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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.

The microvasculature of the normal lung contains a pool of sequestered neutrophils, which is markedly enhanced in acute lung inflammation. Lung neutrophil sequestration is determined by the cells' deformability and adhesivity to capillary endothelium, and is a pre-requisite for emigration into the airspaces. We assessed the effect of several pro-inflammatory mediators associated with acute lung inflammation on these factors. Platelet-activating factor, IL-8 and formyl-Met-Leu-Phe (fMLP) induced a marked, but transient reduction in neutrophil deformability. Also, increased surface expression of the beta(2)-integrin and CD11b, and shedding of L-selectin (CD62L) was observed for these stimuli. TNF-alpha in contrast caused a small decrease in cell deformability only after 30 min, and shedding of L-selectin, but no change in CD11b levels. However, TNF-alpha-pretreatment markedly enhanced the fMLP response for cell deformability, CD11b expression and CD62L loss. Moreover, all pre-treatments were found to induce chemokinesis, and all except fMLP, enhanced fMLP-directed chemotaxis. We were able to demonstrate, using specific TNF-alpha receptor antagonists, that the TNF-alpha-induced changes in chemotaxis were mediated through the 55-kDa receptor. Also, inhibitors of the mitogen activated protein (MAP) kinase signaling pathway showed that the p38 MAP kinase pathway was involved for fMLP-directed chemotaxis of TNF-pretreated neutrophils, although activation of the extracellular signal-regulated kinase (ERK) pathway was also seen. These data demonstrate the differential role of pro-inflammatory mediators in controlling neutrophil sequestration and migration, which may orchestrate the severity of the inflammatory response in such respiratory diseases as chronic obstructive pulmonary disease and asthma.

Background: Neutrophils are likely to play a major role in the inflammatory response seen in chronic obstructive pulmonary disease (COPD). This study sought to address the hypothesis that an enhanced neutrophil response to proinflammatory agents in COPD may contribute to their recruitment and activation in the lungs., Methods: Circulating neutrophils were obtained from 10 patients with COPD, eight long term smokers with normal lung function, and eight healthy never smoking controls. The in vitro production of reactive oxygen species (ROS) was measured by the NADPH oxidase method (respiratory burst) and the surface expression of several adhesion molecules (Mac-1, LFA-1 and L-selectin) was measured by flow cytometry. Measurements were obtained under basal conditions and after stimulation with phorbol myristate acetate (PMA) and tumour necrosis factor alpha (TNFalpha). mRNA levels of p22-phox (a subunit of NADPH oxidase) and Mac-1 (CD11b) were also determined by reverse transcriptase polymerase chain reaction (RT-PCR)., Results: Patients with COPD showed enhanced respiratory burst compared with smokers with normal lung function, both under basal conditions (mean (SE) fluorescence intensity (MFI) 15.1 (0.5) v 11.6 (0.5); mean difference -3.4 (95% CI of the difference -5.1 to -1.8), p<0.01) and after PMA stimulation (MFI 210 (7) v 133 (10); mean difference -77 (95% CI of the difference -102 to -52), p<0.01). Mac-1 surface expression was also enhanced in patients with COPD, both under basal conditions (MFI 91 (5) v 45 (3); mean difference -46 (95% CI of the difference -61 to -31), p<0.001) and after stimulation with TNFalpha (MFI 340 (15) v 263 (11); mean difference -77 (95% CI of the difference -119 to -34), p=0.001). These differences were also apparent when patients with COPD were compared with non-smokers (p<0.05). The mRNA levels of p22-phox and Mac-1 (CD11b) were similar in patients with COPD and smokers with normal lung function, suggesting that the observed differences were due to post-transcriptional regulation., Conclusions: These results demonstrate an enhanced neutrophil response to proinflammatory agents in patients with COPD which may contribute to their enhanced recruitment and activation in the lungs of these patients. These findings support those of other studies which have indicated that the neutrophil is likely to play a major role in the pathogenesis of this disease.

