key: cord-0006927-n1w56x2m
authors: Liu, Chien-Ying; Liu, Yun-Hen; Lin, Shu-Min; Yu, Chih-Ten; Wang, Chun-Hua; Lin, Horng-Chyuan; Lin, Chien-Huang; Kuo, Han-Pin
title: Apoptotic neutrophils undergoing secondary necrosis induce human lung epithelial cell detachment
date: 2003-10-03
journal: J Biomed Sci
DOI: 10.1007/bf02256327
sha: efce114427b6890a6af5e70d5f8a8611dcdc6a75
doc_id: 6927
cord_uid: n1w56x2m

Clearance of apoptotic neutrophils by alveolar macrophages plays an important role in the resolution phase of lung inflammation. If not cleared, apoptotic neutrophils are postulated to release histotoxic granular contents. Since numerous cellular proteins are degraded during apoptosis, we sought to determine whether functional serine proteinases are indeed released by apoptosing neutrophils in vitro. In a coculture system, cytokine-activated neutrophils induced detachment in the human epithelial cell line, A549. This process was CD18- and serine proteinase-dependent. Early apoptotic neutrophils induced significant detachment, but live, senescent, resting neutrophils and terminal, secondary necrotic neutrophils had a different effect. This detachment process was CD18-independent but serine proteinase-dependent. Similarly, detachment occurred with primary human small airway epithelial cells. Notably, epithelial cell detachment correlated with the transition of early apoptotic neutrophils to secondary necrosis and with the accumulation of elastase in the supernatant. The membrane integrity of lung epithelial cells was damaged in advance of significant cell detachment. These observations suggest that not only live activated neutrophils but also apoptosing neutrophils can reveal functional elastase activities. Furthermore, the rapidity of the transition emphasizes the importance of the prompt clearance of apoptotic neutrophils before they progress to secondary necrosis at the site of lung inflammation.

The utilization of neutrophilic proteinase is an important host defense mechanism against pathogenic microorganisms [7, 41, 53] . However, abundant literature documents the potential hazards of these proteolytic enzymes in the pathogenesis of inflammatory organ injury. Circulating mature human neutrophils are committed to die, even when their life spans are prolonged by various endogenous and exogenous factors. Apoptotic neutrophils are recognized by professional phagocytes and semiprofessional phagocytic parenchymal cells. Although definitive in vivo studies are lacking, apoptosing neutrophils are postulated to be removed without further augmentation of the existing inflammation [49, 50] . This process is believed to be rapid, efficient and important during the resolution phase of inflammation [10, 19] . Coincident with the highly controlled process of neutrophil apoptosis is the shutdown of the degranulation response [24] . If not phagocytosed, apoptotic neutrophils will undergo secondary necrosis, an end-stage cellular disintegration characterized by breached membrane integrity. Consequently, proteinases residing in apoptotic neutrophils may be passively released and, in turn, contribute to tissue injury.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-a, both common at inflammation sites, accelerate macrophage clearance of neutrophils undergoing apoptosis in vitro [6, 18, 46, 51] . However, several factors associated with chronic inflammatory diseases can markedly inhibit the ability of macrophages to recognize and engulf apoptotic neutrophils. For example, a number of granulocyte enzymes with an extracellular presence in inflammatory diseases are extremely cationic [41] . These cationic molecules, along with a low pH, are common features of chronic inflammation and have been demonstrated to significantly inhibit recognition of apoptotic neutrophils by macrophages [50] . In patients with cystic fibrosis, Pseudornonas aeruginosa products have been reported to inhibit macrophage phagocytosis [36, 37] . Moreover, the ability of alveolar macrophages to phagocytose apoptotic neutrophils was found to be significantly impaired in smoke inhalation injury, a condition associated with a high incidence of both bacterial pneumonia and adult respiratory distress syndrome (ARDS) [26] , and in children suffering recurrent respiratory tract infections [43] . Other investigations have suggested that macrophages which had previously ingested apoptotic cells subsequently have impaired ingestion capacity [ 18] . In this situation, apoptotic neutrophils can undergo secondary necrosis, presumably resulting in the release of histotoxic granular contents [52] . If the proteinases released remain functional, they can then escalate the inflammatory response and exacerbate lung injury. Surprisingly, few studies have examined the release of functional proteinases from apoptotic neutrophils.

