CORRESPONDENCE 6. Sugimura K, Fukumoto Y, Satoh K, Nochioka K, Miura Y, Aoki T, Tatebe S, Miyamichi-Yamamoto S, Shimokawa H. Percutaneous transluminal pulmonary angioplasty markedly improves pulmonary hemodynamics and long-term prognosis in patients with chronic thromboembolic pulmonary hypertension. Circ J 2012;76:485–488. 7. Andreassen AK, Ragnarsson A, Gude E, Geiran O, Andersen R. Balloon pulmonary angioplasty in patients with inoperable chronic thromboembolic pulmonary hypertension. Heart 2013;99:1415–1420. 8. D’Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT, et al. Survival in patients with primary pulmonary hypertension: results from a national prospective registry. Ann Intern Med 1991;115:343–349. 9. Kim NH, Delcroix M, Jenkins DP, Channick R, Dartevelle P, Jansa P, Lang I, Madani MM, Ogino H, Pengo V, et al. Chronic thromboembolic pulmonary hypertension. J Am Coll Cardiol 2013;62(25 Suppl):D92–D99.
Copyright © 2014 by the American Thoracic Society
Abnormalities in Airway Epithelial Junction Formation in Chronic Obstructive Pulmonary Disease To the Editor: Chronic obstructive pulmonary disease (COPD) is a life-threatening disease with an ongoing increase in mortality worldwide. COPD is characterized by airway inflammation, irreversible airway obstruction, and accelerated lung function decline. Cigarette smoking is the major risk factor of COPD. After inhalation, the
airway epithelium is the first line of defense to cigarette smoke. Epithelial barrier function is maintained by the formation of tight junctions, composed of the proteins occludin and claudins 1–5 (1), which are anchored to the cytoskeleton by zona occludens (ZO)-1. Cigarette smoking induces down-regulation of mRNA expression of junctional proteins in airway epithelium (2, 3) and increases mucosal permeability in vivo (4). In addition, we previously demonstrated that cigarette smoke transiently impairs epithelial barrier function in vitro by disruption of occludin and ZO-1 expression (5). To our knowledge, it has not been studied whether the junctional expression of these proteins differs between airway epithelial cells from patients with COPD and healthy individuals, and whether a disease-related defect exists. Pathway analysis with genome-wide gene expression data from our group shows that lung function improvement after inhaled corticosteroid treatment in COPD is associated with up-regulation of genes enriched for epithelial barrier function (6). This suggests that loss of epithelial barrier function is related to lung function decline in COPD. It is currently unknown whether differentiated mucociliary airway epithelium from patients with COPD displays intrinsic abnormalities in tight junction formation. We therefore compared primary bronchial epithelial cells (PBECs) obtained by protease digestion (7) from transplant recipient lungs of six ex-smoking patients with GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage IV COPD (8) (Table 1) with PBECs from non-COPD lungs of eight transplant donors, three being reported to be nonsmokers, and from the others no data were available.
Table 1: Characteristics of Subjects
Subject COPD 1 COPD 2 COPD 3 COPD 4 COPD 5 COPD 6 COPD 7* COPD 8* COPD 9* COPD 10* COPD 11* COPD 12* Control smoker Control smoker Control smoker Control smoker Control smoker Nonsmoker 1* Nonsmoker 2* Nonsmoker 3* Nonsmoker 4* Nonsmoker 5* Nonsmoker 6*
1* 2* 3* 4* 5*
Age (yr)
Sex
Smoking Status (Years of Cessation)
Pack-Years
FEV1%pred
FEV1/FVC%
58 55 61 62 55 49 64 64 55 69 67 71 65 55 52 43 70 65 73 44 58 37 57
M F F M F M F F F F F F M M M F M M M M M F M
Ex (2) Ex (9) Ex (24) Ex (26) Ex (9) Ex (7) Ex (12) Ex (8) Ex (5) Ex (7) Ex (3) Ex (ND) Current Current Current Current Current Non Non Non Non Non Non
35 72 35 18 18 11 35 45 20 25 30 ND 11 30 35 18 50 0 0 0 0 0 0
16 14 19 25 19 20 28 40 26 25 32 30 105 108 100 98 117 111 129 111 89 103 122
18 29 23 27 52 22 37 36 25 39 50 35 78 74 75 77 68 75 82 75 69 77 78
Definition of abbreviations: COPD = chronic obstructive pulmonary disease; Ex = ex-smoker; F = female; FEV1%pred = forced expiratory volume during the first second as percentage of predicted; M = male; ND = not determined; Non = nonsmoker. *Cells derived by bronchial brushing. Supported by the Dutch Royal Academy of Sciences (KNAW).
