The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.     

Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments.     

Role of actin cytoskeleton in LPS-induced NF-kappaB activation and nitric oxide production in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and is known to cause actin cytoskeleton reorganization in a variety of cells including macrophages. Actin cytoskeleton dynamics influence many cell signaling pathways including the NF-kappaB pathway. LPS is also known to induce the expression of many pro-inflammatory genes via the NF-kappaB pathway. Here, we have investigated the role of actin cytoskeleton in LPS-induced NF-kappaB activation and signaling leading to the expression of iNOS and nitric oxide production. Using murine macrophages, we show that disruption of actin cytoskeleton by either cytochalasin D (CytD) or latrunculin B (LanB) does not affect LPS-induced NF-kappaB activation and the expression of iNOS, a NF-kappaB target gene. However, disruption of actin cytoskeleton caused significant reduction in LPS-induced nitric oxide production indicating a role of actin cytoskeleton in the post-translational regulation of iNOS.     

Endotoxaemia in rats: role of NO, PAF and TXA2 in pulmonary neutrophil sequestration and hyperlactataemia.

OBJECTIVE AND DESIGN: The involvement of PAF, TXA2 and NO in LPS-induced pulmonary neutrophil sequestration an hyperlactataemia was studied in conscious rats. As pharmacological tools WEB 2170 (PAF receptor antagonist, 20 mg/kg), camongarel (inhibitor of TXA2 synthase, 30 mg/kg), N(G)-nitro L-arginine methyl ester (L-NAME -- non-selective nitric oxide synthase inhibitor, 30 mg/kg) were used. METHODS: Plasma lactate and NO2-/NO3- levels as well as myeloperoxidase (MPO) activity in lung tissue were measured one and five hours after administration of LPS (4 mg/kg(-1)). RESULTS: LPS induced a twofold increase in plasma lactate levels and nearly 10-fold increase in plasma NO2-/NO3- levels five but not one hour after LPS administration. However, LPS-induced increase in pulmonary MPO activity was seen at both time intervals. Neither WEB 2170 nor camonagrel changed one or five hours responses to LPS (lactate, NO2-/NO3-, MPO). L-NAME potentiated LPS-induced rise in MPO activity in the lung and this potentiation was not affected by WEB 2170 or camonagrel. L-NAME supressed plasma NO2-/NO3- response and substantially potentiated plasma lactate response to LPS and both effects were partially reversed by WEB 2170 or camonagrel. CONCLUSIONS: In summary, we demonstrated that PAF and TXA 2 play a role in overproduction of lactate during endotoxaemia in NO-deficient rats. However, these lipids do not mediate endotoxin-induced sequestration of neutrophils in the lung.     

Roles of histone acetylation modification in basal and inducible expression of hsp26 gene in D. melanogaster

Src-suppressed C kinase substrate (SSeCKS) plays a role in membrane-cytoskeletal remodeling to regulate mitogenesis, cell differentiation, and motility. Previous study showed that lipopolysaccharide (LPS) induced a selective and strong expression of SSeCKS in the vascular endothelial cells of lung. Here we show that LPS stimulation elevated expression of SSeCKS mRNA and protein in Rat pulmonary microvascular endothelial cell (RPMVEC). LPS potentiated SSeCKS phosphorylation in a time- and dose-dependent manner, and partly induced translocation of SSeCKS from the cytosol to the membrane after LPS challenge. The PKC inhibitor, Calphostin C, significantly decreased LPS-induced phosphorylation of SSeCKS, inhibited SSeCKS translocation and actin cytoskeleton reorganization after LPS challenge, suggesting that PKC may play a role in LPS-induced SSeCKS translocation and actin rearrangement. We conclude that SSeCKS is located downstream of PKC and that SSeCKS and PKC are both necessary for LPS-induced stress fiber formation. Chun Cheng and Haiou Liu are contributed equally to this work.     

On the Mechanism of Blood Plasma Filtration by Neutrophils in the Capillary System

Changes in the ratio between the diameters of capillaries and neutrophils, neutrophil stiffening, and their adhesion to vascular endothelium lead to the retention and accumulation of leukocytes in the pulmonary capillary system. In experiments, these phenomena are induced by different methods, from injection of microorganisms into the blood to breathing pure oxygen for 24 h. They may result in capillary occlusion with neutrophils and platelets and damage to blood vessels and tissues. The hypothesis is discussed that blood is filtered by neutrophils accumulated in capillaries, which ensures the contact of neutrophils with microorganisms no later than 5–10 min after the microorganisms enter the blood circulation. Neutrophils are retained, at least temporarily, in the places in the capillary system where relatively wide capillaries branch into narrower capillaries.     

Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans

Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.     

Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries

Although the lung is known to be a major site of neutrophil margination, the anatomic location of these sequestered cells within the lung is controversial. To determine the site of margination and the kinetics of neutrophil transit through the pulmonary microvasculature, we infused fluorescein isothiocyanate-labeled canine neutrophils into the pulmonary arteries of 10 anesthetized normal dogs and made fluorescence videomicroscopic observations of the subpleural pulmonary microcirculation through a window inserted into the chest wall. The site of fluorescent neutrophil sequestration was exclusively in the pulmonary capillaries with a total of 951 labeled cells impeded in the capillary bed for a minimum of 2 s. No cells were delayed in the arterioles or venules. Transit times of individual neutrophils varied over a wide range from less than 2 s to greater than 20 min with an exponential distribution skewed toward rapid transit times. These observations indicate that neutrophil margination occurs in the pulmonary capillaries with neutrophils impeded for variable periods of time on each pass through the lung. The resulting wide distribution of transit times may determine the dynamic equilibrium between circulating and marginated neutrophils.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms

The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-alpha-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization.     

