Greg Hodge

Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells

Chronic obstructive pulmonary disease is a highly prevalent, complex disease, usually caused by cigarette smoke. It causes serious morbidity and mortality and costs the global community billions of dollars per year. While chronic inflammation, extracellular matrix destruction and increased airway epithelial cell apoptosis are reported in chronic obstructive pulmonary disease, the understanding of the basic pathogenesis of the disease is limited and there are no effective treatments. We hypothesized that the accumulation of apoptotic airway epithelial cells chronic obstructive pulmonary disease in could be due to defective phagocytic clearance by alveolar macrophages. There have been no previous studies of the phagocytic capacity of alveolar macrophages in chronic obstructive pulmonary disease using physiologically relevant apoptotic airway epithelial cells as phagocytic targets. We developed a phagocytosis assay whereby cultured 16HBE airway epithelial cells were induced to apoptosis with ultraviolet radiation and stained with mitotracker green. Alveolar macrophages from bronchoalveolar lavage from eight control and six chronic obstructive pulmonary disease subjects were analysed following 1.5 h incubation with apoptotic airway epithelial cells, then staining with macrophage marker anti CD33. CD33+/mitotracker green + events (i.e., alveolar macrophages which had phagocytosed apoptotic airway epithelial cells) were analysed using flow cytometry. Phagocytosis of polystyrene microbeads was investigated in parallel. A significantly reduced proportion of alveolar macrophages from chronic obstructive pulmonary disease subjects ingested apoptotic airway epithelial cells compared with controls (11.6 ± 4.1% for chronic obstructive pulmonary disease versus 25.6 ± 9.2% for control group). Importantly, the deficiency was not observed using polystyrene beads, suggesting that the failure to resolve epithelial damage in chronic obstructive pulmonary disease may result, at least partially, from specific defects in phagocytic ability of alveolar macrophages to ingest apoptotic airway epithelial cells.

Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation

There is heterogeneity in the propensity of smokers to develop chronic obstructive pulmonary disease (COPD), and improved treatment strategies are hindered by limited understanding of COPD pathogenesis, especially as distinct from the effects of smoking per se. Although apoptosis is essential for tissue homeostasis, increased apoptosis may cause tissue damage and inflammation. This study addressed whether airway T-lymphocytes and airway epithelial cells (AEC) show an increased likelihood of undergoing apoptosis in COPD and if this was related to smoking. Apoptosis (7-amino-actinomycin D, Annexin, single-stranded DNA and caspase), Bcl-2, Bax and p53 were assessed in cells obtained from bronchial bushing and bronchoalveolar lavage from ex- and continuing smokers with COPD, and nonsmoking controls, using flow cytometry. A mean 87% increase in apoptosis of AEC and a 103% increase in T-lymphocyte apoptosis were found in COPD. There were no significant differences in apoptosis of AEC between current and ex-smokers with COPD. Apoptosis may contribute to chronic obstructive pulmonary disease pathogenesis, and continued excess apoptosis after smoking cessation may offer a new target for therapeutic interventions. Whether the persistence of increased apoptosis after smoking cessation results from changes in the pulmonary milleau after years of noxious insult, or whether some individuals have a natural predisposition toward increased apoptosis and possible development of chronic obstructive pulmonary disease remains to be determined.

Azithromycin improves macrophage phagocytic function and expression of mannose receptor in chronic obstructive pulmonary disease.

RATIONALE Defective efferocytosis (phagocytic clearance of apoptotic cells) in the airway may perpetuate inflammation via secondary necrosis in chronic obstructive pulmonary disease (COPD). We have previously reported that low-dose azithromycin improved alveolar macrophage (AM) phagocytic function in vitro. OBJECTIVES We investigated collectins (mannose-binding lectin [MBL] and surfactant protein [SP]-D) and mannose receptor (MR) in COPD and their possible role in the azithromycin-mediated improvement in phagocytosis. METHODS In vitro effects of azithromycin on AM expression of MR were investigated. MBL, SP-D, and MR were measured in patients with COPD and control subjects. Azithromycin (250 mg orally daily for 5 d then twice weekly for 12 wk) was administered to 11 patients with COPD. Assessments included AM phagocytic ability and expression of MR, MBL, SP-D, bronchial epithelial cell apoptosis, pulmonary function, C-reactive protein, blood/BAL leukocyte counts, cytokine production, and T-cell markers of activation and phenotype. MEASUREMENTS AND MAIN RESULTS Azithomycin (500 ng/ml) increased MR expression by 50% in vitro. AM MR expression and levels of MBL and SP-D were significantly reduced in patients with COPD compared with control subjects. In patients with COPD, after azithromycin therapy, we observed significantly improved AM phagocytic ability (pre: 9.9%; post: 15.1%), reduced bronchial epithelial cell apoptosis (pre: 30.0%; post: 19.7%), and increased MR and reduced inflammatory markers in the peripheral blood. These findings implicate the MR in the defective phagocytic function of AMs in COPD and as a target for the azithromycin-mediated improvement in phagocytic ability. CONCLUSIONS Our findings indicate a novel approach to supplement existing therapies in COPD.

Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages

Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng·mL-1) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, αvβ3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease.

Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation.

Cord blood has been used successfully for stem cell transplantation in several haematological conditions: Fanconis anaemia, leukaemia and Wiskott‐Aldrich syndrome. On account of the low incidence of GVHD observed following cord blood transplantation, it has been suggested that cord blood be used for HLA‐matched, or perhaps one or two antigens mismatched, and unrelated stem cell transplantation. Based on an extensive immunophenotype‐functional correlation, we determined that cord blood contains mainly immature unprimed T lymphocytes that are predominantly suppressor cells. Recent findings suggest that dysregulated production of cytokines (IL‐1, IL‐2, TNFα) plays a role in GVHD. We showed that T cells in cord blood express receptors for IL‐2, TNFα, but no receptors for IL‐1. Similarly, NK cells, one of the effector cells of GVHD, express receptors for TNFα and γIFN but do not express receptors for IL‐1, nor IL‐2R α‐chain (CD25) although IL‐2R β‐chain is expressed. The potential for activation of T lymphocytes and NK cells therefore exists in the context of bone marrow transplantation. However, the high number of suppressor cells in cord blood most likely modulate the activation of lymphocytes and NK cells thereby minimizing GVHD.

Increased intracellular T helper 1 proinflammatory cytokine production in peripheral blood, bronchoalveolar lavage and intraepithelial T cells of COPD subjects

The role of T cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) is not yet certain, although varying reports have shown increases in T helper 1 (Th1) and/or Th2 cytokines in peripheral blood and bronchoalveolar lavage (BAL). No studies have examined cytokine production by intraepithelial T cells obtained by bronchial brushing (BB). Intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from smoker and ex‐smoker COPD patients, COPD patients receiving inhaled corticosteroids and smoker and non‐smoker control subjects was studied using multi‐parameter flow cytometry. CD4 : CD8 inversion was noted in the peripheral blood of smoker and ex‐smoker COPD groups, in BAL and BB from smoker controls and BAL of COPD smokers. There was an increase in intracellular CD8+ T cell Th1 proinflammatory cytokines in some COPD groups in the peripheral blood and in CD8+ T cell tumour necrosis factor (TNF)‐α in some COPD groups and smoker controls in BAL and BB. There was an increase in proinflammatory cytokines in COPD smokers compared with ex‐smokers and a decrease in COPD smokers receiving inhaled corticosteroids in the airways. There was a negative correlation between forced expiratory volume in 1 s (FEV1) and the percentage of BAL and intraepithelial CD8+ T cells producing TNF‐α. COPD patients exhibit systemic inflammation as evidenced by increased intracellular Th1 proinflammatory cytokines in blood, BAL and intraepithelial CD8+ T cells, whereas smoker controls showed localized Th1 response in the lung only. Systemic therapeutic targeting of TNF‐α production by CD8+ T cells may improve morbidity in COPD patients while targeting of TNF‐α in the lung may prevent smokers progressing to COPD.

Synthesis and Surface Expression of CD14 by Human Endothelial Cells

ABSTRACT Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14). By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface. Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes. Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages. Evidence for synthesis of CD14 by HUVEC was provided by the finding that l-[35S]methionine was incorporated into CD14. In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis. Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14). Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions. This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria.

Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine.

Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)-derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, FcγR1), M2 markers (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen-presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.

Increased Airway Granzyme b and Perforin in Current and Ex-Smoking COPD Subjects

Increased bronchial epithelial cell apoptosis and CD8+ T-cell numbers in the blood and airways have been reported in COPD. These cells can induce apoptosis via the granzyme-b/perforin-mediated pathway. We hypothesized that increased levels of granzyme-b/perforin would be detected in COPD, contributing to apoptosis and tissue damage. Intracellular granzyme-b/perforin were measured in blood-derived T-cells and natural killer (NK) cells from COPD subjects (30 current and 30 ex-smokers), 20 asymptomatic current-smokers and 30 never-smokers, and bronchoalveolar lavage (BAL)-derived T-cells from a cohort of these subjects using flow cytometry. Soluble granzyme-b was determined by ELISA. In blood, there was an increased percentage of T-cells expressing intracellular granzyme-b/perforin for both COPD groups but not asymptomatic smokers (versus never-smokers). Soluble granzyme-b was undetectable. In BAL, soluble granzyme-b levels and the percentage of T-cells expressing intracellular granzyme-b/perforin were increased in both COPD groups and asymptomatic smokers. There was a significant correlation between granzyme-b expression in BAL and apoptosis of bronchial epithelial cells. Most circulating NK cells expressed granzyme-b/perforin, with the median fluorescence intensity of staining increased in both COPD groups and asymptomatic smokers. Granzyme-mediated apoptosis may thus be one mechanism of lung injury in COPD. The changes that persist despite smoking cessation in COPD likely reflect pathophysiological changes in COPD as opposed to the effects of smoking per se.

Posttransplant Bronchiolitis Obliterans Syndrome Is Associated with Bronchial Epithelial to Mesenchymal Transition

Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells.