Journal of Critical Care (2013) 28, 111.e9–111.e15
Expression of acute-phase cytokines, surfactant proteins,and epithelial apoptosis in small airways of human acuterespiratory distress syndrome☆,☆☆
Ruy Camargo Pires-Neto PhDa,⁎, Maina Maria Barbosa Morales PhDa,Tatiana Lancas PhDa, Nicole Inforsato MDa, Maria Irma Seixas Duarte PhDa,Marcelo Britto Passos Amato PhDb, Carlos Roberto Ribeiro de Carvalho PhDb,Luiz Fernando Ferraz da Silva PhDa, Thais Mauad PhDa, Marisa Dolhnikoff PhDa
aDepartment of Pathology, Experimental Air Pollution Laboratory-LIM05, Sao Paulo University Medical School,01246903 Sao Paulo, BrazilbPulmonary Division, Heart Institute (InCor), Sao Paulo University Medical School, 01246903 Sao Paulo, Brazil
0h
Keywords:ARDS;Respiratory epithelium;Apoptosis;Cytokines;Surfactant protein
☆ Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).☆☆ The authors have no conflicts of interest to declare.
⁎ Corresponding author. Department of Pathology, Sao Paulo University Medical School, 01246903 Sao Paulo, SP, Brasil. E-mail address: [email protected] (R.C. Pires-Neto).
Airway dysfunction has been increasingly recognized asan important contributor to pulmonary impairment inpatients with acute respiratory distress syndrome (ARDS)[1]. Acute respiratory distress syndrome is characterized bythe abrupt onset of hypoxemia with diffuse pulmonaryinfiltrates and an accumulation of a protein-rich pulmonaryedema that causes reduction in lung compliance, alveolarcollapse, and ventilation-perfusion mismatch [2]. Further-more, increased lung resistance, expiratory flow limitations,and dynamic hyperinflation have also been reported, whichare partially attributed to airway closure [1,3].
Animal models of acute lung injury (ALI) have shownthat, in addition to damage to the parenchyma in ALI/ARDS,small airway injuries are characterized by bronchiolarepithelial necrosis and sloughing and by rupture ofalveolar-bronchiolar attachments [4-8]. The loss of mechan-ical alveolar/airway interdependence, airway epithelial inju-ry, interstitial edema, and alveolar collapse may all contributeto distal airway instability [1,7]. We have recently reportedthat in humans who died with ARDS, small airway changeswere characterized by wall thickening with inflammation,extracellular matrix remodeling, and epithelial denudation[9]. Importantly, the degree of airway epithelial denudation inthese patients was associated with disease severity.
Previous studies have suggested a role for distal airwayepithelium injury in the pathophysiology of human ALI/ARDS [10,11]. The epithelium lining the airways modulatesairway function by secreting a large number of moleculessuch as surfactant components and inflammatory mediators[12]. Changes in the composition and function of thesurfactants released by the airways are observed in differentpulmonary diseases, such as asthma and chronic bronchitis.Dysfunction of airway surfactants can be associated with theimpairment of host defenses and distal airway stability [13].So far, there is little information on how these secretoryfunctions of the small airways are altered in ARDS. Amongthe inflammatory mediators involved in lung injury inARDS, interleukins (IL) 6 and IL-8 were increased in bothserum and bronchoalveolar lavage (BAL) [14,15], and theserum interleukins levels were associated with increasedmortality and morbidity [14]. The expression of theseinterleukins in airway epithelial cells has not been addressedin ARDS.
Alveolar cell apoptosis is increased in patients with ARDSand is likely to contribute to alveolar injury; the Fas/Fasligand (FasL) system, a surface receptor and its natural ligand,seems to play a central role in this process [16]. We havepreviously reported that bronchiolar epithelium injury anddenudation are present in humans with ARDS and areassociated with disease severity [9]; the mechanism of airwaycell injury in these patients is not known. We hypothesizedthat apoptosis could be an important mechanism of epithelialcell death not only in the alveolar epithelium but also in thedistal airways in ARDS.
In this study, we assessed small airway alterations thatcould be involved in pulmonary inflammation and surfactantdysfunction in ARDS. For this purpose, we measured airwayexpression of the inflammatory cytokines IL-6 and IL-8, theairway expression of the surfactant proteins (SP) A and SP-B, and an index of airway epithelial apoptosis of patientswith ARDS submitted to autopsy and compared the resultswith those of control subjects.