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro‐inflammatory mediators. We examined mechanisms of LPS‐stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage‐like J774 cells. A wound‐healing assay revealed that LPS also activated directed cell movement that was followed by TNF‐α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5‐bisphosphate. Fractionation of Triton X‐100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin‐regulatory proteins, paxillin on tyrosine 118 by 80% and N‐WASP on serine 484/485 by 20%, and these events preceded activation of NF‐κB. LPS‐induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N‐WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS‐stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF‐α production suggesting that LPS‐induced cell motility is not required for TNF‐α release. J. Cell. Biochem. 113: 80–92, 2012. © 2011 Wiley Periodicals, Inc.     

Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.     

Role of LPS-induced microfilament depolymerization in MIP-2 production from rat pneumocytes

We have previously demonstrated that lipopolysaccharide (LPS) induces production of macrophage inflammatory protein-2 (MIP-2), a C-X-C chemokine for neutrophil recruitment and activation, in primary cultured rat lung alveolar epithelial cells. We have also demonstrated that LPS depolymerizes microfilaments in rat alveolar epithelial cells. To determine whether the polymerization status of microfilaments affects LPS-induced MIP-2 production, we treated rat alveolar epithelial cells with cytochalasin D (CytoD), a microfilament-disrupting agent, before and during LPS stimulation. A lower concentration (0.1 microM) of CytoD inhibited LPS-induced MIP-2 production without affecting microfilament polymerization. In contrast, LPS-induced MIP-2 production was enhanced by a higher concentration (10 microM) of CytoD, which disrupted the filamentous structure of actin. Jasplakinolide (1 nM to 1 microM), a polymerizing agent for microfilaments, decreased LPS-induced MIP-2 secretion. Jasplakinolide (1 microM) also blocked LPS-induced depolymerization of microfilaments. These results suggest that, in alveolar epithelial cells, LPS-induced MIP-2 production is at least partially regulated by microfilament depolymerization.     

Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production,cell migration and apoptosis

Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.     

Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation

Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.     

Distinct adhesive properties of granulocytes and monocytes to endothelial cells under static and stirred conditions

Adherence of neutrophils and monocytes to endothelium is an important event in the sequence of leukocyte responses to inflammatory disease. Double-color flow microfluorimetry analysis was used to determine neutrophil and monocyte adherence to suspended endothelial cells under stirred conditions. The static adherence to endothelial cell monolayers was simultaneously determined. In both assays, neutrophils behaved in a similar way. Basal adherence of neutrophils was very low. Activation by PMA or by the chemoattractants platelet-activating factor and FMLP induced rapid binding, primarily mediated by CR3. Nonactivated neutrophils showed CD18-dependent (lymphocyte function-associated Ag-1 and CR3) and CD18-independent adherence to endothelial cells when the endothelial cells were prestimulated with rIL-1 beta. In contrast to neutrophils, nonactivated monocytes adhered avidly to resting endothelial cells. This adherence was partly CD18 dependent and partly CD18 independent. Whereas monocyte adherence under stirred conditions strongly increased upon activation by PMA, a significant decrease in adherence was found under static conditions, which was prevented by cytochalasin B. This decrease was limited to a distinct CD18-independent binding mechanism, and absent under stirred conditions. We conclude that monocytes adhere with (at least) three binding mechanisms to endothelial cells, a CD18-dependent and two CD18-independent mechanisms.     

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability".

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.     

Effects of CD18 deficiency on the emigration of murine neutrophils during pneumonia.

We hypothesized that CD18 deficiency would impair the ability of neutrophils to emigrate from pulmonary blood vessels during certain pneumonias. To directly compare the abilities of wild-type (WT) and CD18-deficient neutrophils to emigrate, mice with both types of leukocytes in their blood were generated by reconstituting the hemopoietic systems of lethally irradiated C57BL/6 mice with mixtures of fetal liver cells from WT and CD18-deficient mice. Percentages of CD18-deficient neutrophils in the circulating and emigrated pools were compared during experimental pneumonias. Similar percentages were observed in the blood and bronchoalveolar lavage fluid 6 or 24 h after intratracheal instillation of Streptococcus pneumoniae, demonstrating that no site on the CD18 molecule was required for either its adhesive or its signaling functions during neutrophil emigration. However, 6 h after instillation of Escherichia coli LPS or Pseudomonas aeruginosa, the percentage of CD18-deficient neutrophils in the bronchoalveolar lavage fluid was only about one-fourth of that observed in the blood. This difference persisted for at least 24 h after instillation of E. coli LPS. Thus, neutrophil emigration elicited by the Gram-negative stimuli E. coli LPS or P. aeruginosa was compromised by deficiency of CD18. These data, based on comparing WT and gene-targeted CD18-deficient neutrophils within the same animals, provide evidence for molecular pathways regulating neutrophil emigration, which could not be appreciated in previous studies with pharmacological blockade or genetic deficiency of CD18.     

Inflammatory response of tracheobronchial epithelial cells to endotoxin

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.