Correspondence
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CORRESPONDENCE
Figure 1. (See figure legend on following page)
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American Journal of Respiratory and Critical Care Medicine Volume 189 Number 11 | June 1 2014
CORRESPONDENCE PBECs were cultured at the air–liquid interface (ALI) to induce mucociliary differentiation (9), reflecting bronchial epithelium in vivo. To assess tight junction integrity, we analyzed junctional localization of occludin and ZO-1 by immunofluorescence. In addition, we studied barrier function by conventional transepithelial electrical resistance (TER) measurements and electrical cell–substrate impedance sensing (ECIS) (10, 11). Finally, we studied the integrity of bronchial epithelium in lung sections from the same patients with COPD and from non-COPD control subjects by immunohistochemical analysis of occludin and ZO-1. In control-derived ALI cultures, ZO-1 was organized to the membrane throughout the insert, mainly bordering individual cells. A comparable pattern was present in COPD-derived cultures (Table 1, COPD patients 1–6), although areas with discontinuous ZO-1 staining were more frequently observed (Figure 1A). Similar results were observed for occludin (Figure 1A). Importantly, the extent of tight junction disruption was not related to years of smoking cessation. Western blotting revealed that the total levels of ZO-1 and occludin were not significantly different between the groups (Figure 1B), suggesting that their junctional localization, but not transcriptional regulation, is disrupted in COPD-derived cultures. This was not accompanied by significantly lower resistance levels in COPD-derived cultures, as assessed by conventional TER measurement (Figure 1C). In addition, we assessed epithelial permeability as a measure of tight junction function, using fluorescein isothiocyanate–labeled dextran (12) in ALI cultures from the same subjects. Although permeability related well to TER values (Figure 1D), no significant difference was observed between COPD and control-derived cultures. It is worth noting, however, that TER measurements are relatively insensitive to changes in cell–cell contacts compared with ECIS measurements, enabling accurate real-time resistance monitoring (10). Because ECIS does not allow measurements in ALI-cultured cells on Transwell membranes, we used submerged epithelial cultures (13) derived by bronchial brushings in additional patients with COPD (Table 1, patients 7–12). We also included PBECs derived by bronchial brushings from six nonsmoking and five smoking age-matched control subjects (Table 1). PBECs derived by bronchial brushings did not successfully differentiate during ALI
culture, although during submerged culture these cells displayed similar growth rates as cells derived from lung tissue by enzymatic treatment (data not shown). Depriving cells of the growth factors bovine pituitary extract, epidermal growth factor, epinephrine, hydrocortisone, retinoic acid, and triiodothyronine resulted in a marked increase in epithelial resistance in control-derived cultures (Figure 1E), indicating cell–cell contact formation (10). Importantly, this was hardly observed in COPD-derived cultures, which displayed significantly lower resistance levels than cultures from nonsmokers and smoking control subjects (Figure 1E), further reflecting an intrinsic inefficiency to form cell–cell contacts. We did not observe differences in mRNA expression of basal epithelial markers cytokeratin-5 and p63, or in the differentiated epithelial cell marker cytokeratin-18, as assessed by quantitative polymerase chain reaction (TaqMan) after 2-week ALI culture (data not shown), indicating that the inefficient tight junction formation in COPD epithelium is not likely related to a more basal cell phenotype, as has been suggested for asthma (14). We also studied whether the integrity of epithelial