2. Methods
This study was approved by the review board for humanstudies of the São Paulo University Medical School(CAPPesq-FMUSP). The study is retrospective and usedarchived material from routine autopsies performed at theAutopsy Service of Sao Paulo University Medical School.Consent for performing autopsy was obtained from the nextof kin of all the subjects involved in the study.
2.1. Study population
Lung tissue from 31 patients with ARDS submitted toautopsy at Sao Paulo University Medical School between2004 and 2007 was retrospectively included in the study.Inclusion criteria were a clinical diagnosis of ARDS definedaccording to the American-European Consensus criteria[17], histologic findings of diffuse alveolar damage [18], anabsence of chronic lung diseases, and sufficient archivedautopsy material (at least 3 small airways per patient) foranalysis. Eleven nonsmoker, nonventilated patients who diedof nonpulmonary causes, without previous lung diseaseswere used as controls. Control subjects showed normal lungsat gross and microscopic examination. We have character-ized this population in a previous study [9].
The following clinical data were obtained from medicalcharts: age, sex, predisposing cause of ARDS, cause ofdeath, days of ARDS evolution (time interval betweenARDS diagnosis and death), partial pressure of oxygen(PaO2), plateau pressure, positive end-expiratory pressure(PEEP), and the ratio of PaO2 to fraction of inspired oxygen(FIO2) assessed at the time of the clinical diagnosis.
2.2. Tissue processing and histologic analysis
Paraffin blocks of lung tissue collected during routineautopsywere retrieved from the archives of the Department ofPathology of Sao Paulo University Medical School. In theroutine autopsies, 3 to 4 fragments of lung tissue werecollected from any regions of altered lung parenchyma. Innormal lungs, 1 fragment of lung tissue was collected fromeach lobe. The tissue had been previously fixed in 10%buffered formalin for 24 hours, routinely processed andparaffin embedded. Sections 5 μm thick were stainedwith hematoxylin and eosin for histologic diagnoses of
111.e11Expression of acute‐phase cytokines in human ARDS
diffuse alveolar damage and identification of small airways.Small airways were defined as those with a diameter of2.0 mm or less in a transversal section (short/long diameterratio N0.6) [19].
The following parameters were analyzed in the smallairways: epithelial expression of SP-A and SP-B, epithelialapoptosis, and the airway expression of inflammatorycytokines. Apoptosis was assessed with caspase 3 expres-sion, Fas/FasL epithelial expression and the peroxidaseterminal deoxynucleotidyl transferase-mediated deoxyuri-dine-triphosphatase nick-end labeling (TUNEL) assay [20].The airway expression of inflammatory cytokines wasassessed as the epithelial expression of IL-6 and IL-8 andas the density of IL-positive inflammatory cells within theairway wall.
The following proteins were identified with immunohis-tochemistry as previously described [19]: SP-A, SP-B, IL-6,IL-8, caspase 3, Fas, and FasL. Briefly, sections weredeparaffinized, and a 0.3% hydrogen peroxide solution wasapplied for 35 minutes to inhibit endogenous peroxidaseactivity. Antigen retrieval (except for SP-A) was performedwith a citrate solution for 45 minutes. Sections wereincubated with the primary antibody overnight at 4°C (SP-A, 1:1200, DAKO, Glostrup, Denmark; SP-B, 1:400,Labvision, Fremont, Calif; IL-6, 1:20, R&D, Minneapolis,Minn; IL-8, 1:100, R&D; Fas, 1:1200, Santa Cruz Bio-technologies, Santa Cruz, Calif; FasL, 1:100, Santa CruzBiotechnologies; Caspase 3, 1:500, Novocastra, Newcastle,UK). Novolink Polymer (Novocastra) was used as thesecondary antibody and 3,3’-diaminobenzidine (SigmaChemical Co, St Louis, Mo) as the chromogen. The sectionswere counterstained with Harris hematoxylin. For negativecontrols, the primary antibody was replaced by phosphate-buffered saline.