We investigated the expression of adhesion molecules in circulating neutrophils (lymphocyte function-associated antigen-1 [LFA-1], Mac-1, and L-selectin) and endothelial cells (soluble intercellular adhesion molecule-1[sICAM-1]) in 23 patients with stable chronic obstructive pulmonary disease (COPD), 18 subjects with exacerbated COPD, and 23 healthy volunteers. Also, in these circulating neutrophils, we assessed the expression of two G protein subunits (Galphas and Galphai1/2). Compared with control subjects, patients with stable COPD showed increased expression of Mac-1 (p < 0.001) and lower levels of sICAM-1 (p = 0.002); LFA-1 and L-selectin expression was similar in patients and control subjects. During exacerbations, compared with stable patients, the expression of Mac-1 and LFA-1 was reduced (p < 0.001). Finally, the expression of Galphas (but not Galphai1/2) was also reduced (p < 0.001) in circulating neutrophils of patients with COPD, irrespective of the clinical condition of the patient. These results indicate that in patients with COPD: (1) the expression of some neutrophil adhesion molecules (Mac-1) is abnormal, and that this pattern changes during exacerbations; (2) there may be a form of endothelial dysfunction, as suggested by the low sICAM-1 levels; (3) the expression of G protein subunit (Galphas) in circulating neutrophils is downregulated, irrespective of their clinical conditions. Overall, these results indicate the presence of significant systemic abnormalities in COPD.

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.

To investigate whether salbutamol inhibits platelet-activating factor (PAF)-induced neutrophil sequestration in the lungs, we studied eight nonatopic, nonsmoking, healthy subjects (six men; aged 27.0 +/- 1.5 (SE) yr) with PAF-induced bronchial response. Prior to PAF challenge (24 micrograms), they inhaled either salbutamol (300 micrograms) or placebo in a randomized, double-blind, crossover manner two weeks apart. Respiratory system resistance (Rrs), arterial blood gases, and neutrophil counts were measured 4, 8, 12 and 30 min after PAF. Neutrophil kinetics in the lungs were assessed by tracking autologous 99mTc-erythrocytes and 111in-neutrophils. Compared with salbutamol, arterial blood neutrophil counts fell (p < 0.04) maximally at 4 min after PAF, followed by a mild rebound neutrophilia, whereas Rrs increased (p < 0.01) and Pao2 decreased (p < 0.05) at 4 min only. The intrapulmonary activity of 111in-neutrophils after pretreatment with placebo was higher compared with salbutamol (1.98 +/- 0.15 versus 1.33 +/- 0.23 cps/mCi/pixel) (p < 0.01) although both their initial sequestration (first-pass) and subsequent washout were not significantly different. Inhaled salbutamol blocks pulmonary neutrophil sequestration and lung function abnormalities following PAF challenge in humans.

Background: Increased epithelial permeability of the airspaces occurs commonly in the lungs of cigarette smokers. It is likely to be important in augmenting the inflammatory response in the airspaces and hence may have a role in the pathogenesis of emphysema. It has previously been shown that intratracheal instillation of cigarette smoke condensate induces increased epithelial permeability in vivo in rats and in vitro in epithelial cell monolayers, associated with a disturbance in the lung antioxidant, glutathione (GSH). The aim of this study was to assess the role of neutrophils, GSH, and tumour necrosis factor (TNF) in the increased epithelial permeability following intratracheal instillation of cigarette smoke condensate., Methods: Epithelial permeability of the airspaces was measured in rat lungs as the passage of intratracheally instilled 125-iodine labelled bovine serum albumin (BSA) into the blood. The permeability of a monolayer of human type II alveolar epithelial cells to 125I-BSA was also measured., Results: Cigarette smoke condensate produced a 59.7% increase in epithelial permeability over control values peaking six hours after instillation and returning to control values by 24 hours. Depletion of neutrophils and, to a lesser extent, macrophages by an intraperitoneal injection of antineutrophil antibody did not influence the increased epithelial permeability induced by cigarette smoke condensate. Although instillation of human recombinant TNF alpha produced an increase in epithelial permeability in the rat lung from 0.62 (0.61)% to 1.27 (0.08)%, only a trivial amount of TNF alpha was detected in bronchoalveolar lavage (BAL) fluid in vivo or in culture medium from BAL leucocytes obtained from animals treated with cigarette smoke condensate (94.9 (28.8) units/ml). Furthermore, antiTNF antibody did not abolish the increased epithelial permeability produced by cigarette smoke condensate. The role of GSH was assessed by measuring the changes in both the reduced (GSH) and oxidised form (GSSG) in lung tissue and in BAL fluid. One hour after instillation of cigarette smoke condensate there was a marked fall in the GSH content in the lung (from 809.8 (31.8) to 501.7 (40.5) nmol/g) in association with increased GSSG levels (from 89.8 (2.7) to 148.7 (48.8) nmol/g). This was followed by a return of GSH levels to control values, with a concomitant decrease in GSSG levels six hours after instillation. GSH levels in BAL fluid fell dramatically following cigarette smoke condensate (from 2.56 (0.30) to 0.31 (0.21) nmol/ml) and this fall was sustained up to six hours after instillation of cigarette smoke condensate., Conclusions: These studies suggest that neutrophils and TNF do not have a major role in the increased epithelial permeability induced by cigarette smoke condensate. However, the data support a role for the depletion of the antioxidant glutathione in the increased epithelial permeability caused by cigarette smoke condensate.

