Violet Mukaro

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.

Role of increased CD8/CD28null T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease‐modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self‐maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8+ T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7‐1/CTLA4, 4‐1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co‐stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28null cells in COPD by measuring their production of proinflammatory cytokines, co‐stimulatory molecules, granzyme and perforin. A smoke‐exposed murine model was applied to investigate the relative expression of CD8/CD28null T cells in blood, lung tissue and airway. CD8/CD28null cells were increased in both current‐ and ex‐smoker COPD groups; these cells expressed significantly more interferon (IFN)‐γ, OX40, 4‐1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28+ T cells. There were no changes in CD4/CD28null T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28null T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co‐stimulatory molecules by CD8/CD28null T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.

Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response.

The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3–5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.

Increased proteinase inhibitor-9 (PI-9) and reduced granzyme B in lung cancer: Mechanism for immune evasion?

Cytotoxic CD8(+) T-cells mount immune responses to cancer via cytotoxic pathways including granzyme B. Cancer cells are also known to develop immune evasion mechanisms. We hypothesised that lung cancer cells would over-express the granzyme B-inhibitor, proteinase inhibitor-9 (PI-9) and down-regulate granzyme B expression by neighbouring CD8(+) T-cells. We investigated PI-9 expression in lung cancer cell lines, and primary lung cancer cells obtained at curative lung resection from cancer patients with/without chronic obstructive pulmonary disease (COPD). Granzyme B and PI-9 expression was also determined in CD8(+) T-cells from the cancer and non-cancer areas of resected lung tissue and from bronchoalveolar lavage (BAL). We then evaluated the effects of conditioned media from lung cancer cell lines on granzyme B expression and the cytotoxic activity of CD8(+) T-cells. PI-9 was highly expressed in lung cancer cell lines. Increased PI-9 expression was also observed in primary cancer cells vs. epithelial cells from non-cancer tissue or bronchial brushing-derived normal primary large airway epithelial cells. Expression significantly correlated with cancer stage. Significantly reduced granzyme B was noted in CD8(+) T-cells from cancer vs. non-cancer tissue. Granzyme B production by CD8(+) T-cells was reduced in the presence of conditioned media from lung cancer cell lines. Our data suggest that lung cancer cells utilise their increased PI-9 expression to protect from granzyme B-mediated cytotoxicity as an immune evasion mechanism, a function that increases with lung cancer stage.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective:  Natural killer (NK) and natural killer T (NKT)‐like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro‐inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

IntroductionWhile consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation.MethodsIn a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content.ResultsErythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content.ConclusionThe data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

Airway clearance of apoptotic cells in COPD.

Apoptosis of bronchial epithelial cells and the phagocytic clearance of these cells by alveolar macrophages (a process termed efferocytosis) are integral processes leading to repair of airway epithelial injury. Efferocytosis allows for the removal of apoptotic material with minimal inflammation and prevents the development of secondary necrosis and ongoing inflammation. Defective efferocytosis and the increased presence of apoptotic cells have been identified in the airways of subjects with chronic obstructive pulmonary disease (COPD). There are three major potential causes for this accumulation of apoptotic cells: (i) increased apoptosis per se as a result of an increase in apoptotic mediators, (ii) defects in the recognition of apoptotic cells by AM and (iii) failure to clear the unwanted cells by the process of efferocytosis. The implications of these processes in COPD and novel treatment strategies aimed at improving clearance of apoptotic cells form the focus of the present review.

Inhibition of the Lipopolysaccharide-Induced Stimulation of the Members of the MAPK Family in Human Monocytes/Macrophages by 4-Hydroxynonenal, a Product of Oxidized Omega-6 Fatty Acids

The compound 4-hydroxynonenal (4-HNE) is the major aldehyde formed during lipid peroxidation of omega-6-polyunsaturated fatty acids and has been suggested to regulate inflammatory responses because it inhibits tumor necrosis factor (TNF) mRNA production in the human monocytic cell line THP-1. Here we demonstrate that 4-HNE inhibits TNF and interleukin-1beta production in human monocytes in response to lipopolysaccharide. The main action of 4-HNE occurred at the pretranscriptional level; there was no effect on TNF mRNA production or stability when 4-HNE was added after stimulation. The mechanism of action of 4-HNE appears to be downstream of lipopolysaccharide-receptor binding. In the human monocytic MonoMac 6 cell line, 4-HNE caused selective inhibition of the activity of the mitogen-activated protein kinases p38 and ERK1/ERK2, but not JNK. However, in monocytes, the activities of all three kinases were inhibited, suggesting that the effects of 4-HNE were exerted at points upstream of ERK1/ERK2 and JNK as the levels of the phosphorylated kinases were reduced. In contrast, p38 phosphorylation was not inhibited, suggesting that 4-HNE affects kinase activity. 4-HNE also inhibited nuclear factor-kappaB activation in monocytes. In view of the roles of p38, ERK1/ERK2, JNK, and nuclear factor-kappaB in inflammation, the data suggest that 4-HNE, at nontoxic concentrations, has anti-inflammatory properties, most likely through an effect on these signaling molecules, and could lead to the development of novel treatments for inflammatory diseases.

Lectins Offer New Perspectives in the Development of Macrophage-Targeted Therapies for COPD/Emphysema

We have previously shown that the defective ability of alveolar macrophages (AM) to phagocytose apoptotic cells (‘efferocytosis’) in chronic obstructive pulmonary disease/emphysema (COPD) could be therapeutically improved using the C-type lectin, mannose binding lectin (MBL), although the exact mechanisms underlying this effect are unknown. An S-type lectin, galectin-3, is also known to regulate macrophage phenotype and function, via interaction with its receptor CD98. We hypothesized that defective expression of galectin/CD98 would be associated with defective efferocytosis in COPD and that mechanisms would include effects on cytoskeletal remodeling and macrophage phenotype and glutathione (GSH) availability. Galectin-3 was measured by ELISA in BAL from controls, smokers and current/ex-smokers with COPD. CD98 was measured on AM using flow cytometry. We assessed the effects of galectin-3 on efferocytosis, CD98, GSH, actin polymerisation, rac activation, and the involvement of PI3K (using β-actin probing and wortmannin inhibition) in vitro using human AM and/or MH-S macrophage cell line. Significant decreases in BAL galectin-3 and AM CD98 were observed in BAL from both current- and ex-smoker COPD subjects vs controls. Galectin 3 increased efferocytosis via an increase in active GTP bound Rac1. This was confirmed with β-actin probing and the role of PI3K was confirmed using wortmannin inhibition. The increased efferocytosis was associated with increases in available glutathione and expression of CD98. We provide evidence for a role of airway lectins in the failed efferocytosis in COPD, supporting their further investigation as potential macrophage-targeted therapies.