Higher TGF-β with lower CD124 and TSLP, but no difference in PAR-2 expression in bronchial biopsy of bronchial asthma patients in comparison with COPD patients.

RESEARCH ARTICLE

Higher TGF-b With Lower CD124 and TSLP, But No Difference in PAR-2 Expression in Bronchial Biopsy of Bronchial Asthma Patients in Comparison With COPD Patients Radoslav Mateˇj, MD, PhD,*w Martina Vasˇa´kova´, MD, PhD,z Jaromı´r Kukal, PhD,y Martina Sˇterclova´, MD, PhD,z and Toma´sˇ Oleja´r, MD, PhD*8

Abstract: Chronic obstructive pulmonary disease (COPD) and bronchial asthma (BA) are 2 severe respiratory disorders with different predominated immunopathologies. There are several “novel molecules” from different families that are proposed as part of the etiopathogenesis of COPD and BA. Proteinaseactivated receptor 2 (PAR-2), thymic stromal lymphoprotein (TSLP), interleukin-4 and its receptor (CD124), Yin-Yang 1 (YY1), and transforming growth factor beta (TGF-b) have been previously shown to be involved in the pathophysiology of both these diseases. We investigated PAR-2, TSLP, CD124 (interleukin-4R), TGF-b, and YY1 immunohistochemical expression in endobronchial and transbronchial biopsies from 22 BA patients and 20 COPD patients. Immunostaining for the above-mentioned antigens was quantified using a modified semiquantitative scoring system and statistically evaluated. The values of TGF-b in the epithelial cells (P = 0.0007) and TGF-b in the submucosa (P = 0.0075) were higher in the BA samples, whereas values of CD124 (P = 0.0015) and TSLP (P = 0.0106) were higher in the COPD samples. No statistically significant differences between the groups were recorded for PAR-2 and YY1. Airway inflammatory reaction diversity in BA and COPD seems to be disease specific; however, there are also shared mechanisms involved in the pathophysiology of both diseases. Key Words: COPD, bronchial asthma, TGF-b, TSLP, CD124, PAR-2 (Appl Immunohistochem Mol Morphol 2014;22:543–549)

Received for publication March 27, 2013; accepted July 13, 2013. From the Departments of *Pathology and Molecular Medicine; zRespiratory Diseases, Thomayer Teaching Hospital; wDepartment of Pathology, Third Faculty of Medicine, Charles University; yFaculty of Nuclear Sciences and Physical Engineering, Czech Technical University; and 8Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Partly supported by grants NS10423-3/2009 and NT 13433-4/2012 of the Grant Agency of Ministry of Health of the Czech Republic. The authors declare no conflict of interest. Reprints: Toma´sˇ Oleja´r, MD, PhD, Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins

Appl Immunohistochem Mol Morphol

I

n chronic obstructive pulmonary disease (COPD), the Th1 immune reaction predominates, whereas in bronchial asthma (BA) Th2 immunity is primarily involved.1 There are several “novel molecules” from different families that are proposed as part of the etiopathogenesis and development of COPD and BA. These molecules modulate different inflammatory properties and subsequently the clinical and morphologic appearance of both diseases. Proteinase-activated receptor 2 (PAR-2) is activated by, among others, trypsin, mast cell tryptase, Alternaria alternata serine proteinases, and the fungal asthma-gene.2 In addition, German cockroach frass proteinases, another widely discussed asthma-gene, are thought to be involved in allergic asthma and to involve PAR-2 activation.3,4 Thymic stromal lymphoprotein (TSLP) is known to induce Th2-naive T-cell differentiation by dendritic cell (DC) maturation.5,6 Its expression correlates well with clinical severity in asthma patients7 and has been shown to be induced by proteinases through PAR-2 activation.8 However, DCs are not the only primary target of this cytokine. It has been demonstrated that DCs can also produce TSLP and this production is augmented by interleukin (IL)-4.9 Moreover, selective stimulation of the IL-4 receptor (CD124) contributes to airways’ smooth muscle hypersensitivity.10 TSLP Alternaria-induced production is also enhanced by IL-4.8 However, the overexpression of PAR-2 and TSLP has also been reported in COPD/cigarette smoke-exposed tissues in animal models and has been seen during human observations as well.11–14 This demonstrates an important role for proteinases in the development of different inflammatory processes in the bronchial walls. Moreover, there is strong evidence that many other enzymes and their receptors are involved in these complex processes, which raises the question whether the role is primary or unspecific, secondarily induced. In contrast, it has been recently reported that transforming growth factor beta (TGF-b) stimulates PAR-2 production in human lung fibroblasts15 and demonstrates its role in the pathophysiology of idiopathic pulmonary fibrosis. All published data indicate a close relationship between lung fibroblasts and DC trafficking, airway inflammation, and fibrosis mainly by integrin avb8-mediated TGF-b activation.16 This finding

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could be linked to secondarily induced PAR-2 in the etiology of BA and COPD. A potential role for the YinYang 1 (YY1) involves a gene expression regulator that participates in idiopathic pulmonary fibrosis17 and is overexpressed secondary to TGF-b stimulation in BA and COPD. This observation merits further investigation. Taken together and based on previously published experimental data, airway inflammatory reactions seem to be a perpetual cycle involving subsequent activation of TGF-b-PAR-2-TSLP-IL-4, where excess of IL-4 in BA patients switches the Th2 inhibitory effect of TGF-b to a proinflammatory reaction18 through the PAR-2-TSLP pathway. A simplified scheme is presented in Figure 1. In the present study, we investigated the PAR-2, TSLP, CD124 (IL-4R), TGF-b, and YY1 immunohistochemical expression in parallel groups of BA and COPD endoscopic bronchial biopsy samples and seek potential differences in the expression of these molecules during the etiopathogenesis of BA and COPD. In addition, we seek to contribute to the elucidation of pathophysiologic pathways described in clinical practice.

METHODS Subjects A total of 42 patients were enrolled into the study. They were all outpatients of the Department of Respiratory Diseases at Thomayer University Hospital, Prague. All of them underwent a bronchoscopic investigation for differential diagnosis between asthma and COPD, for unexplained cough or for differential diagnosis of difficult-to-treat asthma. Before bronchoscopy, all pa-


tients gave their informed consent. A definite diagnosis of COPD was established in 20 patients and were staged as GOLD I and II. The mean age of the COPD group was 50.5 years (range, 25 to 75 y), 10 men and 10 women. The other 22 patients were clinically diagnosed as suffering from asthma. The mean age was 48.2 years (range, 25 to 80 y), 10 men and 12 women. All received a comprehensive clinical investigation, which included medical history, physical findings, and functional investigations including spirometry, body plethysmography, diffusion capacity, bronchomotor tests, laboratory investigations, and chest x-ray. Detailed information regarding subject distribution relative to smoking and treatment history is presented in Table 1. A bronchoscopic examination was conducted under local anesthesia. During the procedure, routine samples were taken for microbiology. Bronchial samples and lung tissue were taken using transbronchial biopsies and endobronchial excisions. The endobronchial biopsies (usually 3 samples) were taken from the ridges between the lobar and segmental bronchi, usually on the right side, using bronchoscopic flexible forceps introduced by the working channel of the bronchoscope. The transbronchial biopsies (TBB) were conducted with the same forceps after introducing the instrument into peripheral parts of the bronchial tree, usually the right lower lobe. Usually, 3 transbronchial biopsy samples were taken while pushing the opened jaws of the forceps against the lung tissue, then closing the forceps and tearing and extracting small samples of the tissue. All the samples were stored in formaldehyde for further histopathologic examination. All patients signed informed consent before entering the study and the study protocol was approved by the Central Ethics Committee of Thomayer Hospital and the Institute for Clinical and Experimental Medicine. In addition, all data were analyzed with respect for patient privacy.

TABLE 1. Overview of Subject Distribution Related to the Smoking and Treatment History

FIGURE 1. Simplified scheme of the cytokine network involved in bronchial asthma and chronic obstructive pulmonary disease. AAM indicates activated alveolar macrophages; B, Blymphocyte; DC, dendritic cells; Eos, eosinophils; IL, interleukin; IgE, immunoglobulin E; MC, mast cells; PAR-2, proteinase-activated receptor 2; SMC, smooth muscle cells; Th2, Th2-lymphocyte; TSLP, thymic stromal lymphoprotein; TGF-b, transforming growth factor beta. Blue arrow indicates activation pathway; red arrow, inhibitory pathway. The scheme was composed using Servier Powerpoint image bank (http:// www.servier.com).

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Smoking Current smokers Ex-smokers Nonsmokers Data not available Disease onset Childhood Adult Data not available Treatment Inhalation steroids (IS) Systemic steroids (SS) IS+SS Omalizumab (0)+IS O+IS+SS No treatment Data not available

COPD

Asthma

4 10 4 2

1 12 4 3

NA NA NA

11 6 3

3 1 1 NA NA 14 1

4 1 6 3 4 2 0

COPD indicates chronic obstructive pulmonary disease; NA, not available.