Neutrophil (PMN) sequestration in the pulmonary microvasculature precedes the migration of these cells into the airspaces in inflamed lungs. Intratracheal instillation of the heat-killed organism Corynebacterium parvum in the rat induces an alveolitis in which PMN constitute 70 to 80% of the total cell count in the bronchoalveolar lavage (BAL). This acute alveolitis results in increased sequestration in the pulmonary microvasculature of 51Cr-labeled PMN when compared with control lungs. The aims of this study were to confirm this increased pulmonary PMN sequestration using unlabeled cells and to assess the function and adhesion molecule expression of such sequestered PMN. We counted the number of PMN and erythrocytes obtained by pulmonary vascular lavage (PVL) and compared the ratio of these two cell types in PVL and peripheral blood (PB) as a measure of the sequestration of PMN in the pulmonary vasculature. Compared with control animals, PVL in C. parvum-treated rats had higher PMN counts, which could not be accounted for by the PB leukocytosis. Sequestration of PMN in the pulmonary microvasculature depends on several factors, including the upregulation of adhesion molecules on both PMN and endothelial surfaces and the ability of the cells to deform when passing through the microcirculation. Cells obtained from the PVL were less deformable than PB cells in control but not in C. parvum-treated animals. The expression of the CD18 integrin on PMN obtained from the PVL of C. parvum-treated animals was increased compared with cells from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: The transit of neutrophils through the pulmonary microvasculature is prolonged compared with red blood cells and is increased further during cigarette smoking and in exacerbations of chronic obstructive pulmonary disease. The increased residence time (sequestration) of neutrophils in the pulmonary capillaries in these conditions may be the first step leading to the accumulation of cells within the lung interstitium and in the bronchoalveolar space, so potentiating lung damage. A rat model has been developed to investigate the factors which may influence neutrophil transit through the lung microvasculature., Methods: Intratracheal instillation of the heat killed organism Corynebacterium parvum was used to induce an acute neutrophil alveolitis. Neutrophils and red blood cells were isolated from donor rats, labelled with two distinct radioisotopes, and then reinjected into recipient rats to assess their transit through the pulmonary circulation. To ascertain whether peripheral blood neutrophils were minimally altered by the isolation procedure their functional status in vitro was compared with that of inflammatory neutrophils in a number of assays commonly used as descriptors of neutrophil activation. The influence of neutrophil activation on the accumulation of cells in the lungs was assessed by comparing the lung sequestration of control neutrophils, isolated from peripheral blood, with that of inflammatory neutrophils obtained from bronchoalveolar lavage of inflamed rat lungs. Lung sequestration of neutrophils was defined as the fold increase in the ratio of neutrophils labelled with chromium-51 to red blood cells labelled with technetium-99m in lung tissue compared with the same ratio in peripheral blood., Results: Sequestration of peripheral blood neutrophils occurred in control rat lungs as shown by a 17.5 (2.1) fold increase in the ratio of neutrophils to red blood cells in the pulmonary circulation compared with the ratio of these cells in the peripheral circulation. When inflammatory neutrophils, obtained by bronchoalveolar lavage from C parvum-treated animals, were injected into control rats, the increase was 90.6 (11.0) fold. Induction of an inflammatory response in the lung tissue of the recipient rat also caused an increase in the sequestration of control neutrophils compared with the same cells in control rat lungs which was, however, less marked than when inflammatory neutrophils were used (34.7 (4.7) fold). The mean (SE) pressure developed on filtration of inflammatory neutrophils in vitro through a millipore filter (7.53 (0.2) cm H2O) was greater than that of peripheral blood neutrophils (1.18 (0.2) cm H2O). Increased filtration pressure indicates a decrease in cell deformability and suggests that this may be a contributory factor to the increased sequestration of inflammatory neutrophils in the pulmonary vasculature., Conclusions: This study shows that there is sequestration of neutrophils in the pulmonary vasculature in normal rat lungs which increases in acute lung inflammation and when inflammatory neutrophils are injected into control animals. In this model changes in the neutrophil, such as cell deformability, may have a more important role in inducing increased neutrophil sequestration than the inflammatory response in the lungs.

