Limits...
Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

van 't Wout EF, Dickens JA, van Schadewijk A, Haq I, Kwok HF, Ordóñez A, Murphy G, Stolk J, Lomas DA, Hiemstra PS, Marciniak SJ - Hum. Mol. Genet. (2013)

Bottom Line: Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling.Moreover, the mechanism of NF-κB activation has not yet been elucidated.Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

Show MeSH

Related in: MedlinePlus

Z α1-antitrypsin expression enhances NF-κB signalling in lung epithelial cells. (A) Schematic diagram of culturing primary bronchial epithelial cells at an air (apical)–liquid (basal) interface. Once differentiated, epithelium is a pseudo-stratified cell layer composed of ciliated cells, goblet cells and basal cells. (B) Meso scale discovery® of apically and basally secreted cytokines and chemokines (IL-8, IL-6, TNFα, IL-1β, MCP-1 and IP-10). Cells were treated with oncostatin M (100 ng/ml; OSM) and TNFα/IL1β (both 20 ng/ml; OSM-mix) as indicated for 48 h before harvesting apical washes and basal medium (mean, n = 6). (C) NF-κB luciferase activity of undifferentiated MM and ZZ cells. Submerged cells were cultured for 24 h and then transfected with luciferase reporters for 6 h and left 16 h with OSM or TNFα (20 ng/ml) as indicated. NF-κB reporter activity is corrected for Renilla (mean, n = 3–4). (D) Basal IL-8 mRNA expression levels of undifferentiated primary bronchial epithelial cells measured by qPCR (mean, n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus—with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4007119&req=5

DDT487F1: Z α1-antitrypsin expression enhances NF-κB signalling in lung epithelial cells. (A) Schematic diagram of culturing primary bronchial epithelial cells at an air (apical)–liquid (basal) interface. Once differentiated, epithelium is a pseudo-stratified cell layer composed of ciliated cells, goblet cells and basal cells. (B) Meso scale discovery® of apically and basally secreted cytokines and chemokines (IL-8, IL-6, TNFα, IL-1β, MCP-1 and IP-10). Cells were treated with oncostatin M (100 ng/ml; OSM) and TNFα/IL1β (both 20 ng/ml; OSM-mix) as indicated for 48 h before harvesting apical washes and basal medium (mean, n = 6). (C) NF-κB luciferase activity of undifferentiated MM and ZZ cells. Submerged cells were cultured for 24 h and then transfected with luciferase reporters for 6 h and left 16 h with OSM or TNFα (20 ng/ml) as indicated. NF-κB reporter activity is corrected for Renilla (mean, n = 3–4). (D) Basal IL-8 mRNA expression levels of undifferentiated primary bronchial epithelial cells measured by qPCR (mean, n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus—with a two-way repeated-measurements ANOVA (Bonferroni post hoc).

Mentions: It is well-recognized that overexpression of Z α1-antitrypsin activates the NF-κB response leading to pro-inflammatory cytokine release (20,21,30). We therefore asked whether expression of Z α1-antitrypsin regulated by its endogenous promoter in airway epithelial cells could also activate this pathway. Primary bronchial epithelial cells were differentiated into mucin-producing, ciliated epithelial cell layers (Fig. 1A), and a multiplex ELISA (Meso Scale Discovery®, Rockville, MD, USA) of apical washings (air exposed) and basal (liquid exposed) conditioned medium for IL-8, IL-6, TNFα, IL-1β, monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein-10 (IP-10) was performed (Fig. 1B). This revealed that resting ZZ-differentiated primary bronchial epithelial cells secreted more IL-8 basally when compared with MM cells (P < 0.01). After combined stimulation with oncostatin M (OSM), TNFα and IL-1β (OSM-mix), the ZZ differentiated primary bronchial epithelial cells showed significantly higher release of MCP-1 (P < 0.01), IP-10 (P < 0.05) and IL-1β (P < 0.01) compared with MM controls. The reduced secretion of IL-8 most likely reflects the known inhibitory effect of OSM on IL-1β-induced IL-8 release (31). To determine whether this enhanced release of cytokines was mediated by increased NF-κB signalling, submerged cultures of patient-derived ZZ primary bronchial epithelial cells were induced to express increased levels of α1-antitrypsin by treatment with OSM and NF-κB activity was assayed using a luciferase reporter system (Fig. 1C) (29). Low levels of basal NF-κB luciferase activity were detected in MM primary bronchial epithelial cells, whereas ZZ cells showed significantly higher activity at baseline (P < 0.05 compared with MM; Fig. 1C). When α1-antitrypsin production was increased by treatment with OSM, the NF-κB activity in ZZ primary bronchial epithelial cells increased significantly (P < 0.001) and the difference between MM and ZZ cells increased still further (P < 0.01). Stimulation with TNFα, a cytokine known to induce NF-κB activation, showed the same difference between MM and ZZ cells (P < 0.05). The same effect was seen in Tet-On A549 cells overexpressing either M or Z α1-antitrypsin (Supplementary Material, Fig. S1). To test whether the baseline difference in NF-κB activity was related to the transfection of the reporter constructs, we measured transcription of the NF-κB-dependent chemokine IL-8, which confirmed basal levels of inflammatory signalling were higher in ZZ primary bronchial epithelial cells compared with controls (P < 0.05; Fig. 1D).Figure 1.


Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

van 't Wout EF, Dickens JA, van Schadewijk A, Haq I, Kwok HF, Ordóñez A, Murphy G, Stolk J, Lomas DA, Hiemstra PS, Marciniak SJ - Hum. Mol. Genet. (2013)

Z α1-antitrypsin expression enhances NF-κB signalling in lung epithelial cells. (A) Schematic diagram of culturing primary bronchial epithelial cells at an air (apical)–liquid (basal) interface. Once differentiated, epithelium is a pseudo-stratified cell layer composed of ciliated cells, goblet cells and basal cells. (B) Meso scale discovery® of apically and basally secreted cytokines and chemokines (IL-8, IL-6, TNFα, IL-1β, MCP-1 and IP-10). Cells were treated with oncostatin M (100 ng/ml; OSM) and TNFα/IL1β (both 20 ng/ml; OSM-mix) as indicated for 48 h before harvesting apical washes and basal medium (mean, n = 6). (C) NF-κB luciferase activity of undifferentiated MM and ZZ cells. Submerged cells were cultured for 24 h and then transfected with luciferase reporters for 6 h and left 16 h with OSM or TNFα (20 ng/ml) as indicated. NF-κB reporter activity is corrected for Renilla (mean, n = 3–4). (D) Basal IL-8 mRNA expression levels of undifferentiated primary bronchial epithelial cells measured by qPCR (mean, n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus—with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007119&req=5

DDT487F1: Z α1-antitrypsin expression enhances NF-κB signalling in lung epithelial cells. (A) Schematic diagram of culturing primary bronchial epithelial cells at an air (apical)–liquid (basal) interface. Once differentiated, epithelium is a pseudo-stratified cell layer composed of ciliated cells, goblet cells and basal cells. (B) Meso scale discovery® of apically and basally secreted cytokines and chemokines (IL-8, IL-6, TNFα, IL-1β, MCP-1 and IP-10). Cells were treated with oncostatin M (100 ng/ml; OSM) and TNFα/IL1β (both 20 ng/ml; OSM-mix) as indicated for 48 h before harvesting apical washes and basal medium (mean, n = 6). (C) NF-κB luciferase activity of undifferentiated MM and ZZ cells. Submerged cells were cultured for 24 h and then transfected with luciferase reporters for 6 h and left 16 h with OSM or TNFα (20 ng/ml) as indicated. NF-κB reporter activity is corrected for Renilla (mean, n = 3–4). (D) Basal IL-8 mRNA expression levels of undifferentiated primary bronchial epithelial cells measured by qPCR (mean, n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus—with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
Mentions: It is well-recognized that overexpression of Z α1-antitrypsin activates the NF-κB response leading to pro-inflammatory cytokine release (20,21,30). We therefore asked whether expression of Z α1-antitrypsin regulated by its endogenous promoter in airway epithelial cells could also activate this pathway. Primary bronchial epithelial cells were differentiated into mucin-producing, ciliated epithelial cell layers (Fig. 1A), and a multiplex ELISA (Meso Scale Discovery®, Rockville, MD, USA) of apical washings (air exposed) and basal (liquid exposed) conditioned medium for IL-8, IL-6, TNFα, IL-1β, monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein-10 (IP-10) was performed (Fig. 1B). This revealed that resting ZZ-differentiated primary bronchial epithelial cells secreted more IL-8 basally when compared with MM cells (P < 0.01). After combined stimulation with oncostatin M (OSM), TNFα and IL-1β (OSM-mix), the ZZ differentiated primary bronchial epithelial cells showed significantly higher release of MCP-1 (P < 0.01), IP-10 (P < 0.05) and IL-1β (P < 0.01) compared with MM controls. The reduced secretion of IL-8 most likely reflects the known inhibitory effect of OSM on IL-1β-induced IL-8 release (31). To determine whether this enhanced release of cytokines was mediated by increased NF-κB signalling, submerged cultures of patient-derived ZZ primary bronchial epithelial cells were induced to express increased levels of α1-antitrypsin by treatment with OSM and NF-κB activity was assayed using a luciferase reporter system (Fig. 1C) (29). Low levels of basal NF-κB luciferase activity were detected in MM primary bronchial epithelial cells, whereas ZZ cells showed significantly higher activity at baseline (P < 0.05 compared with MM; Fig. 1C). When α1-antitrypsin production was increased by treatment with OSM, the NF-κB activity in ZZ primary bronchial epithelial cells increased significantly (P < 0.001) and the difference between MM and ZZ cells increased still further (P < 0.01). Stimulation with TNFα, a cytokine known to induce NF-κB activation, showed the same difference between MM and ZZ cells (P < 0.05). The same effect was seen in Tet-On A549 cells overexpressing either M or Z α1-antitrypsin (Supplementary Material, Fig. S1). To test whether the baseline difference in NF-κB activity was related to the transfection of the reporter constructs, we measured transcription of the NF-κB-dependent chemokine IL-8, which confirmed basal levels of inflammatory signalling were higher in ZZ primary bronchial epithelial cells compared with controls (P < 0.05; Fig. 1D).Figure 1.

Bottom Line: Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling.Moreover, the mechanism of NF-κB activation has not yet been elucidated.Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

Show MeSH
Related in: MedlinePlus