Neutrophils express several serine proteinases, including neutrophil elastase, cathepsin G and proteinase 3. Among these, elastase is the major proteinase, and has been demonstrated to be critical in neutrophil-mediated tissue injury and extracellular proteolysis [1, 8, 34, 40] . Previous studies showed that serine proteinase inhibitors blocked A549 cell detachment induced by activated neutrophils, and that exogenous elastase restored this detachment [3] . Because the disruption and desquamation of airway and lung epithelial cells have been widely observed in a variety of immunological, infectious and inflammatory pulmonary diseases [2, 8, 10, 23] , we used lung epithelial cell detachment as a bioassay in this study [9] to investigate the effect of early apoptotic neutrophils on airway epithelial cells and the role of elastase activity in causing the effect. Because CD 18 is a f~2 integrin subunit critical to the firm adhesion and the subsequent degranulation response of activated neutrophils [5, 57] , and because apoptotic neutrophils have been demonstrated to accumulate CD 18 on the cell surface [16] , we also investigated the role of the integrin CD 18 in the cytotoxic effect of apoptosing neutrophils.

Secretory leukocyte proteinase inhibitor (SLPI), TNF-a and GM-CSF were purchased from R&D Systems (Minneapolis, Minn., USA). The caspase inhibitor zVAD-fmk and the calpain inhibitors zLLY-fmk and PF150606 were purchased from Calbiochem (San Diego, Calif., USA). Human CD 18 blocking monoclonal antibody (mAb) 60.3 is a mnrine IgG2a which was obtained from Serotec (Raleigh, N.C., USA) [44] . All culture medium and additives were purchased from BioWhittaker (Walkersville, Md., USA) unless otherwise specified.

Human neutrophils were isolated from freshly drawn heparinized blood by dextran sedimentation and centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia Biotech, Uppsala, Sweden), as previously described [17] . Additionally, contaminating erythrocytes were lysed using 0.9% buffered ammonium chloride. Purified neutrophils of varying concentrations were then suspended in RPMI with or without 10% heat-inactivated fetal bovine serum (FBS; HyClone Sterile, Logan, Utah, USA), depending on assay conditions. Isolated neutrophils were consistently > 98 % pure according to Lukostat staining (Fisher Scientific, Pittsburgh, Pa., USA), and > 99 % viable according to the trypan blue exclusion test. nologies, Carlsbad, Calif., USA), 1.9 rm~ sodium pyruvate and 0.1 mM nonessential amino acids at 37 °C in a 5% CO2 incubator. The cells were passaged at 80-100°/o confluence using 0.25% trypsin. Human primary small airway epithelial cells (SAECs) were purchased from Clonetics (Walkersville, Md., USA). The SAECs were expanded and maintained in SAEC basal medium supplemented with bovine pituitary extract, hydrocortisone, human recombinant epidermal growth factor, epinephrine, insulin, retinoic acid, triiodothyronine, gentamicin, amphotericin B and bovine serum albumin (termed SAEC growth medium) at 37°C in a 5% C02 incubator. To minimize the phenotypic changes in epithelial cells, SAECs were used in a maximum of 3 passages. Screening tests for Mycoplasma contamination at the beginning and at the end of this study showed no positive reaction. All cells used in this study grew well for the determined times.