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Histology Formalin-fixed and paraffin-embedded slices, 5 mm thick, from endoscopic samples were routinely stained with hematoxylin and eosin for confirmation of the histopathologic pattern associated with the clinical diagnosis. Representative histopathologic samples from both diseased groups were selected based on the registry of the Department of Pathology and Molecular Medicine, Thomayer Teaching Hospital, Prague, in accordance with the following rules: (1) COPD: compliance with clinical diagnosis, increased number of goblet cells, mixed neutrophil/lymphoplasmacytic submucosa infiltration, and squamous metaplasia of the epithelial surface of the bronchial mucosa. (2) BA: compliance with clinical diagnosis, thickening of the epithelial basement membranes, the presence of large numbers of eosinophils in a mixed submucosa infiltration, smooth muscle hyperplasia, and bronchial gland hypertrophy in the bronchial walls.

Immunohistochemistry Microscopic tissue slices, 5 mm thick, were deparaffinized, rehydrated, and pretreated with a citrate buffer (pH 7.6) or high pH Tris/HCl buffer (pH 9.0) 3 5 minutes in a microwave oven. Endogenous peroxidase was blocked with a water solution containing 0.01% sodium azide and 1% hydrogen peroxide. Nonspecific positivity was blocked using rabbit serum in TBS (150 mL/10 mL) for 30 minutes. Primary antibodies against the following antigens were used: (1) PAR-2 (1:250, rabbit polyclonal, recognizes N-terminal, extracellular part of the molecule; Millipore, Billerica, MA); (2) CD124 (1:200, mouse monoclonal, recognizes cytoplasmic domain of IL-4R; Millipore); (3) TGF-b (1:300, mouse monoclonal, raised against the C terminus of the human TGF-b protein; Abcam, Cambridge, UK); (4) YY1 (1:100, goat polyclonal, recognizes the full length of the human YY1; Santa Cruz Biotechnology, Santa Cruz, CA); and (5) TSLP (1:500, rabbit polyclonal, raised against the part of human TSLP near the C-terminus of the molecule; Novus Biologicals, Littleton, CO). The slices were then incubated overnight at 41C. Detection of immunostaining was carried out using an Envision kit and diaminobenzidine was used as a chromogen. Slides incubated with only the secondary antibody and with nonspecific isotype-matched primary antibodies were used as specificity controls. Mayer hematoxylin was used as a nuclear counterstain. As a negative control, slides of bronchial endoscopic samples were incubated with nonspecific isotype-matched primary antibodies and with secondary antibody only.

Immunostaining Quantification A modified, semiquantitative scoring system (immunoreactive score; IRS) according to Remmele and Schicketanz19 was used to allow a reproducible evaluation of protein-staining levels in the epithelial and stromal components of immunohistochemically stained tissue sections. Stained sections were scored independently by 2 experienced pathologists. The IRS was calculated acr


Different TGF-b, CD124 and TSLP Expression in BA and COPD

cording to the following formula: IRS = staining intensity (0 to 3) percentage of positive cells or structures (0, 0% to 10%; 1, 10% to 25%; 2, 26% to 50%; 3, 51% to 75%; 4, 76% to 100%). Possible scores ranged from a minimum of 0 to a maximum of 12.

Statistics Two statistical samples for every stochastic variable were investigated using 3 tests: (i) normality test belonging to the Gaussian normal distribution, (ii) F test of variance equity, and (iii) the 2-sample 2-sided t test of mean value equity. The critical level was set to P = 0.05. All the calculations were done using MATLAB 7.8.0 Statistical Toolbox (Mathworks Inc., 2009).

RESULTS Immunohistochemical positivity was investigated for CD124, TGF-b, TSLP, YY1, and PAR-2 in all samples from both the groups. TGF-b in the epithelial cells (TGF-b epi) and the submucosa (TGF-b sub) and PAR-2 in the epithelial cells (PAR-2 epi) and the submucosa (PAR-2 sub) were evaluated separately. Figure 2 demonstrates representative samples corresponding to median IRS values recorded in the 2 groups. TGF-b and PAR-2 are produced by different cell types in different locations and can be expressed with different intensity in mucosal and submucosal elements with a wide range of possible actions; thus, separate evaluations were done for epithelial and stromal staining. Only CD124, TSLP, and YY1 mucosal staining was evaluated in accordance with the expectation of possible epithelial effects. Figure 3 demonstrates IRS values recorded for all individual samples in the 2 groups of patients. All statistical samples passed the normality test and were nonsignificant P > 0.05. The F test also exhibited nonsignificant variance ratios with P > 0.05 for every variable. Thus, the standard t test was used to indicate mean value differences between COPD and BA. Statistically significant differences were seen in TGF-b epi (P = 0.0007), CD124 (P = 0.0015), TGF-b sub (P = 0.0075), and TSLP (P = 0.0106). The remaining differences were nonsignificant with P > 0.05. Summarized score results are presented in Table 2.