junctions is similarly compromised in situ in the airways of patients with COPD. We compared the localization and expression of junctional proteins in lung sections from the same patients with COPD included for bronchial epithelial cell isolation (Table 1, patients 1–6) and non-COPD control subjects. ZO-1 was frequently localized at the brush border of ciliated columnar bronchial epithelium in the lungs of non-COPD control subjects, with moderate to intense staining (Figure 1F). Bronchial epithelium from patients with COPD showed weaker intensity of ZO-1 with a less continuous staining pattern at the brush border than control epithelium (Figure 1F). Occludin was expressed more strongly throughout the epithelium, with less pronounced apical localization. Similar to ZO-1, weaker expression of occludin was observed in patients with COPD, with reduced membrane localization (Figure 1F). These data indicate that bronchial epithelium from ex-smoking patients with severe COPD displays structural changes with respect to the integrity of cell–cell contacts. Together, our data indicate that cultured bronchial epithelial cells from ex-smoking patients with COPD display abnormalities
Figure 1. Tight junctions are disrupted in the bronchial epithelium of patients with chronic obstructive pulmonary disease (COPD). (A) Primary bronchial epithelial cells of patients with COPD (open symbols, n = 6) and non-COPD control subjects (solid symbols, n = 8) were cultured at the air–liquid interface (ALI; 2 wk), fixed with acetone, and analyzed for zona occludens (ZO)-1 (red), occludin (green), and 49,6-diamidino-2-phenylindole (blue) by immunofluorescence. Representative images are shown. The staining intensity and localization were graded blindly and independently by two researchers (means) as follows: 0, absence of staining; 1, weak staining or moderate staining with no or discontinued staining at the brush border/disrupted surrounding of the cells; 2, moderate staining or intense staining with no or discontinued staining at the brush border/disrupted surrounding of the cells; 3, intense or very intense staining with occasional discontinued staining at the brush border/disrupted surrounding of the cells; 4, very intense staining mainly at the brush border/cell junctions. Medians are indicated. (B) Total cell lysates were prepared, and ZO-1 and occludin were detected by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Densitometry was performed, and levels of ZO-1 and occludin were related to GAPDH and the ratios (medians) are shown. (C) Transepithelial electrical resistance (mean 6 SEM) was measured weekly in ALI-derived cultures from patients with COPD (n = 5) and control subjects (n = 6). (D) Permeability of cultures to 4-kD fluorescein isothiocyanate (FITC)–dextran (2 mg/ml) applied to the apical surface of cultures and incubated for 24 hours was compared in ALI-derived cultures from the same patients with COPD (n = 5) and control subjects (n = 6). Values of all individual wells related to the transepithelial electrical resistance levels and mean levels per subject are shown (horizontal bar indicates median). (E ) Primary bronchial epithelial cells from patients with COPD (n = 6), nonsmoking control subjects (n = 6), and smoking control subjects (n = 5) were grown submerged to confluence in electrical cell–substrate impedance sensing (ECIS) arrays for 3 days and growth factor deprived for 24 hours. Resistance values (mean 6 SEM) were measured at 400 Hz by ECIS. (F ) Airway sections from patients with COPD (n = 6) and non-COPD subjects (n = 6 or 7) were analyzed for ZO-1 (frozen sections) and occludin (paraffin sections) by immunohistochemical staining. Representative images are shown. The expression of ZO-1 and occludin was analyzed and graded as described for A and medians are indicated. Significance was tested by the Mann–Whitney U test and repeated measures analysis of variance for ECIS experiments.