A549 cells were plated in 96-well plates at 4 x 10 4 cells/well in 100 gl of minimum essential Eagle medium supplemented with 10% FBS. Following overnight culturing, confluent cells were labeled with 20 ttM CellTracker Green CMFDA (Molecular Probes, Eugene, Oreg., USA) for 30 rain at 37 °C and in darkness. Cells were washed twice and incubated in fresh medium for another 30 rain at 37 °C. At this point, the culture medium was replaced with 200 gl of superuatant from neutrophils cultured in phenol red-free, serum-free RPMI-1640 medium. Alternatively, fresh or cultured neutrophils resuspended at varying concentrations in fresh medium were added to the A549 cell monolayer. Following 6 h of coincubation, the fluorescence intensity (FI) in the wells was measured using CytoFluor (PerSeptive Biosystems, Framingham, Mass., USA) (with excitation at 485 nm and emission at 530 rim). Detached and loosely adherent cells were then removed by gently washing the plate 3 times with phenol redfree medium. Next, the remaining fluorescence in the wells was determined. After subtracting background fluorescence (phenol redfree medium alone without cells), detachment was computed using the following formula: FI in test well after washing/FI in test well before washing 1 -FI in ~~ingJ ! x 100%, where the control well contained the A549 monolayer with RPMI-1640 supplemented with 10% FBS for A549 cells and SAEC growth medium for SAECs. Detachment of the SAEC monolayer was determined similarly, except that SAEC growth medium was used.

For spontaneous apoptosis, freshly isolated neutrophils were suspended in RPMI-1640 supplemented with 10% FBS and incubated at a concentration of 5 x 106 cells/ml in 6-well tissue culture plates for 16 h. Apoptotic neutrophils were isolated using a MACS apoptotie cell isolation kit (Miltenyi Biotech, Auburn, Calif., USA) according to the instructions of the manufacturer. Briefly, neutrophits in culture were resuspended in 80 gl of binding buffer and 20 gl of annexin V-conjugated paramagnetic microbeads/107 cells. Cells were then incubated for 15 rain at 6 °C. After a single washing, cells resuspended in 1 ml of binding buffer were loaded onto a Midi-MACS column, and the column was then irrigated with binding buffer. Both the annexin V-selected (with a magnet) and flow-through fractions were collected. To ensure quality control, the purity of each fraction was determined in parallel with experiments by double labeling with annexin V-FITC (PharMingen, San Diego, Calif., USA) and propidium iodide (Molecular Probes). Briefly, neutrophils in each fraction were washed once with phosphate-buffered saline (PBS). Cells were then incubated with annexin V-FITC and propidium iodide in binding buffer containing 10 mMHEPES, 1.5 mMMgC12 and 2.5 mMCaC12 in normal saline for 15 min at room temperature, and analyzed by flow cytometry (Epics XL, Coulter, Hialeah, Fla., USA).

Elastase activity in cell-free superuatant was assessed by a modified method described previously [13] using methoxysuccinyt-Ala-Ala-Val-7-amid-4-methyl-coumarin (Calbiochem). Sample activity was then compared with a standard curve determined using known concentrations of purified human neutrophil elastase (Calbiochem).

To determine the cell membrane integrity of the adherent lung epithelial cells, propidium iodide staining was used [9] . At each evaluation point, floating cells were removed. The remaining adherent cells were washed twice with PBS and stained with propidium iodide (5 gg/ml in RPMI-1640) for 5 rain at 37°C. After 2 gentle washes, cells were examined with fluorescence microscopy using a 590-nm filter and then trypsinized for flow cytometric analysis (with excitation at 488 nm and emission at 585 nm). The percentage ofpropidium iodide-stained cells in the gated lung epithelial cell region on flow cytometric plots was determined in order to quantify the damage to cell membrane integrity.

Data are expressed as the mean + SEM. For normally distributed data, a t test was used to evaluate differences between sets. For nonnormally distributed data, the Mann-Whitney U test was used. GraphPad Prism (version 2.0 I, GraphPad Software, San Diego, Calif., USA) was used for all statistical analyses. Statistical significance was defined as p < 0.05. 