Pulmonary capillary transit times were examined in patients who required lung resection by use of 99mTc-labeled macroaggregates (99Tc-MAA) and chromium-labeled erythrocytes (51Cr-RBC) to measure regional blood flow and volume in the resected lung. Cell flow (cells.ml-1.s-1) to each resected lung sample was determined by multiplying the number of polymorphonuclear leukocytes (PMN) per milliliter of circulating blood by the blood flow to that sample. Capillary blood volume was obtained by multiplying the morphometrically determined fraction of pulmonary blood in capillaries by the total 51Cr-RBC volume in each sample. Cell concentrations (cells/ml) in capillary blood were calculated morphometrically, and capillary transit times were obtained by dividing cell concentration by cell flow. The results show that PMN transit times were 60-100 times longer than the RBC transit times, with a 22% overlap between their distributions. We conclude that PMN are concentrated with respect to RBC in pulmonary capillary blood because of differences in their transit times and that these long transit times provide an opportunity for PMN-endothelial interactions.

The potential for neutrophils to obstruct microvessels was evaluated by measuring transit of individual neutrophils through 8-microns pores in an automated cell transit analyzer (CTA) or into micropipettes (4-8 microns ID). Stimulation in vitro by the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine. (fMLP), cigarette smoke, or purified antineutrophil cytoplasm antibodies greatly increased flow resistance, but the response varied in its dependence on time and pore diameter. Cigarette smoke or fMLP caused rapid loss of cellular deformability, although observations were complicated by changes in cell shape: progressive bipolar shape formation (after treatment with fMLP) could facilitate entry into larger pores (approximately 8 microns), whereas blebs induced by cigarette smoke caused bridging of these pores with cell immobilization. These processes led to an underestimation of the changes in deformability by the CTA. Neutrophils responded slowly to the antineutrophil cytoplasm antibodies (approximately 30 min), with a greater increase in flow resistance evaluated by a micro-pipette (4-6 microns ID) than by the CTA. We conclude that the effect of neutrophil stimulation on flow through capillary-sized vessels is potentially great (with resistance typically increased 10-fold or even complete blockage) but may depend on the vascular and cellular geometry and may be local or disseminated, depending on the rate of the rheological response.

Neutrophils within the lungs are considered to play an important role in the pathogenesis of pulmonary emphysema. We have studied the intravascular distribution of reinjected autologous 111In-labeled neutrophils in lung specimens resected 10 min after reinjection from 10 patients undergoing surgery for peripheral bronchogenic tumors. An excess of neutrophils relative to that expected for the 99mTc-labeled erythrocyte blood volume was confirmed in all specimens (range, 3- to 136-fold). In seven specimens which were completely examined, this excess displayed a skewed distribution, with a median neutrophil sequestration of 20-fold excess, and correlated with local blood volume (r = -0.51; p < 0.001). There was also a significant correlation between alveolar wall surface area per unit volume of lung (AWUV) and neutrophil excess, when randomly selected tissue blocks from each specimen were analyzed (r = 0.34, n = 51, p = 0.012). This same trend was demonstrated when whole specimen median values were considered (r = 0.64, n = 7, p = 0.07). Thus in areas of the lungs with lower AWUV values (increasing microscopic emphysema), fewer neutrophils were present. These studies add further support to the view that emphysema per se is not associated with an increased sequestration of pulmonary intravascular neutrophils.