Correspondence
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CORRESPONDENCE with reduced capacity to form epithelial junctions during ALI differentiation in vitro. Of importance, this cannot merely be attributed to cigarette smoking, but rather appears to be a diseaserelated effect. Disrupted expression of tight junction proteins was also observed in lung sections of the same patients with COPD, indicating that the disruption of epithelial barrier as previously observed on cigarette smoking is persistent on smoking cessation. This may be due to the compromised ability of the bronchial epithelium of patients with COPD to (re)constitute cell–cell contacts and to redifferentiate into a functionally intact epithelium upon damage, for example, by cigarette smoking. Upon epithelial differentiation, efficient tight junction assembly may be crucial for the formation of a polarized epithelial layer (15). Tight junctions regulate paracellular permeability and functionally segregate the basolateral from the apical compartment, which is a requirement for full epithelial polarization and epithelial homeostasis (16). Loss of epithelial integrity may facilitate transepithelial crossing of noxious gases as well as pathogens. This may have important implications, because viral and bacterial infections have been implicated in nearly 50% of COPD exacerbations (17). Therefore, there is a great need to further investigate the mechanisms underlying impaired epithelial barrier function to improve mucosal barrier function in COPD. This may improve insight in strategies to improve barrier function in COPD, a promising novel target in the treatment of COPD. n Author disclosures are available with the text of this letter at www.atsjournals.org. Irene H. Heijink, Ph.D. Jacobien A. Noordhoek, B.Sc. Wim Timens, M.D. Antoon J. M. van Oosterhout, Ph.D. Dirkje S. Postma, M.D. University of Groningen Groningen, The Netherlands
Copyright © 2014 by the American Thoracic Society
Our Enlightened Understanding of the Risks of Persistent Delirium
References 1. Holgate ST. Epithelium dysfunction in asthma. J Allergy Clin Immunol 2007;120:1233–1244, quiz 1245–1246. 2. Shaykhiev R, Otaki F, Bonsu P, Dang DT, Teater M, Strulovici-Barel Y, Salit J, Harvey BG, Crystal RG. Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo. Cell Mol Life Sci 2011;68:877–892. 3. Milara J, Peiro´ T, Serrano A, Cortijo J. Epithelial to mesenchymal transition is increased in patients with COPD and induced by cigarette smoke. Thorax 2013;68:410–420. 4. Hogg JC. Bronchial mucosal permeability and its relationship to airways hyperreactivity. Eur J Respir Dis Suppl 1982;122:17–22. 5. Heijink IH, Brandenburg SM, Postma DS, van Oosterhout AJ. Cigarette smoke impairs airway epithelial barrier function and cell–cell contact recovery. Eur Respir J 2012;39:419–428. 6. van den Berge M, Steiling K, Timens W, Hiemstra PS, Sterk PJ, Heijink IH, Liu G, Alekseyev YO, Lenburg ME, Spira A, et al. Airway gene expression in COPD is dynamic with inhaled corticosteroid treatment and reflects biological pathways associated with disease activity. Thorax 2014;69:14–23. 7. Hackett TL, Shaheen F, Johnson A, Wadsworth S, Pechkovsky DV, Jacoby DB, Kicic A, Stick SM, Knight DA. Characterization of side population cells from human airway epithelium. Stem Cells 2008;26: 2576–2585. 8. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS; GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease.
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NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 2001;163: 1256–1276. 9. Heijink IH, Postma DS, Noordhoek JA, Broekema M, Kapus A. House dust mite–promoted epithelial-to-mesenchymal transition in human bronchial epithelium. Am J Respir Cell Mol Biol 2010;42:69–79. 10. Heijink IH, Brandenburg SM, Noordhoek JA, Postma DS, Slebos DJ, van Oosterhout AJ. Characterisation of cell adhesion in airway epithelial cell types using electric cell–substrate impedance sensing. Eur Respir J 2010;35:894–903. 11. Wegener J, Keese CR, Giaever I. Electric cell–substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 2000;259: 158–166. 12. Xiao C, Puddicombe SM, Field S, Haywood J, Broughton-Head V, Puxeddu I, Haitchi HM, Vernon-Wilson E, Sammut D, Bedke N, et al. Defective epithelial barrier function in asthma. J Allergy Clin Immunol 2011;128:549–556, e1–e12. 13. Heijink I, van Oosterhout A, Kliphuis N, Jonker M, Hoffmann R, Telenga E, Klooster K, Slebos DJ, ten Hacken N, Postma D, et al. Oxidantinduced corticosteroid unresponsiveness in human bronchial epithelial cells. Thorax 2014;69:5–13. 14. Hackett TL, Singhera GK, Shaheen F, Hayden P, Jackson GR, Hegele RG, Van ES, Bai TR, Dorscheid DR, Knight DA. Intrinsic phenotypic differences of asthmatic epithelium and its inflammatory responses to RSV and air pollution. Am J Respir Cell Mol Biol 2011;45: 1090–1100. 15. Kubota H, Chiba H, Takakuwa Y, Osanai M, Tobioka H, Kohama G, Mori M, Sawada N. Retinoid X receptor a and retinoic acid receptor g mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation. Exp Cell Res 2001;263:163–172. 16. Dragsten PR, Handler JS, Blumenthal R. Asymmetry in epithelial cells: is the tight junction a barrier to lateral diffusion in the plasma membrane? Prog Clin Biol Res 1982;91:525–536. 17. Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2355–2365.