When cultured in serum-free media, approximately 50% of the fresh neutrophils lost their membrane integrity with positive trypan blue staining within 36 h, and 95% of cells within 72 h ( fig. 1 ). Fresh neutrophils, when cocultured on a monolayer of the human lung epithelial cell line A549, were found to induce epithelial detachment within 24 h ( fig. 2A, B) . Moreover, the time required for comparable detachment to occur was significantly reduced to 6 h if the neutrophils had previously been cultured for 36 h ( fig. 2C ), suggesting a process related to neutrophil senescence and possibly cell death. Next, the underlying mechanism was examined to determine whether it was soluble factor-dependent. As shown in figure 3A, supernatant from neutrophils cultured for 36 h induced A549 cell detachment after 6 h of coculturing. Supernatant from neutrophils cultured for 72 h also caused significant detachment. In parallel experiments, freshly isolated neutrophils or neutrophils cultured for 72 h and resuspended in fresh serum-free medium induced minimal detachment. In contrast, neutrophils cultured for 36 h induced significant A549 cell detachment in a cell number-dependent manner without exogenous neutrophil-activating stimuli ( fig. 3B ).

There was prominent A549 cell detachment within 6 h when freshly isolated neutrophils were activated with TNF-a ( fig. 4 ). In this coculture system, detachment was noted to be almost completely abrogated by mAb 60.3, a CD 18-blocking antibody. To determine whether detachment induced by resting cultured neutrophils requires CD18 engagement, the effect of mAb 60.3 on A549 celt Annexin-FITC Fig. 4 . Detachment of A549 cells by cultured neutrophils is CD18independent. Neutrophils (5 x 106/ml) were cultured in serum-free medium for 36 h and then collected and centrifuged. Cells were resuspended in the same volume of serum-free fresh medium. Two hundred microliters of cell suspension was added to each well (1 x 106 cell equivalents/well) and cocultured with A549 cells in the presence or absence ofmAb 60.3 (10 gg/ml). Fresh neutrophils (1 x 106/ well) stimulated with 10 ng/ml TNF-a were also cocultured with A549 cells in both the presence and absence ofmAb 60.3 (10 gg/ml). Detachment was measured after 6 h of incubation. Results are expressed as the mean + SEM of 4 experiments performed in triplicate. *p < 0.01 compared with medium alone. ** p < 0.01 compared with neutrophils treated with TNF-a alone. detachment was tested. Figure 4 confirms the suspicion that A549 cell detachment induced by resting neutrophils cultured for 36 h was not affected by mAb 60.3.

Neutrophils incubated in vitro undergo apoptosis constitutively. Flow cytometric analysis of the approximately 50% trypan blue-positive neutrophils cultured for 36 h revealed that over 90% of cells were annexin V positive. Thus, we hypothesized that apoptotic neutrophils, if allowed to undergo secondary necrosis, would contribute to A549 cell detachment. To test this hypothesis, neutrophils cultured for 16 h were separated into an annexin V-selected fraction and a flow-through fraction, each of which was then cocultured with A549 cells for 6 h. Neutrophils in the annexin V-selected fraction mainly occurred early in apoptosis (>80% annexin V positive, propidium iodide negative, fig. 5A ) and induced significant detachment within 6 h of coculturing in a cell number-dependent manner. Meanwhile, >85% of neutrophils in the flowthrough fraction were alive (annexin V negative, propid-ium iodide negative, fig. 5B ) and had a minimal effect at best ( fig. 6A ). Utilizing human primary-lung epithelial cells, SAECs, also revealed significant detachment ( fig. 6B ). This detachment was not affected by mAb 60.3 ( fig. 7) . However, the apoptosing neutrophil-induced detachment of airway epithelial cells was completely abrogated by exogenous SLPI, a specific inhibitor of serine proteinases ( fig. 7A) . Additionally, the supernatant from the apoptosing neutrophils was able to induce A549 cell detachment, which could be attenuated by SLPI (fig. 7B ).

