Uncontrolled asthmatics have increased FceRI+ and TGF-ß-positive MCTC mast cells and collagen VI in the alveolar parenchyma.

BACKGROUND: Asthma has been associated with increased collagen deposition in both conducting airways and alveolar parenchyma. Mast cells (MCs) are key effector cells in asthma and have the ability to affect collagen synthesis. However, the link between clinical control and changes in bronchial and alveolar MC phenotypes and specific collagens in controlled and uncontrolled asthma remains unknown. OBJECTIVE: To investigate MC phenotypes in correlation with deposition of specific collagen subtypes in patients with controlled and uncontrolled asthma as well as to healthy controls. METHODS: The tissue expression of IgE+ , FcεRI+ and TGF-ß+ MCs, as well as immunoreactivity of collagen I, III and VI, was assessed using immunohistochemistry on bronchial and transbronchial biopsies from controlled asthmatics (n = 9), uncontrolled asthmatics (n = 16) and healthy controls (n = 8). RESULTS: In the alveolar parenchyma, the total number of MCs, as well as the number of FcεRI+ MCs and pro-fibrotic TGF-ß+ MCTC, was significantly increased in uncontrolled asthma compared to both controlled asthma and healthy controls. The proportion of TGF-ß+ MCTC correlated positively to an increased immunoreactivity of alveolar collagen VI but not collagen I and III. Collagen VI was increased in the alveolar parenchyma of uncontrolled asthmatics compared to controlled asthmatics. Controlled asthmatics had an increased deposition of alveolar collagen I. In bronchi, the immunoreactivity of collagen I was increased in both controlled and uncontrolled asthmatics while collagen III was increased only in controlled asthmatics. CONCLUSIONS: Patients with uncontrolled atopic asthma have an altered pro-fibrotic MCTC phenotype in the alveolar parenchyma that is associated with alveolar collagen VI. The present data thus support distal lung mast cell and matrix changes as histopathological features of asthma that may be of particular clinical relevance in patients who have remaining symptoms despite conventional inhaler therapy.

Inducible nitric oxide synthase expression is increased in the alveolar compartment of asthmatic patients.

INTRODUCTION: Increased exhaled nitric oxide (NO) levels in asthma are suggested to be through inducible NO synthase (iNOS). The aim of this study was to investigate the expression of iNOS in bronchoalveolar lavage (BAL) cells and tissue from central and peripheral airways and compare it with the exhaled bronchial and alveolar NO levels in patients with asthma vs a control group. METHODS: Thirty-two patients with asthma (defined as controlled or uncontrolled according to Asthma Control Test score cut-off: 20) and eight healthy controls were included. Exhaled NO was measured, and alveolar concentration and bronchial flux were calculated. iNOS was measured in central and peripheral lung biopsies, as well as BAL cells. Bronchoalveolar lavage macrophages were stimulated in vitro, and iNOS expression and NO production were investigated. RESULTS: Expression of iNOS was increased in central airway tissue and the alveolar compartment in uncontrolled as compared to controlled asthmatics and healthy controls. There were no differences, however, in iNOS mRNA levels in total BAL cells in uncontrolled as compared to controlled asthma. Bronchoalveolar lavage cell mRNA levels of iNOS or iNOS expression in central and alveolar tissue did not relate to alveolar NO, nor to bronchial flux of NO. In vitro stimulation with leukotriene D4 increased iNOS mRNA levels and NO production in cultured BAL macrophages. CONCLUSION: The levels of both bronchial and alveolar iNOS are increased in uncontrolled as compared to controlled asthma. However, levels of iNOS in BAL macrophages were not reflected by alveolar NO. Both central and distal iNOS levels may reflect responsiveness to steroid treatment.

IL-33 is related to innate immune activation and sensitization to HDM in mild steroid-free asthma.

BACKGROUND: IL-33 represents a potential link between the airway epithelium and induction of a Th2-type inflammatory response in asthma. However, the association with markers of eosinophilic airway inflammation has not previously been reported in patients with steroid-free asthma. AIM: To describe the relationship between airway IL-33 and markers of eosinophilic airway inflammation, as well potential triggers of IL-33, in mild, steroid-free asthma. METHODS: IL-33 mRNA expression and IL-33 immunoreactivity were measured in bronchial biopsies from patients with asthma untreated with inhaled steroids and healthy individuals. Furthermore, fractional exhaled nitric oxide (FeNO) and eosinophils in sputum and BAL were measured, as well as airway hyperresponsiveness to mannitol and methacholine. Epithelial integrity was assessed by computerized image analysis on haematoxylin-stained sections, and TLR mRNA expression by PCR. RESULTS: A total of 23 patients with asthma and 10 healthy individuals were examined (age: 24 years (20-40); females: 53%). The level of IL-33 mRNA expression was significantly higher in patients with asthma compared to healthy individuals (Median (IQR) 1.12 (0.78) vs. 0.86, P = 0.04). There was a positive correlation between IL-33 mRNA expression and the level of FeNO (r = 0.56, P = 0.01), whereas there was no association with airway or blood eosinophils. IL-33 expression was unrelated to loss of epithelial integrity, but correlated with an increased expression of TLR2 and TLR4 (TLR2: r = 0.47, P = 0.04; TLR4: 0.68, P < 0.001), as well allergy to house dust mites (HDMs). CONCLUSION: In mild untreated asthma, the expression of IL-33 mRNA in bronchial mucosa is related to innate immune activation and allergic sensitization to HDM, rather than epithelial damage, and correlates with FeNO. These findings suggest that in mild allergic asthma, IL-33 may represent a link between innate immune activation and FeNO production.