To the Editor: Patel and colleagues (1) add a very important new piece to the delirium literature puzzle, which begs two important questions. First, what is the prognostic distinction between a small but important subgroup of patients manifesting rapidly reversible delirium (RRD, n = 12, 12%), defined as delirium symptoms that resolved within 2 hours after cessation of sedative/analgesic medications (often called spontaneous awakening trials [SATs]) versus the much larger group of patients in whom delirium persisted beyond 2 hours of an SAT (comprising persistent delirium [PD] and mixed delirium [MxD], n = 75, 74%)? Consider how many times every day you have patients whose brains remain delirious (“inattentive”) 2 hours after stopping sedation. This simple finding, which was present in three out of every four patients, portended a threefold higher likelihood of death (1-yr mortality of 66% for PD vs. 20% for RRD). In fact, each additional day of “persistent delirium” (again, delirium just 2 or more hours after SAT) was associated with a 14% increased risk of death at 1 year, a risk 4% higher per day than 10% indicated in previous investigations (2, 3). This strengthens our understanding of the
American Journal of Respiratory and Critical Care Medicine Volume 189 Number 11 | June 1 2014
Copyright © 2014 by the American Thoracic Society
Abnormalities in Airway Epithelial Junction Formation in Chronic Obstructive Pulmonary Disease To the Editor: Chronic obstructive pulmonary disease (COPD) is a life-threatening disease with an ongoing increase in mortality worldwide. COPD is characterized by airway inflammation, irreversible airway obstruction, and accelerated lung function decline. Cigarette smoking is the major risk factor of COPD. After inhalation, the
airway epithelium is the first line of defense to cigarette smoke. Epithelial barrier function is maintained by the formation of tight junctions, composed of the proteins occludin and claudins 1–5 (1), which are anchored to the cytoskeleton by zona occludens (ZO)-1. Cigarette smoking induces down-regulation of mRNA expression of junctional proteins in airway epithelium (2, 3) and increases mucosal permeability in vivo (4). In addition, we previously demonstrated that cigarette smoke transiently impairs epithelial barrier function in vitro by disruption of occludin and ZO-1 expression (5). To our knowledge, it has not been studied whether the junctional expression of these proteins differs between airway epithelial cells from patients with COPD and healthy individuals, and whether a disease-related defect exists. Pathway analysis with genome-wide gene expression data from our group shows that lung function improvement after inhaled corticosteroid treatment in COPD is associated with up-regulation of genes enriched for epithelial barrier function (6). This suggests that loss of epithelial barrier function is related to lung function decline in COPD. It is currently unknown whether differentiated mucociliary airway epithelium from patients with COPD displays intrinsic abnormalities in tight junction formation. We therefore compared primary bronchial epithelial cells (PBECs) obtained by protease digestion (7) from transplant recipient lungs of six ex-smoking patients with GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage IV COPD (8) (Table 1) with PBECs from non-COPD lungs of eight transplant donors, three being reported to be nonsmokers, and from the others no data were available.