Recent studies confirm data that for over a century have suggested that the lungs are an important source of noncirculating neutrophils. Many factors control how neutrophils pass through the normal lungs, including the unique and complex structure of the pulmonary capillary bed, local hemodynamic factors, neutrophil deformability, and neutrophil-endothelial interactions. Alterations in these factors are likely to influence neutrophil traffic in the lungs in disease processes where neutrophil-induced lung injury has been implicated. In recent years experimental approaches using in vitro techniques have been used to study neutrophil function. Such studies, together with in vivo studies in whole animals and to a limited extent in humans, will help to elucidate the important mechanisms in neutrophil sequestration in the acute and chronically inflamed lung. Novel avenues of therapeutic intervention in neutrophil traffic through the lungs may then be possible. However, whether interference with this cell, which forms a major defense mechanism in the lungs, is advisable, remains a matter for debate.

Peripheral blood neutrophils were harvested and exposed acutely in vitro to physiologically attainable levels of cigarette smoke. The adherence of radiolabeled neutrophils subsequently to alveolar epithelial cell monolayers was measured. In contrast to control cells, smoke-exposed neutrophils were significantly less adherent and failed to increase their adherence following stimulation with phorbol ester or f-met-leu-phe (fMLP). Flow cytometric analysis of the cell surface adhesion protein CD18 demonstrated no significant change in expression following in vitro smoke exposure and, furthermore, no increase in surface CD18 of smoke-exposed cells following consecutive fMLP stimulation was demonstrated. Acute in vivo cigarette smoking of up to 4 cigarettes also did not alter peripheral blood neutrophil CD18 expression. Cell spreading and chemokinesis, but not chemotaxis, was also impaired following in vitro smoke exposure. These data suggest that acute cigarette smoke may impair the crucial neutrophil functions of adherence and movement. However, the chronic effects of cigarette smoke exposure may clearly differ.

Acute cigarette smoking delays neutrophils within the pulmonary circulation in some smokers. Evidence from an in-vitro Micropore filter model of the pulmonary capillaries indicates that this may be due to a smoke induced decrease in cell deformability. In order to determine whether changes in cell shape are associated with the observed decrease in neutrophil deformability following smoke exposure, cell morphology, using scanning electron microscopy, and morphometric measurements, made using transmission electron microscopy, were performed on aliquots of neutrophils harvested from whole blood in non-smoking subjects before and after exposure in vitro to cigarette smoke. Smoke exposure increased the maximum diameter and circumference of neutrophils, without changing their area. There was also a change in the maximum to minimum cell diameter ratio, which indicated that the cells had become less spherical. Scanning electron microscopy showed that smoke exposed cells had developed blebbing of their surface membranes, suggestive of an oxidative injury to the cell membrane rather than the shape changes associated with cell activation. These changes in the morphology and morphometry of smoke exposed neutrophils may contribute to the reduction in cell deformability induced by cigarette smoke.