Protein epithelial expression was assessed using Image-Pro Plus 4.1 for Windows image analysis software (MediaCybernetics, Silver Spring, Md) on a personal computerconnected to a digital camera coupled to a microscope (LeicaDMR, Leica Microsystems Wetzlar GmbH, Wetzlar, Ger-many). The image analysis system measured the mean colordensity of the immunohistochemical staining in the smallairway epithelium, which represents the mean intensity of thestaining within the positive area (range, 0-255) [21]. Theexpression of SP-A, SP-B, IL-6, IL-8, Fas, and FasL in theepithelium was calculated as the product of the area ofpositive staining and mean density, normalized by thecorresponding epithelial basement membrane length [22].This index of protein expression takes into account both theintensity and the area of staining. The densities of IL-6– andIL-8–positive cells in the small airway were calculated as thenumber of positive cells within the airway wall normalizedby the basal membrane length (cells per micrometer). Theapoptosis index by TUNEL assay and caspase 3 expressionwas calculated as the number of epithelial-positive cellsdivided by the corresponding basal membrane length (cellsper micrometer).
At least 3 small airways were analyzed per patient byan investigator blinded to the study group. The entireairway circumference was analyzed for each variable, at a200× magnification.
2.3. Clinical correlations
Because lung injury can be observed after a few hours ofinjurious mechanical ventilation [4], we investigated thepossible association of airway changes with mechanicalventilation and disease severity in patients with ARDS whodied within 48 hours after diagnosis (n = 16). For thispurpose, we correlated the histologic parameters with theventilatory parameters (mean values of plateau pressure andPEEP) and the PaO2/FIO2 value obtained at the moment of theclinical diagnosis.
To evaluate the airway changes over the course of thedisease, we correlated the different histologic parameterswith the time interval between ARDS diagnosis and death(days of ARDS evolution).
Statistical analysis was performed using the statisticalsoftware SPSS 15.0 (SPSS, Chicago, Ill). According to thedistribution of each variable, either the Student t test or theMann-Whitney U test was used to compare data between theARDS and control groups. The association betweenhistologic and clinical data was analyzed by Pearson orSpearman rank test. Data are presented as mean ± SD ormedian (interquartile range). The level of significance wasset at P b .05.
3. Results
3.1. Study population
Demographic and clinical data of the ARDS (n = 31) andcontrol (n = 11) groups are presented in Table 1. The mainpredisposing factors for ARDS were pneumonia (42%) andsepsis (35%). Median values of plateau pressure and PEEP inpatients with ARDS were 28 (10) cm H2O and 11 (8) cmH2O, respectively. Individual values of ventilatory parame-ters, PaO2/FIO2 values, and days of ARDS evolution werepreviously reported [9]. Ventilatory management was basedon a protective low-tidal volume strategy (4-6 mL/kg ofpredicted body weight) [23]. None of the subjects in thecontrol group were mechanically ventilated.
3.2. Image analysis
Adequate samples (lung tissue containing at least 3small transversely cut airways) were available for 18 to 31patients with ARDS and 6 to 11 control subjects,depending on the protein studied. All available smallairways for each patient were analyzed (Table 2). A totalof 1798 distal airways were analyzed (mean of 6 airways
Table 2 Proteins expression and TUNEL assay in distalairways of patients with ARDS and controls
ARDS Controls P
Epithelial expression of 211.5 (386.9) 131.2 (227.5) NSSP-A (μm2/μm) × density n = 112 n = 45Epithelial expression of 29.3 (138.1) 35.48 (56.5) NSSP-B (μm2/μm) × density n = 136 n = 54Epithelial expression of 132.2 (344.5) 208.9 (514.6) NS
111.e12 R.C. Pires-Neto et al.
per histologic parameter in each patient). The meanperimeters of the small airways for the ARDS groupand control group were 1.55 ± 0.14 and 1.45 ± 0.19 mm(P = .273), respectively.
Table 2 and Fig. 1 show image analysis data for theARDS and control groups. The ARDS group showedsignificantly higher expression of IL-8 in the airwayepithelium (P = .006) and a higher number of IL-6– (P =
Table 1 Clinical data and ventilatory parameters of patientswith ARDS and controls
Data are given as median (interquartile range) or n (percentage). Foreach patient, plateau pressure and PEEP correspond to mean values inthe first 48 hours. The PaO2/FIO2 corresponds to values assessed at thetime of clinical diagnosis. M indicates male; F, female; NA,nonapplicable; PaO2/FIO2, ratio of arterial oxygen tension to the fractionof inspired oxygen.
