perhaps by direct toxic action. Cigarette smokers have increased numbers of neutrophils present in their lower respiratory tract. Acute exposure to cigarette smoke initiates a superoxide-dependent mechanism that, through NF-kappa B activation and IL-8 expression, induces infiltration of neutrophils into the airways in vivo. The alveolar macrophage is one potential source of NF-kappa B activation and IL-8 production after acute exposure to cigarette smoke. Manipulation of NF-kappa B by antioxidants in vivo may be useful in limiting biologic processes such as pro-inflammatory cytokine production, which may lead to neutrophil accumulation in the lung.     

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.     

Inflammatory potential of neutrophils detected in sickle cell disease

An early event in the inflammatory response is neutrophil recruitment to endothelium in response to chemotactic stimulation, which in turn activates CD18-integrin, which anchors neutrophils to the vessel wall under the shear force of blood flow. Activated neutrophils circulating in sickle cell disease (SCD) patients may significantly contribute to vascular occlusions (VOC) as neutrophils adherent to inflamed endothelium recruit sickle red blood cells inducing VOC. To elucidate the mechanisms by which neutrophils may participate in VOC in SCD, CD18-integrin expression and function in fresh blood samples of non-crisis patients were measured by flow cytometry. CD11b/CD18 membrane expression was approximately 70% higher on unstimulated SCD neutrophils than controls, which correlated with a 1-fold higher rate of adhesion to ligand. Unstimulated SCD neutrophils expressed approximately 30,000 active CD18 per cell, while controls expressed approximately 6,000. Stimulation with a low concentration of IL-8 (0.1 nM) upregulated 100% more active CD18 and induced 60% more adhesion of SCD than control neutrophils. These data demonstrate that neutrophils from SCD patients constitutively express active CD18 in blood and respond with enhanced sensitivity to chemokine activation of adhesion, thus increasing their propensity for exuberant adhesion.