DISCUSSION Considering the variability within the 2 cohorts, with regard to smoking history, disease onset in BA patients and drug intake history, only in histologically wellconfirmed subjects, fulfilling the morphologic criteria of BA and chronic bronchitis, underwent analysis. In our immunohistochemical study, we demonstrated statistically significant higher production of TGF-b in the bronchial epithelial cells and bronchial submucosa of BA patients. These results also correlate with previously published data reporting higher serum levels of TGF-b1 in BA patients compared with COPD patients.20 The difference between these 2 groups can be probably explained by the different response of T cells to www.appliedimmunohist.com |

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FIGURE 2. Immunohistochemical positivity illustrates median immunoreactive score values in representative bronchial asthma samples: A, CD124 positivity—median value 3; B, PAR2 positivity—median value 6 in epithelial cells and 4 in the submucosa; C, TGF-b positivity in the epithelial cells—median value 8; D, TGF-b positivity in the submucosa—median value 4.5; E, TSLP positivity in the submucosa—median value 4; F, YY1 positivity in the submucosa—median value 2.25; and in chronic obstructive pulmonary disease samples: G, CD124 positivity—median value 4; H, PAR-2 positivity—median value 4 in the epithelial cells and in the submucosa; I, TGF-b positivity in the epithelial cells—median value 4; J, TGF-b positivity in the submucosa—median value 3.25; K, TSLP positivity in the submucosa—median value 6; L, YY1 positivity in the submucosa—median value 3. Original magnification 200. PAR-2 indicates proteinase-activated receptor 2; TGF-b, transforming growth factor beta; TSLP, thymic stromal lymphoprotein; YY1, Yin-Yang 1.

TGF-b. IL-2 and IL-4 stimulation contributes to T-cell resistance to TGF-b suppression in asthma patients, but not in COPD or healthy subjects.18 Increased TGF-b in BA patients (compared with COPD), from this point of view, may result from insufficient negative feedback, secondary to an inappropriate reaction of T cells resistant to


this chemokine. In BA patients, TGF-b is produced by local epithelial and mesenchymal structures. TGF-b probably suppresses immunopathologic reactions, but resistant T cells do not respond and a perpetual cycle leads to ever-increasing levels of TGF-b. Whether this reaction is responsible for widening of the epithelial basement membranes is a reasonable question, as this cytokine is known to induce fibroblast differentiation into myofibroblasts accompanied by the excessive synthesis of stromal proteins.21 Subject distribution, related to the steroid intake history, in our cohorts was not statistically evaluable. However, the high number of nontreated subjects with COPD (total 14) suggests that steroid intake does not decrease TGF-b levels in asthma subjects (total treated 18). In contrast, the CD124 (IL-4R) and TSLP expression was statistically significantly higher in COPD patients. Higher production of CD124 and TSLP in COPD patients probably comes from different tissue responsiveness in the 2 etiological diseases and must be related to the above-mentioned T-cell IL-4-induced resistance to TGF-b inhibition. Polymorphisms in IL-4 and IL-4R genes could represent asthma susceptible gene variations in certain populations22–24 and positive feedback resulting in the higher expression of CD124 would be expected. However, involvement of this cytokine and its receptor is probably a common and shared mechanism in both BA and COPD. The mechanism likely involves “alternatively activated macrophages” dependent on the effects of IL-4 and IL-13, resulting in the production of IL-13 with subsequent production of mucus and airway hypersensitivity.25 From this point of view, we can raise the question of whether our results relate to the higher production of mucus and increased number of goblet cells in COPD patients that contributes to differential diagnosis between investigated nosology units; however, the exact mechanism of action needs further investigation. On the basis of our results, the predicted involvement of IL-4 and IL-4R gene polymorphisms cannot be related to the higher CD124 expression in BA samples compared with COPD samples. r





FIGURE 3. Dot-plot illustration of individual immunoreactive score values for both BA and COPD. A, CD124; B, PAR-2 epithelial positivity; C, PAR-2 submucosal positivity; D, TGF-b epithelial positivity; E, TGF-b submucosal positivity; F, TSLP positivity; and G, YY1 positivity. BA indicates bronchial asthma; PAR-2, proteinase-activated receptor 2; COPD, chronic obstructive pulmonary disease; TGF-b, transforming growth factor beta; TSLP, thymic stromal lymphoprotein; YY1, Yin-Yang 1.