To determine whether early apoptotic neutrophils release functional elastase, we examined elastase activity in supernatant from the annexin V-selected fraction cultured in vitro, and the data revealed that elastase activity continually increased with time ( fig. 8A ). Parallel to the determination of elastase activity, the membrane integrity of cells was evaluated using trypan blue. The release of functional elastase activity from apoptosing neutrophils was temporally correlated with the progression to second- ary necrosis (fig. 8B ). In separate dose-response experiments using purified human neutrophil elastase, it was observed that a threshold concentration of 1.25 pg/ml was required to induce significant A549 cell detachment within 6 h. In our experiments, elastase activity in the supernatant of the annexin V-selected fraction reached this level after only 4 h. Although the annexin V-negatively selected flow-through fraction also released elastase, the level of its activity was below the threshold concentration ( fig. 8A, B) .

To further explore the effect of early apoptotic neutrophils on adherent lung epithelial cells, the annexin Vselected fraction ofneutrophils cultured for 16 h was coincubated with A549 cells for 6 h. As shown in figure 9 , the percentage of propidium iodide-stained adherent A549 cells increased with time, in advance of significant cell detachment, suggesting that early apoptotic neutrophils Apoptotic Neutrophils Induce Epithelial Cell Detachment are able to breach the ceil membrane integrity and possibly, in turn, cause cell detachment.

Our study demonstrates that early apoptotic neutrophils induce lung epithelial cell detachment. This detachment is strictly serine proteinase-dependent and CD18independent. It also correlates with an increase in elastase activities in the extracellular milieu and the toss of membrane integrity in adherent lung epithelial cells.

A healthy human adult turns over almost 1 × 109 neutrophils/kg of body mass daily [ 11 ] . Once recruited to an inflammatory site, neutrophit life spans were demonstrated to be extended ex vivo [ 14, 35] . Notwithstanding, mature neutrophils are committed to undergoing apoptosis. Apoptosis is an intracellular remodeling process with initially limited autoproteolysis and oligonucleosomal cleavage which is accompanied by downrcgulation ofneutrophil inflammatory responses [29, 30, 59] . This process eventually leads to cellular disintegration, with the breach in cellular membrane integrity (secondary necrosis). Although histotoxic neutrophilic granular contents have been widely stated to be released under this scenario, an exhaustive literature search yielded surprisingly few peerreviewed original studies that characterized the release of functional proteinase activities from these shrinking, autoproteolysing neutrophits. Utilizing annexin V-conjugated paramagnetic beads that selectively bind to exposed cell surface phosphatidylserine residues present on apoptotic but not live neutrophils [22, 31, 55] , this study revealed more accelerated release of functional elastase activity from predominantly early apoptotic neutrophils, compared to that from predominantly live, senescent, resting neutrophils. Furthermore, the timing of this release correlates with the progression of early apoptotic neutrophils to secondary necrosis and lung epithelial cell detachment in vitro. Thus, this study clearly confirms that resting apoptosing neutrophils release functional elastase activity in vitro.

Early apoptotic neutrophils differ from live, selectively activated neutrophils in that despite increased cell surface CD 18, they do not require the participation of the 132 integrin, CD 18, for the adhesion and release of granule contents [16] . On the other hand, SLPI, one of the endogenous serine proteinase-specific antiproteinase defenses, abrogates both live, activated neutrophil-and apoptosing neutrophil-induced lung epithelial cell detachment. Importantly, exogenous SLPI does not protect the membrane integrity of apoptosing neutrophils.

There are relatively few investigations which have quantified the occurrence of apoptotic neutrophils in the course of acute inflammatory lung diseases in vivo. Thus, the role of dying neutrophils in lung injury is unclear. In patients with ARDS, apoptotic neutrophils in bronchoalveolar lavage fluid are not abundantly identifiable morphologically but are present during the first few days of the initial onset of disease [35] . This phenomenon may be consistent with the prevailing concept that apoptotic neu- trophils are rapidly recognized and ingested by professional and semiprofessional phagocytic cells [49, 50] . Alternatively, the rapid transition from early apoptosis to apoptotic body-tbrmation, secondary necrosis and eventual total cellular disintegration may lead to a certain degree of underestimation. Furthermore, current histochemical techniques and in vitro phagocytosis assays do not distinguish whether the clearance of dying neutrophils occurs before or after the breach of membrane integrity.