We have previously demonstrated a reduction in the deformability of neutrophils, exposed to whole particulate cigarette smoke in vitro, by measuring their ability to filter through a micropore membrane with pore dimensions similar to those of the average pulmonary capillary segment. In this study, we exposed neutrophils to the vapor phase of cigarette smoke and investigated the mechanism of the reduction in neutrophil filterability. Although both stimulated neutrophils and smoke-exposed neutrophils demonstrated an increase in filtration pressures, and thus a reduction in cell deformability, compared with control untreated cells, the spontaneous release of the reactive oxygen intermediates hydrogen peroxide and the superoxide anion was depressed following in vitro smoke exposure and there was no shape change to suggest that smoke-exposed cells were activated. The presence of erythrocytes, plasma, or the antioxidants albumin and glutathione prevented the reduction in cell filterability following smoke exposure, suggesting that in vitro smoke exposure, in our system, was mediated by oxidants. Indeed, the increase in filtration pressures, produced by smoke, could be mimicked by the addition of the oxidant hypochlorous acid. The cytoskeletal inhibitors cytochalasin B and D improved the filterability of smoke-exposed cells, suggesting that smoke may change neutrophil deformability through an effect on the actin component of the cytoskeleton. By contrast, colchicine, a specific inhibitor of the microtubules, had no effect. Preincubation with a monoclonal antibody to the CD18 antigen, to block this major neutrophil adhesive glycoprotein, did not alter the filtration pressure developed by stimulated or smoke-exposed neutrophils, suggesting that increased adhesivity was not the mechanism of the increase in filtration pressures observed following smoke exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of peripheral blood neutrophils to injure elements of the alveolar septum was assessed. Experiments consisted of activating previously isolated neutrophils (PMNs) with a soluble (phorbol myristate acetate, PMA) and a particulate (zymosan) trigger, and measuring detachment of 51Cr-labeled epithelial cells. In addition, the mechanisms of epithelial injury were investigated by including anti-proteinase and antioxidants in the system. Untriggered PMNs produced only slight detachment injury to epithelial cells at two effector-target cell ratios, this effect being dramatically increased after triggering with PMA; zymosan caused triggering but less than was produced by PMA. Alpha-1-protease inhibitor produced a decrease in detachment when both PMA- and zymosan-triggered cells were the effectors; superoxide dismutase did not significantly reduce detachment. The ability of triggered PMNs to cause proteolysis of fibronectin, as measured in a radiolabeled fibronectin matrix degradation assay, was related to their triggerability in the detachment assay, zymosan being largely ineffective in triggering enhanced proteolysis. These findings suggest that fibronectin is important in maintaining the integrity of the alveolar epithelial surfaces. Furthermore, in inflamed alveoli, activated PMNs can release proteinases, which cause degradation of this matrix component leading to compromise of the alveolar epithelial barrier.

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.

Human peripheral blood neutrophils were exposed in vitro, in a tonometer, to two different fractions of cigarette smoke-designated particulate phase and vapor phase. The proteolytic activity of the cells following exposure was assessed by measuring their elastase release and ability to degrade fibronectin. At levels of smoke exposure that were physiologically attainable, neither smoke fraction caused an increase in elastase release or fibronectin degradation. In most experiments, fibronectin proteolysis was suppressed by smoke exposure--an effect that was reversible on treatment with phorbol myristate acetate. These data provide evidence that the proteolytic activity of neutrophils is not enhanced by a direct effect of cigarette smoke on these cells.

N-acetylcysteine (NAC) is rapidly de-acetylated in vivo to cysteine (CYSH), a precursor of glutathione (GSH) which is an antioxidant in cells and body fluids. We investigated the effect of oral administration of N-acetyl cysteine for 5 days on the spontaneous and stimulated generation of hydrogen peroxide (H2O2) and superoxide anion (O2-) from human and rat phagocytic leucocytes. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage (BAL) in control rats and rats given NAC in their drinking water. Neutrophils (PMNL) were harvested from whole blood in normal nonsmoking volunteers before and after NAC was given by mouth. The stimulated release of H2O2 and O2 from both rat AM and human PMN was not changed by administration of NAC. However, a small but significant increase was observed in both the spontaneous generation of O2- from rat AM and the spontaneous generation of H2O2 from human PMNL. Administration of NAC significantly increased cysteine levels in human plasma and rat BAL, but the levels in human PMNL and rat AM after NAC did not differ from control levels. GSH levels were not altered significantly by NAC.