IL-6 (μm2/μm) × density n = 118 n = 51Epithelial expression of 152.67 (162.2) 25.21 (59.02) .006IL-8 (μm2/μm) × density n = 161 n = 59Airway wall IL-6+ cell 25.5 (14.7) 13.2 (16.7) .004density (cells*10−3/μm) n = 118 n = 51Airway wall IL-8+ cell 12.7 ± 10.7 1.6 ± 1.8 b.001density (cells*10−3/μm) n = 161 n = 59Epithelial expression of 523.8 ± 457.9 779.5 ± 524.1 NSFas (μm2/μm) × density n = 131 n = 48Epithelial expression of 757.7 ± 319.4 746.2 ± 276.0 NSFasL (μm2/μm) × density n = 139 n = 39Caspase 3 + epithelial 0.0 (0.3) 0.0 (1.0) NScells (cells *103/μm) n = 135 n = 46TUNEL+ epithelial 4.5 ± 4.6 8.6 ± 5.2 .065cells (cells * 104/μm) n = 101 n = 34
A mean of 6 airways per histologic parameter was analyzed in eachpatient. NS indicates nonsignificant; n, number of analyzed airways ineach parameter per group.
.004) and IL-8–positive (P b .001) inflammatory cells in theairway walls when compared with the control group. Therewere no differences in epithelial expression of SP-A, SP-B,IL-6, caspase 3, Fas, and FasL between the 2 groups.Although there was no significant difference in the numberof TUNEL-positive epithelial cells between the groups, weobserved a trend toward higher numbers of apoptotic cells inthe control group (P = .06).
3.3. Clinical correlations
The density of IL-8–positive inflammatory cells showed anegative correlation with PaO2/FIO2 (r = –0.56; P = .024).There was a significant correlation between days of ARDSevolution and airway epithelium Fas expression (r = 0.46;P = .034) and a negative correlation between days of ARDSevolution and the density of IL-6–positive cells in the airwaywalls (r = –0.64; P b .001). There was no correlationbetween ventilatory and histologic parameters.
4. Discussion
The main finding of this study was a greater inflammatoryresponse in the small airways of patients with ARDS than inpatients who died of nonpulmonary causes. Our results alsoshowed that there was no difference in the apoptotic indexbetween the ARDS and control groups.
Fig. 1 Representative photomicrographs of small airways of the control group (A and C) and patients with ARDS (B and D) stained withanti–IL-8 (A and B) and anti–IL-6 (C,D). There is higher expression of IL-8 in the airway epithelium and a higher number of IL-6– and IL-8–positive inflammatory cells (arrows) in the airway walls in the ARDS group. L, lumen; EP, respiratory epithelium; scale bar, 50 μm.
111.e13Expression of acute‐phase cytokines in human ARDS
In normal conditions, the functions of the small airwayepithelium include providing a mechanical barrier to inhaledparticles from the environment; host defense, including therecruitment and modulation of inflammatory cells; and thedirect repair of injury to the airways [24]. In airway diseasessuch as chronic obstructive pulmonary disease and asthma,the alterations in the structure and function of the respiratoryepithelium are well recognized [25]; however, little is knownabout the changes in the airway epithelium in ARDS.Experimental data show an increase in inflammatoryparameters in the small airways in animal models of bothARDS and ventilator-induced lung injury [4,8]. In asurfactant-depleted model of ALI in rats, Tsuchida et alshowed that airway epithelial injury associated withperibronchiolar inflammation was generalized in the lungs,affecting both atelectatic and nonatelectatic regions [8]. Ourresults are in line with those experimental data and showedthat the small airways are involved in the overall lunginflammation and cytokine production in ARDS. Interleukin6 is one of the first acute-phase cytokines released in sepsis[26]; the chemotatic cytokine IL-8 is related to neutrophilrecruitment that is associated with massive lung damage inARDS [27]. Previous studies have shown that IL-6 serumlevels can vary during the course of ARDS, decreasing in thefirst days compared with baseline values [14]. Although thisis not a consistent finding in human ARDS [15], we observed
the same dynamics of IL-6 expression within the airwayinflammation cells, with higher expression of IL-6 in patientswho died within the first days of disease evolution.