One recent study observed that numerous, persistent apoptotic neutrophils are present in lung abscesses formed in experimental pneumonia in vivo [28] . This important observation implies that the balance between neutrophil apoptosis and their phagocytic clearance can be disturbed. Indeed, this possibility is supported by two further ex vivo studies that specifically verified the functional impairment of alveolar macrophages, both in clearing apoptotic neutrophils in children suffering from recurrent respiratory tract infections, and in an animal model of experimental smoke inhalation injury [26, 43] . Although this study used a lung epithelial cell detachment assay as an in vitro bioassay to characterize the serine proteinases released from dying neutrophils, the biological effects of neutrophil proteinases are wide-ranging. For example, besides a direct role of neutrophil proteinases in experimental lung injury [23, 38, 45] , elastase can activate neutrophil matrix metalloproteinase-9 [21] , which is capable of solubilizing the human alveolar extracellular matrix [ 15, 42] and is increased in epithelial lining fluid from patients with ARDS [38] . Cathepsin G can also augment inflammation through platelet activation via pmteinase-activated receptor-4 activation [47] . Furthermore, neutrophil elastase and a-defensins can stimulate lung epithelial cell production of IL-8 and/or GM-CSF in vitro [39, 54] . Conceivably, during the resolution phase of an acute inflammatory insult, functional proteinases released from apoptosing neutrophils that have escaped timely phagocytic clearance may perpetuate inflammation and contribute to tissue injury.

Numerous in vitro investigations have suggested that neutrophils contribute not only to host defenses against pathogenic organisms but also to defense against tissue injury. NeutrophiI proteinases are among the essential causes of these dual effects. Moreover, the realization that robust, endogenous antiproteinase systems are generally in place further leads to the conclusion that maintaining a certain proteinase/antiproteinase balance in vivo is a biological necessity. The current paradigm of a self-limiting, neutrophil-mediated inflammatory" process entails not only the timely recruitment of neutrophils capable of active phagocytosis and degranulation, but also their subsequent efficient removal from inflammatory sites [25] . The appreciation that multiple recognition mechanisms exist, whereby effete granulocytes are eliminated by both professional and semiprofessional phagocytes, further indicates the critical importance of this process [ 12, 20, 48, 56] . However, confirmatory in vivo experiments that directly test the importance of neutrophil apoptosis in the resolution phase of this paradigm are not yet available.

Our data showed that the cell membrane integrity of A549 cells was significantly disrupted in advance of detachment. The molecular mechanisms of detachment of A549 cells or airway epithelial cells are complex and not completely known. The disruption of cell-matrix adhesion and cell-cell tight junctions, and the induction of apoptosis-necrosis have been observed on or in A549 cells [3, 9, 10, 27, 32, 33] . The mechanisms are inducer-specific in part. The disruption and desquamation of airway and lung epithelial cells resulting from cellular damage can increase the permeability of the lung parenchyma to fluid and cells [2, 4, 58] . Many biological cascades follow, resulting in acute airway narrowing and pulmonary inflammatory edema or chronic airway remodeling and lung fibrosis [2, 4, 58] . Related symptoms, including dyspnea, cough and wheezing, can occur. The pulmonary function test will reveal airway obstruction, limited lung expansion and decreased gas diffusion [2, 4, 48] .

In conclusion, this study demonstrates that apoptosing neutrophils release, at least, functional elastase activity during secondary necrosis and induce a cytotoxic effect on airway epithelial cells. As this process is rapid, our results indicate that neutrophil apoptosis may not necessarily be benign, and emphasize the importance of removal of these disintegrating cells promptly by phagocytic cells. Clearly, further study is needed to explore the molecular mechanisms of epithelial celt detachment induced by apoptotic neutrophils and to document the occurrence of neutrophil apoptosis and the kinetics of apoptotic neutrophil removal in various disease states. Finally, our results suggest that a therapeutic strategy for inducing granulocyte apoptosis en masse so as to attenuate active lung inflammation should consider the simultaneous enhancement of clearance mechanisms for apoptosing granulocytes.

Effects of human neutrophil elastase and Pseudomonas aen~ginosa proteinases on human respiratory epithelium

Cotton dust-mediated lung epithelial injury

The injurious effect of neutrophils on pneumocytes in vitro

Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro

ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release

Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse ceil-mediated immune functions

Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis

Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis

A novel cell culture model for studying ischemiareperfusion injury in lung transplantation

Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo

Neutrophil kinetics in man

Human CD14 mediates recognition and phagocytosis of apoptotic cells

Evaluation of PAF antagonists using human neutrophils in a microtiter plate assay

Cytokinemediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation

Asbestos fibre length-dependent detachment injury to alveolar epithelial cells in vitro: Role of a fibronectin-binding receptor

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

Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque

Previous uptake of apoptotic neutrophils or ligation of integrin receptors downmodulates the ability of macrophages to ingest apoptotic neutrophils

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF

A receptor for phosphatidylserine-specific clearance of apoptotic cells

Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury

Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action

A comparison of alpha 1-proteinase inhibitor me-thoxysuccinyI-Ala-Ala-Pro-Val-chloromethylketone and specific beta-lactam inhibitors in an acute model of human polymorphonuclear leukocyte elastase-induced tung hemorrhage in the hamster

Dransfield I. Molecular characterization of the surface of apoptotic neutrophils: Implications for functional downregulation and recognition by phagocytes

Granulocyte apoptosis and its role in the resolution and control of lung inflammation

Impaired alveolar macrophage function in smoke inhalation injury

Neutrophil adherence to lung epithelial cells induce interleukin-8 release

Differential patterns of apoptosis in resolving and nonresolving bacterial pneumonia

Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis

Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Lipopolysaccharide enhances substance Pmediated neutrophil adherence to epithelial cells and cytokine release

Endogenous nitric oxide inhibits neutrophil adherence to lung epithelial cells to modulate interleukin-8 release

Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules

Neutrophil apoptosis in the acute respiratory distress syndrome

Effects of Pseudomonas aeruginosa rhamnolipids on human monocyte-derived macrophages

Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo

In vivo suppression of immune complex-induced alveolitis by secretory leukopmteinase inhibitor and tissue inhibitor of metalloproteinases 2

Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial ceil line

Direct cytotoxicity of polymorphonuclear leukocyte granule proteins to human lung-derived cells and endothelial cells

The cell biology ofleukocyte-mediated proteolysis

Mechanisms of neutrophil damage to human alveolar extracellular matrix: The role of serine and metalloproteases

Alveolar macrophages of children suffering from recurrent infections of respiratory tract are less efficient in eliminating apoptotic neutrophils

CD18 adhesion blockade decreases bacterial clearance and neutrophil recruitment after intrapulmonary E. coli, but not after S. aureu~

Effects of cystic fibrosis airway secretions on rat lung: Role of neutrophil elastase

Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis

Cathepsin G activates protease-activated receptor-4 in human platelets

Vitronectin receptor-mediated phagocytosis of ceils undergoing apoptosis

Glomerular mesangial cells and inflammatory macrophages ingest neutrophils undergoing apoptosis

Phagocytosis of aged human neutrophils by macrophages is mediated by a novel 'charge-sensitive' recognition mechanism

Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages

Calpain and calpastatin regulate neutrophil apoptosis

Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G

Effect ofneutrophil serine proteinases and defensins on lung epithelial cells: Modulation of cytotoxicity and IL-8 production

A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V

Convery CM. Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils

Cross-linking ofCD18 in human neutrophils induces an increase ofintracellular free Ca 2+, exocytosis of azurophilic granules, quantitative up-regulation of CD 18, shedding of Lselectin, and actin polymerization

Neutrophils induce damage to respiratory epithelial cells infected with respiratory syncytiaI virus

Haslett C. Impairment of function in aging neutrophils is associated with apoptosis

The authors would like to thank the National Science