Table 1: Characteristics of Subjects
Subject COPD 1 COPD 2 COPD 3 COPD 4 COPD 5 COPD 6 COPD 7* COPD 8* COPD 9* COPD 10* COPD 11* COPD 12* Control smoker Control smoker Control smoker Control smoker Control smoker Nonsmoker 1* Nonsmoker 2* Nonsmoker 3* Nonsmoker 4* Nonsmoker 5* Nonsmoker 6*
1* 2* 3* 4* 5*
Age (yr)
Sex
Smoking Status (Years of Cessation)
Pack-Years
FEV1%pred
FEV1/FVC%
58 55 61 62 55 49 64 64 55 69 67 71 65 55 52 43 70 65 73 44 58 37 57
M F F M F M F F F F F F M M M F M M M M M F M
Ex (2) Ex (9) Ex (24) Ex (26) Ex (9) Ex (7) Ex (12) Ex (8) Ex (5) Ex (7) Ex (3) Ex (ND) Current Current Current Current Current Non Non Non Non Non Non
35 72 35 18 18 11 35 45 20 25 30 ND 11 30 35 18 50 0 0 0 0 0 0
16 14 19 25 19 20 28 40 26 25 32 30 105 108 100 98 117 111 129 111 89 103 122
18 29 23 27 52 22 37 36 25 39 50 35 78 74 75 77 68 75 82 75 69 77 78
Definition of abbreviations: COPD = chronic obstructive pulmonary disease; Ex = ex-smoker; F = female; FEV1%pred = forced expiratory volume during the first second as percentage of predicted; M = male; ND = not determined; Non = nonsmoker. *Cells derived by bronchial brushing. Supported by the Dutch Royal Academy of Sciences (KNAW).
Correspondence
1439
CORRESPONDENCE
Figure 1. (See figure legend on following page)
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American Journal of Respiratory and Critical Care Medicine Volume 189 Number 11 | June 1 2014
CORRESPONDENCE PBECs were cultured at the air–liquid interface (ALI) to induce mucociliary differentiation (9), reflecting bronchial epithelium in vivo. To assess tight junction integrity, we analyzed junctional localization of occludin and ZO-1 by immunofluorescence. In addition, we studied barrier function by conventional transepithelial electrical resistance (TER) measurements and electrical cell–substrate impedance sensing (ECIS) (10, 11). Finally, we studied the integrity of bronchial epithelium in lung sections from the same patients with COPD and from non-COPD control subjects by immunohistochemical analysis of occludin and ZO-1. In control-derived ALI cultures, ZO-1 was organized to the membrane throughout the insert, mainly bordering individual cells. A comparable pattern was present in COPD-derived cultures (Table 1, COPD patients 1–6), although areas with discontinuous ZO-1 staining were more frequently observed (Figure 1A). Similar results were observed for occludin (Figure 1A). Importantly, the extent of tight junction disruption was not related to years of smoking cessation. Western blotting revealed that the total levels of ZO-1 and occludin were not significantly different between the groups (Figure 1B), suggesting that their junctional localization, but not transcriptional regulation, is disrupted in COPD-derived cultures. This was not accompanied by significantly lower resistance levels in COPD-derived cultures, as assessed by conventional TER measurement (Figure 1C). In addition, we assessed epithelial permeability as a measure of tight junction function, using fluorescein isothiocyanate–labeled dextran (12) in ALI cultures from the same subjects. Although permeability related well to TER values (Figure 1D), no significant difference was observed between COPD and control-derived cultures. It is worth noting, however, that TER measurements are relatively insensitive to changes in cell–cell contacts compared with ECIS measurements, enabling accurate real-time resistance monitoring (10). Because ECIS does not allow measurements in ALI-cultured cells on Transwell membranes, we used submerged epithelial cultures (13) derived by bronchial brushings in additional patients with COPD (Table 1, patients 7–12). We also included PBECs derived by bronchial brushings from six nonsmoking and five smoking age-matched control subjects (Table 1). PBECs derived by bronchial brushings did not successfully differentiate during ALI