Interleukin 6 is also overexpressed in airway diseases andhas been associated with airway remodeling in asthma [28].We have previously shown that patients who died withARDS present a large number of inflammatory cells andremodeling of the airways [9]. We now report that theseairway inflammatory cells showed higher expression of IL-6and IL-8 compared with controls and that the airway densityof IL-8+ cells was associated with ARDS severity assessedby PaO2/FIO2 values. We suggest that airway expression ofboth IL-6 and IL-8 may be involved in the mechanism ofairway injury and remodeling observed in these patients.
Although SP-A participates in innate defense andregulates the release of surfactants by type II cells, SP-B'smain function is reducing surface tension [12,13,29].Pulmonary surfactants in the airways are secreted by Claracells and involved in airway stability and prevention of fluidaccumulation in the lumen, act as a barrier, and haveimportant immunomodulatory properties [13]. We hypoth-esized that the expression of SP-A and SP-B would bedecreased in the airway epithelium of patients with ARDS,which could play a role in the mechanism of distal airwaycollapse. We did not find any differences in the small airwayepithelium expressions of either SP-A or SP-B between the 2
111.e14 R.C. Pires-Neto et al.
groups. Studies analyzing BAL of patients at risk and in theacute onset of ARDS have shown diminished concentrationsof SP-A and SP-B [30,31]. Our results could either indicatethat changes in airway surfactant expression do notcontribute to local airway alterations in ARDS, or if thesmall airway does contribute to the decrease in the levels ofSP observed in BAL, it is likely due to the extensive damageand sloughing of the epithelium [9]. Furthermore, althoughthere was no difference in the amount of airway surfactantproteins between the groups, it is also possible that airwaysurfactant function was altered in these patients.
The relative contribution of apoptosis or necrosis inepithelial cells death in ARDS is unknown [16]. Elevatedconcentrations of soluble Fas and FasL have been detected inBAL fluids from patients with ALI/ARDS [32,33]. Albertineet al showed increased immunoexpression of Fas and FasL inalveolar epithelial cells in patients who died with ALI/ARDS. The rationale that the Fas/FasL system has a potentialrole in airway epithelial apoptosis is based on the observationthat BAL fluids obtained from patients with ARDS induceddistal lung epithelial cell apoptosis and that this apoptosiswas inhibited by blocking the Fas/FasL system [33]. In thisstudy, the index of airway epithelial apoptosis was assessedwith caspase 3 expression, Fas/FasL epithelial expression,and the TUNEL assay. None of these analyses showed asignificant difference between patients with ARDS andcontrols. The results show no evidence that apoptosis is amajor mechanism of airway epithelial cell death in thesepatients with ARDS patients, corroborating previous exper-imental data that suggest that bronchiolar injury inexperimental models of ALI might be secondary to epithelialnecrosis [4,7]. Furthermore, experimental studies show thatlow levels of lung injury are associated with high levels ofapoptosis, whereas increased lung injury is associated withdecreased apoptosis and increased necrosis [34-36]. Thus,epithelial necrosis might be an important mechanism ofairway epithelial cell death in our patients due to the severityof acute lung injury.
Some limitations of the present study should beaddressed. It is well known that pulmonary injury inARDS is heterogeneously distributed within the lungs;however, the retrospective character of the study did notallow us to systematically assess regional differences inairway injury. Because adequate samples were not availablefor all the stainings, some of the analyses did not include allthe patients, what could have decreased the strength of thecorrelations. Furthermore, we did not evaluate lungs withalterations exclusively related to mechanical ventilation.Although there was no correlation between the ventilatoryand histologic parameters, it is not possible to exclude thepossibility that mechanical ventilation is associated to theobserved airway changes. We believe that these changes areprobably the result of both the primary pulmonary insultleading to ARDS and a ventilator-induced injury.
In conclusion, our results showed that the distal airwayswere involved in ARDS lung inflammation with higher
expressions of proinflammatory interleukins in both airwayepithelial and inflammatory cells compared with controls.Our results show no evidence that apoptosis is a majormechanism of airway epithelial cell death in ARDS.
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