Airway responsiveness to mannitol in asthma is associated with chymase-positive mast cells and eosinophilic airway inflammation.

BACKGROUND: Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol. METHODS: Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR. RESULTS: The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01). CONCLUSION: Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa.

Osteopontin binds and modulates functions of eosinophil-recruiting chemokines.

BACKGROUND: Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines. METHODS: Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated. RESULTS: We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24. CONCLUSIONS: The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

Mapping of TLR5 and TLR7 in central and distal human airways and identification of reduced TLR expression in severe asthma.

BACKGROUND: The toll-like receptors, TLR5 and TLR7, have recently been proposed in asthma immunopathogenesis. While supporting data come from animal or in vitro studies, little is known about TLR5 and TLR7 expression in human asthmatic airways. METHODS: Advanced immunohistochemical mapping of TLR5 and TLR7 was performed on bronchial and transbronchial biopsies from healthy individuals and patients with moderate and severe asthma. RESULTS: TLR5 was identified in multiple structural cells; bronchial epithelium, alveolar type II pneumocytes, plasma cells, macrophages and neutrophils. Contrary to bronchial TLR5, which had a basolateral expression, alveolar TLR5 had polarized apical localization. Patients with severe asthma had decreased total and epithelial TLR5 expression compared to controls and moderate asthmatics (P < 0.001). TLR7 expression was found in several structural cells and asthma-related immune cells. Whereas TLR7 expression was decreased in severe asthmatics (P < 0.001), nerve-associated TLR7 increased (P = 0.035). Within the asthma groups, both TLR5 and TLR7 expression correlated with multiple lung function parameters. CONCLUSIONS: Our results reveal broad expression patterns of TLR5 and TLR7 in the lung and that the expression is decreased in severe asthma. Hence, severe asthmatics may suffer from insufficient TLR signalling during viral or bacterial infections leading to poor and impaired defence mechanisms.

Cultured mast cells from patients with asthma and controls respond with similar sensitivity to recombinant Der p2-induced, IgE-mediated activation.

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22,275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [-0.4795 log ng/ml ± 0.092 (SE)] and controls (-0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma.

Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model.

Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1-targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required.

Alveolar mast cells shift to an FcεRI-expressing phenotype in mild atopic asthma: a novel feature in allergic asthma pathology.

BACKGROUND: A unique feature of alveolar mast cells is their low high-affinity IgE receptor (FcεRI) expression. Recent discoveries in uncontrolled asthma suggest that the appearance of FcεRI-expressing alveolar mast cells may be a novel disease-specific feature of allergic asthma. This study investigates whether increased FcεRI-expressing alveolar mast cells are present in patients with mild allergic asthma or even in non-asthmatic allergic rhinitis patients (AR) who have developed bronchial hyperactivity (BHR). METHODS: Bronchial and alveolar tissues were obtained from healthy controls, AR patients with or without BHR, and AR patients with concurrent asthma. Samples were processed for immunohistochemical identification of MC(T) and MC(TC) and expression of FcεRI and surface-bound IgE. RESULTS: Bronchial mast cell expression of FcεRI was high in all groups. In contrast, in the alveolar tissue, the expression of FcεRI on mast cells was low in healthy controls and in the AR patient groups, whereas a high expression was present in AR patients with concurrent asthma (P = 0.006 compared to controls). The asthmatics had a 29-fold increase in numbers (P = 0.006) and a 19-fold increase in proportion (P = 0.007) of alveolar mast cells that expressed surface-bound IgE. CONCLUSIONS: The present data show that alveolar mast cells in patients with mild atopic asthma, but not atopic patients with AR, have turned into a highly FcεRI- and IgE-expressing phenotype. These data support the hypothesis that increased FcεRI expression on alveolar mast cells is a novel disease-specific feature of allergic asthma that is important for understanding asthma phenotypes and designing new therapeutic strategies.

Allergic eosinophil-rich inflammation develops in lungs and airways of B cell-deficient mice.