In order to study neutrophil traffic in the lungs of humans, we harvested autologous neutrophils and radiolabeled them with indium-111 prior to reinjection. The passage of these [111In]neutrophils through the pulmonary vasculature was compared with that of [99mTc]erythrocytes in normal elderly subjects and in patients with chronic obstructive pulmonary disease (COPD). Neutrophil sequestration within the lungs of seven normal subjects, 10 min after reinjection, correlated with local erythrocyte transit times in the lungs (tau = 0.72, p less than 0.001). This relationship was lost in patients with COPD. In seven patients studied during an acute exacerbation of COPD, neutrophil retention was higher during the first passage through the lungs (mean, 22.0 SD 14.1%) compared with 14 patients studied when their condition was stable (16.3 SD 3.4%, p less than 0.001), or to the normal elderly subjects (13.7 SD 7.0%, p less than 0.001). In addition, the subsequent rate of neutrophil washout from the lungs was slower in patients with acute COPD (1.93 SD 0.66 x 10(-3)s1) than in those with stable disease (3.08 SD 1.8 x 10(-3)s-1, p less than 0.02). Neutrophil retention in the lungs correlated inversely with the extent of emphysema, assessed quantitatively by CT scanning (tau = 0.68, p less than 0.05). Thus, patients presenting with acute exacerbations of COPD have an increased neutrophil burden in the pulmonary vasculature with the potential for increased lung proteolysis.

When in transit in the pulmonary capillaries, neutrophils can interact with the endothelium and gas in the alveoli. Neutrophils play a major role in lung defences and have been implicated in the pathogenesis of lung injury, such as emphysema and the adult respiratory distress syndrome. Moreover, the pulmonary capillaries receive all of the circulating neutrophils. It is surprising therefore, that relatively little is known of the factors which influence the passage of neutrophils through the pulmonary circulation. This article reviews our knowledge of neutrophil kinetics in the lungs and describes techniques using radiolabelled cells to quantify neutrophil traffic in the pulmonary circulation which should help to elucidate the pathogenesis of conditions where neutrophils are thought to play a role in lung injury.

Neutrophils may play a part in the pathogenesis of the centrilobular emphysema associated with cigarette smoking. The capillary bed of the lungs concentrates neutrophils approximately 100-fold with respect to erythrocytes, producing a large pool of marginated cells. We examined the effect of cigarette smoking on the kinetics of this pool of cells, using 99mTc-labeled erythrocytes to measure regional blood velocity and 111In-labeled neutrophils to measure the removal of neutrophils during the first passage through the pulmonary circulation, their subsequent washout from the lungs, and the effect of local blood velocity on the number of neutrophils retained in each lung region. We observed no difference in these measurements between subjects who had never smoked (n = 6) and smokers who did not smoke during the study (n = 12). However, subjects who did smoke during the study (n = 12) had a significantly slower rate of washout of radiolabeled neutrophils from the lung (0.08 +/- 0.04 of the total per minute, as compared with 0.13 +/- 0.06 in smokers who did not smoke during the experiment and 0.14 +/- 0.08 in non-smokers) (P = 0.02). We also observed an increase in the regional retention of labeled neutrophils with respect to blood velocity in 5 of the 12 subjects who smoked during the study, but in none of the other subjects. We conclude that the presence of cigarette smoke in the lungs of some subjects increases the local concentration of neutrophils, and suggest that the lesions that characterize emphysema may be a result of the destruction of lung tissue by neutrophils that remain within pulmonary microvessels.

A range of different particle types cause excessive lung inflammation that is thought to play a role in the various types of pathology they produce. Recently attention has been focused on ultrafine particles and the phenomenon of rat lung overload. The authors and their collaborators have shown previously that the surface area metric drives the overload response. Acute inflammatory response following instillation of particles has also been used to evaluate hazard but has been criticized because of the non-physiological delivery and the problems of local overload. We have instilled a number of low-toxicity dusts of various particle sizes and assessed neutrophil influx into the lung at 18–24 h. The extent of inflammation has been shown to be a function of the surface area instilled. Since ultrafine particles present a ‘special’ case of high surface area, they are relatively inflammogenic. There is no evidence that ultrafine particles of carbon black, titanium dioxide or latex have any special reactivity in addition to their large surface area. We tested whether we could use this approach to model the reactivity of highly toxic dusts. Rats were instilled with either DQ12 quartz or aluminium lactate-treated DQ12 and, as anticipated, the high specific surface toxicity of DQ12 meant that it was much more inflammogenic than was predicted using the relationship described for low-toxicity dusts. By contrast, aluminium lactate-treated DQ12 fell on to the line of ‘low-toxicity’ dusts. This approach presents the possibility of modelling potential toxicity for nuisance dusts based on the inflammatory response of a given instilled surface area dose. [ABSTRACT FROM AUTHOR]