culture, although during submerged culture these cells displayed similar growth rates as cells derived from lung tissue by enzymatic treatment (data not shown). Depriving cells of the growth factors bovine pituitary extract, epidermal growth factor, epinephrine, hydrocortisone, retinoic acid, and triiodothyronine resulted in a marked increase in epithelial resistance in control-derived cultures (Figure 1E), indicating cell–cell contact formation (10). Importantly, this was hardly observed in COPD-derived cultures, which displayed significantly lower resistance levels than cultures from nonsmokers and smoking control subjects (Figure 1E), further reflecting an intrinsic inefficiency to form cell–cell contacts. We did not observe differences in mRNA expression of basal epithelial markers cytokeratin-5 and p63, or in the differentiated epithelial cell marker cytokeratin-18, as assessed by quantitative polymerase chain reaction (TaqMan) after 2-week ALI culture (data not shown), indicating that the inefficient tight junction formation in COPD epithelium is not likely related to a more basal cell phenotype, as has been suggested for asthma (14). We also studied whether the integrity of epithelial junctions is similarly compromised in situ in the airways of patients with COPD. We compared the localization and expression of junctional proteins in lung sections from the same patients with COPD included for bronchial epithelial cell isolation (Table 1, patients 1–6) and non-COPD control subjects. ZO-1 was frequently localized at the brush border of ciliated columnar bronchial epithelium in the lungs of non-COPD control subjects, with moderate to intense staining (Figure 1F). Bronchial epithelium from patients with COPD showed weaker intensity of ZO-1 with a less continuous staining pattern at the brush border than control epithelium (Figure 1F). Occludin was expressed more strongly throughout the epithelium, with less pronounced apical localization. Similar to ZO-1, weaker expression of occludin was observed in patients with COPD, with reduced membrane localization (Figure 1F). These data indicate that bronchial epithelium from ex-smoking patients with severe COPD displays structural changes with respect to the integrity of cell–cell contacts. Together, our data indicate that cultured bronchial epithelial cells from ex-smoking patients with COPD display abnormalities
Figure 1. Tight junctions are disrupted in the bronchial epithelium of patients with chronic obstructive pulmonary disease (COPD). (A) Primary bronchial epithelial cells of patients with COPD (open symbols, n = 6) and non-COPD control subjects (solid symbols, n = 8) were cultured at the air–liquid interface (ALI; 2 wk), fixed with acetone, and analyzed for zona occludens (ZO)-1 (red), occludin (green), and 49,6-diamidino-2-phenylindole (blue) by immunofluorescence. Representative images are shown. The staining intensity and localization were graded blindly and independently by two researchers (means) as follows: 0, absence of staining; 1, weak staining or moderate staining with no or discontinued staining at the brush border/disrupted surrounding of the cells; 2, moderate staining or intense staining with no or discontinued staining at the brush border/disrupted surrounding of the cells; 3, intense or very intense staining with occasional discontinued staining at the brush border/disrupted surrounding of the cells; 4, very intense staining mainly at the brush border/cell junctions. Medians are indicated. (B) Total cell lysates were prepared, and ZO-1 and occludin were detected by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Densitometry was performed, and levels of ZO-1 and occludin were related to GAPDH and the ratios (medians) are shown. (C) Transepithelial electrical resistance (mean 6 SEM) was measured weekly in ALI-derived cultures from patients with COPD (n = 5) and control subjects (n = 6). (D) Permeability of cultures to 4-kD fluorescein isothiocyanate (FITC)–dextran (2 mg/ml) applied to the apical surface of cultures and incubated for 24 hours was compared in ALI-derived cultures from the same patients with COPD (n = 5) and control subjects (n = 6). Values of all individual wells related to the transepithelial electrical resistance levels and mean levels per subject are shown (horizontal bar indicates median). (E ) Primary bronchial epithelial cells from patients with COPD (n = 6), nonsmoking control subjects (n = 6), and smoking control subjects (n = 5) were grown submerged to confluence in electrical cell–substrate impedance sensing (ECIS) arrays for 3 days and growth factor deprived for 24 hours. Resistance values (mean 6 SEM) were measured at 400 Hz by ECIS. (F ) Airway sections from patients with COPD (n = 6) and non-COPD subjects (n = 6 or 7) were analyzed for ZO-1 (frozen sections) and occludin (paraffin sections) by immunohistochemical staining. Representative images are shown. The expression of ZO-1 and occludin was analyzed and graded as described for A and medians are indicated. Significance was tested by the Mann–Whitney U test and repeated measures analysis of variance for ECIS experiments.