Immunoglobulins (Ig), particularly IgE, are believed to be crucially involved in the pathogenesis of asthma and, equally, in allergic models of the disease. To validate this paradigm we examined homozygous mutant C57BL/6 mice, which are B cell deficient, lacking all Ig. Mice were immunized intraperitoneally with 10 micrograms ovalbumin (OVA) plus alum, followed by daily (day 14-20) 30 min exposures to OVA aerosol (OVA/OVA group). Three control groups were run: OVA intraperitoneally plus saline (SAL) aerosol (OVA/SAL group); saline intraperitoneally plus saline aerosol; saline intraperitoneally plus OVA aerosol (n = 6-7). Lung and large airway tissues obtained 24 h after the last OVA or SAL exposure were examined by light microscopy and transmission electron microscopy (TEM). The Ig-deficient mice receiving OVA/ OVA treatment had swollen and discolored lungs and exhibited marked eosinophilia both in large airway subepithelial tissue (49.2 +/- 12.0 cells/mm basement membrane [BM] versus OVA/ SAL control 1.2 +/- 0.3 cells/mm BM; P < 0.001), and perivascularly and peribronchially in the lung (49.3 +/- 9.0 cells/unit area versus OVA/SAL control 2.6 +/- 0.6 cells/unit area; P < 0.001). The eosinophilia extended to the regional lymph nodes. TEM confirmed the subepithelial and perivascular localization of eosinophils. Mucus cells in large airway epithelium increased from 1.5 +/- 0.8 (OVA/SAL mice) to 39.5 +/- 5.7 cells/mm BM in OVA/OVA treated mice (P < 0.001). OVA/SAL mice never differed from the other control groups. Corresponding experiments in wild-type mice (n = 6-7 in each group) showed qualitatively similar but less pronounced eosinophil and mucus cell changes. Macrophages and CD4+ T cells increased in lungs of all OVA/OVA-treated mice. Mast cell number did not differ but degranulation was detected only in OVA/OVA-treated wild-type mice. Immunization to OVA followed by OVA challenges thus cause eosinophil-rich inflammation in airways and lungs of mice without involvement of B cells and Ig.

Novel site-specific mast cell subpopulations in the human lung.

BACKGROUND: Lung mast cells are stereotypically divided into connective tissue (MC(TC)) and mucosal (MC(T)) mast cells. This study tests the hypothesis that each of these subtypes can be divided further into site-specific populations created by the microenvironment within each anatomical lung compartment. METHODS: Surgical resections and bronchial and transbronchial biopsies from non-smoking individuals were obtained to study mast cells under non-inflamed conditions. Morphometric and molecular characteristics of mast cell populations were investigated in multiple lung structures by immunohistochemistry and electron microscopy. RESULTS: MC(T) and MC(TC) coexisted in all compartments, with MC(T) being the prevailing type in bronchi, bronchioles and the alveolar parenchyma and MC(TC) being more abundant in pulmonary vessels and the pleura. Each of the MC(TC) and MC(T) phenotypes could be further differentiated into site-specific populations. MC(TC) were significantly larger in pulmonary vessels than in small airway walls, while the reverse was observed for MC(T). Within each MC(TC) and MC(T) population there were also distinct site-specific expression patterns of the IgE receptor, interleukin-9 receptor, renin, histidine decarboxylase, vascular endothelial growth factor, fibroblast growth factor, 5-lipoxygenase and leukotriene C4 synthase (eg, bronchial MC(T) consistently expressed more histidine decarboxylase than alveolar MC(T)). Renin content was high in vascular MC(TC) but markedly lower in MC(TC) in other compartments. For both MC(TC) and MC(T), the IgE receptor was highly expressed in conducting airways but virtually absent in alveolar parenchyma. CONCLUSIONS: These findings demonstrate novel site-specific subpopulations of lung MC(TC) and MC(T) at baseline conditions. This observation may have important implications in the future exploration of mast cells in a number of pulmonary diseases.

The neutrophil-recruiting chemokine GCP-2/CXCL6 is expressed in cystic fibrosis airways and retains its functional properties after binding to extracellular DNA.

Infections in cystic fibrosis (CF), often involving Pseudomonas aeruginosa, result from a dysregulated airway immunity where one hallmark is the accumulation of necrotic and apoptotic immune cells, in particular neutrophils. In addition, neutrophils actively release DNA, forming neutrophil extracellular traps (NETs) that contain antimicrobial proteins. Altogether, free DNA in complex with actin accumulates in the airway lumen, resulting in highly viscous sputum that provides an anionic matrix, binding cationic antimicrobial proteins. In this study, granulocyte chemotactic protein 2 (GCP-2)/CXCL6, a neutrophil-activating chemokine with bactericidal properties, was detected in the airway epithelium of CF patients and was also present in azurophilic and specific granules of neutrophils. Elastase of neutrophils, but not of P. aeruginosa, completely degraded CXCL6 (chemokine (C-X-C motif) ligand 6). In addition, CXCL6 colocalized with extracellular DNA in both CF sputa and in in vitro-formed NETs. In vitro, CXCL6 bound DNA with a KD of 2,500 nM. Interestingly, both the bactericidal and the receptor-activating properties of CXCL6 (against neutrophils) remained largely unaffected in the presence of DNA. However, the chemotactic properties of CXCL6 were reduced by the presence of DNA. Taken together, CXCL6 is expressed in CF, retaining its functional properties even after binding to the anionic scaffold that extracellular DNA provides in CF.

Eosinophil lysis and free granules: an in vivo paradigm for cell activation and drug development.

Release of cytotoxic granule proteins from activated eosinophil granules is considered to be a key pathogenic mechanism in eosinophilic diseases. Degenerated eosinophils and extracellular eosinophil granules have been repeatedly depicted. The present overview describes evidence that eosinophil lysis and distribution of free eosinophil granules (as opposed to 'classical degranulation') is an important mechanism by which eosinophils affect their surroundings. Here, Carl Persson and Jonas Erjefält summarize how recent reports on the induction of eosinophil lysis in vivo provide a new paradigm for eosinophil activation and thus constitute a novel basis for pharmacological manipulations in eosinophilic diseases.

The mouse trap.

Mouse models of asthma are now being used extensively in drug research. However, the successful unravelling of combinatorial interplays of cells and molecules in the murine airways may not be matched by equally successful demonstrations of an asthma-like pathophysiology. Here, Carl Persson, Jonas Erjefält, Magnus Korsgren and Frank Sundler discuss the fact that major features of asthma may still need to be demonstrated in the airways of allergic mice.

A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality.

Mucosal tissues contain large numbers of memory CD4(+) T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4(+) T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα(+)DR3(+)CD4(+) T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα(+)DR3(+)CD4(+) T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα(+)DR3(+) CD4(+) T cells may contribute to antigen-independent innate responses at barrier surfaces.

Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood.

The origins of allergic asthma, particularly in infancy, remain obscure. Respiratory viral infections and allergen sensitization in early life have been associated with asthma in young children. However, a causal link has not been established. We investigated whether an influenza A infection in early life alters immune responses to house dust mite (HDM) and promotes an asthmatic phenotype later in life. Neonatal (8-day-old) mice were infected with influenza virus and 7 days later, exposed to HDM for 3 weeks. Unlike adults, neonatal mice exposed to HDM exhibited negligible immune responsiveness to HDM, but not to influenza A. HDM responsiveness in adults was associated with distinct Ly6c+ CD11b+ inflammatory dendritic cell and CD8α+ plasmacytoid (pDC) populations that were absent in HDM-exposed infant mice, suggesting an important role in HDM-mediated inflammation. Remarkably, HDM hyporesponsiveness was overcome when exposure occurred concurrently with an acute influenza infection; young mice now displayed robust allergen-specific immunity, allergic inflammation, and lung remodeling. Remodeling persisted into early adulthood, even after prolonged discontinuation of allergen exposure and was associated with marked impairment of lung function. Our data demonstrate that allergen exposure coincident with acute viral infection in early life subverts constitutive allergen hyporesponsiveness and imprints an asthmatic phenotype in adulthood.

Direct evidence of secondary necrosis of neutrophils during intense lung inflammation.

Several pulmonary inflammatory conditions are characterised by infiltration of neutrophils. Normally, neutrophils are silently removed by apoptosis, followed by phagocytosis. However, if phagocytosis fails, apoptotic cells undergo secondary necrosis. Recent findings of increased levels of the pan-necrosis marker lactate dehydrogenase in bronchoalveolar lavage from lipopolysaccharide-exposed mice implies potential involvement of secondary necrosis. Using a similar model, this study aimed to identify the source of lactate dehydrogenase and to search for direct histological evidence of secondary necrosis. Lipopolysaccharide (LPS) was administered to the lungs of BALB/c mice, and bronchoalveolar lavage and tissue samples were collected 4, 12, 24, 36, 48, 60 and 72 h after administration. LPS induced a patchy neutrophil-rich lung inflammation, where the numbers of terminal deoxynucleotide transferase-mediated dUTP nick-end labelling-positive neutrophils were increased at 12 h and onwards. Lavage levels of neutrophils and lactate dehydrogenase increased significantly at 4 and 24 h, respectively. Detailed electron microscopic assessment of neutrophil activation and death modes revealed that up to 14% of the neutrophils were undergoing secondary necrosis, whereas apoptotic or primary necrotic structural cells were rarely found. In summary, this study provides direct evidence that secondary necrosis of neutrophils is a common process during intense lung inflammation. This implies that neutrophil apoptosis may cause rather than resolve airway inflammation.