Correspondence
1441
CORRESPONDENCE with reduced capacity to form epithelial junctions during ALI differentiation in vitro. Of importance, this cannot merely be attributed to cigarette smoking, but rather appears to be a diseaserelated effect. Disrupted expression of tight junction proteins was also observed in lung sections of the same patients with COPD, indicating that the disruption of epithelial barrier as previously observed on cigarette smoking is persistent on smoking cessation. This may be due to the compromised ability of the bronchial epithelium of patients with COPD to (re)constitute cell–cell contacts and to redifferentiate into a functionally intact epithelium upon damage, for example, by cigarette smoking. Upon epithelial differentiation, efficient tight junction assembly may be crucial for the formation of a polarized epithelial layer (15). Tight junctions regulate paracellular permeability and functionally segregate the basolateral from the apical compartment, which is a requirement for full epithelial polarization and epithelial homeostasis (16). Loss of epithelial integrity may facilitate transepithelial crossing of noxious gases as well as pathogens. This may have important implications, because viral and bacterial infections have been implicated in nearly 50% of COPD exacerbations (17). Therefore, there is a great need to further investigate the mechanisms underlying impaired epithelial barrier function to improve mucosal barrier function in COPD. This may improve insight in strategies to improve barrier function in COPD, a promising novel target in the treatment of COPD. n Author disclosures are available with the text of this letter at www.atsjournals.org. Irene H. Heijink, Ph.D. Jacobien A. Noordhoek, B.Sc. Wim Timens, M.D. Antoon J. M. van Oosterhout, Ph.D. Dirkje S. Postma, M.D. University of Groningen Groningen, The Netherlands
Copyright © 2014 by the American Thoracic Society
Our Enlightened Understanding of the Risks of Persistent Delirium
References 1. Holgate ST. Epithelium dysfunction in asthma. J Allergy Clin Immunol 2007;120:1233–1244, quiz 1245–1246. 2. Shaykhiev R, Otaki F, Bonsu P, Dang DT, Teater M, Strulovici-Barel Y, Salit J, Harvey BG, Crystal RG. Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo. Cell Mol Life Sci 2011;68:877–892. 3. Milara J, Peiro´ T, Serrano A, Cortijo J. Epithelial to mesenchymal transition is increased in patients with COPD and induced by cigarette smoke. Thorax 2013;68:410–420. 4. Hogg JC. Bronchial mucosal permeability and its relationship to airways hyperreactivity. Eur J Respir Dis Suppl 1982;122:17–22. 5. Heijink IH, Brandenburg SM, Postma DS, van Oosterhout AJ. Cigarette smoke impairs airway epithelial barrier function and cell–cell contact recovery. Eur Respir J 2012;39:419–428. 6. van den Berge M, Steiling K, Timens W, Hiemstra PS, Sterk PJ, Heijink IH, Liu G, Alekseyev YO, Lenburg ME, Spira A, et al. Airway gene expression in COPD is dynamic with inhaled corticosteroid treatment and reflects biological pathways associated with disease activity. Thorax 2014;69:14–23. 7. Hackett TL, Shaheen F, Johnson A, Wadsworth S, Pechkovsky DV, Jacoby DB, Kicic A, Stick SM, Knight DA. Characterization of side population cells from human airway epithelium. Stem Cells 2008;26: 2576–2585. 8. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS; GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease.
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To the Editor: Patel and colleagues (1) add a very important new piece to the delirium literature puzzle, which begs two important questions. First, what is the prognostic distinction between a small but important subgroup of patients manifesting rapidly reversible delirium (RRD, n = 12, 12%), defined as delirium symptoms that resolved within 2 hours after cessation of sedative/analgesic medications (often called spontaneous awakening trials [SATs]) versus the much larger group of patients in whom delirium persisted beyond 2 hours of an SAT (comprising persistent delirium [PD] and mixed delirium [MxD], n = 75, 74%)? Consider how many times every day you have patients whose brains remain delirious (“inattentive”) 2 hours after stopping sedation. This simple finding, which was present in three out of every four patients, portended a threefold higher likelihood of death (1-yr mortality of 66% for PD vs. 20% for RRD). In fact, each additional day of “persistent delirium” (again, delirium just 2 or more hours after SAT) was associated with a 14% increased risk of death at 1 year, a risk 4% higher per day than 10% indicated in previous investigations (2, 3). This strengthens our understanding of the
American Journal of Respiratory and Critical Care Medicine Volume 189 Number 11 | June 1 2014