TSLP contributes to Th2 immunity by activating DCs, but is also expressed on the bronchial epithelial cells in COPD patients where it contributes to the Th1 pattern26 and the r


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TABLE 2. Summarized Immunoreactive Score Values Recorded in Each Group COPD (average) BA (average) COPD (median) BA (median) COPD (SD) BA (SD) t test (P)

CD124

TGF-b epi

TGF-b sub

TSLP

YY1

PAR-2 epi

PAR-2 sub

4.775 3.1136 4 3 1.8199 1.2241 0.0015

4.875 7.7955 4 8 2.392 2.6053 0.0007

3.375 4.9773 3.25 4.5 1.5959 1.9626 0.0075

5.675 4.1818 6 4 1.3899 2.0369 0.0106

3.45 2.8636 3 2.25 2.0911 2.5681 0.4358

5.075 5.5909 4 6 1.6902 1.4972 0.3122

4.5 4.6591 4 4 1.6583 1.1519 0.7245

Statistically significant values are in bold. BA indicates bronchial asthma; COPD, chronic obstructive pulmonary disease; epi, epithelial cells; PAR-2 indicates proteinase-activated receptor 2; sub, submucosa; TGF-b, transforming growth factor beta; TSLP, thymic stromal lymphoprotein; YY1, Yin-Yang 1.

were observed.7 However, viral (and probably bacterial) stimuli probably increase the expression of TSLP at the protein level in COPD.27 We must question the possible inhibitory effect of TGF-b overproduction on TSLP and CD124 in the context of the natural anti-inflammatory actions of this cytokine relative to the T-cell resistance in BA patients. However, the larger number of subjects with corticosteroid treatment (with anti-inflammatory effects) in the BA cohort must also be taken into account. It is important to remember that all patients, regardless of treatment, had the same histopathologic picture and suffered clinically from a chronic cough and entered the investigation because of difficult-to-treat asthma. More experiments in this field need to be conducted. No significant difference was observed between the 2 groups with regard to the expression of PAR-2. In our understanding, upregulation of this molecule is probably common and shared mechanism utilized in both BA and COPD. In a previous study, Miotto et al11 showed different immunohistochemical distribution and intensity in the bronchial walls between COPD patients who were smokers and those who did not smoke. Their results suggest that smoking could be associated with the activation of PAR-2 and thus directly involved in COPD progression. However, these results are not in contradiction with our observations as the difference between the 2 cohorts with regard to smoking history was not statistically significant. Upregulation of PAR-2 should be expected in BA, as mast cells contained high levels of PAR-2—activating tryptase.28 This pathway was not excluded by our results for the acute phase of the disease, where paroxysmal release of tryptase, secondary to Th2 cytokine stimulation, contributes to hypersensitivity and late-phase inflammation. Despite the role of PAR-2, with regard to inflammation of the respiratory system, PAR-2 is most likely an important part of the complex pathophysiology, in which many different proteinases and antiproteinases influence each other. In addition, no difference was observed in the expression of transcription factor YY1. Some difference was expected as a speculated polymorphism in the TGF-b promoter gene, also associated with BA, could create a putative YY1 transcription factor-binding site29,30 and regulate the Th2 inflammatory reaction and IL-4 gene expression in


T cells.31,32 However, there is no direct experimental study that has evaluated YY1 expression either in BA or COPD patients and the regulatory/stimulatory pathway could be present in both BA and COPD. Finally, in routine endoscopic bronchial biopsy samples, we confirmed certain differences between clinical conditions arising from previously published experimental data. Different expression of TGF-b and IL-4R together with changes of TSLP positivity most probably occurred owing to differences in the reactivity of BA and COPD to TGF-b after IL-4 stimulation; however, this is not clearly explained and should be investigated more deeply in the future. In contrast, there are unspecific and/or shared complex pathways, utilizing different molecules such as PAR-2 or YY1, which play an important role in both the diseases. This complies with our understanding of PAR-2 as a crucial but unspecific enhancer of the pathophysiology of different membrane/ transcellular processes. In summary, airway inflammatory reaction differences in both the diseases is unlikely to be a simple TGF-b-PAR-2-TSLP-IL-4 pathway; instead, it seems that there are complex disease-specific mechanisms (TGF-b, TSLP, IL-4) that utilize shared inflammatory effectors such as PAR-2, among others. ACKNOWLEDGMENT The authors thank Thomas Secrest for revisions on the English version of this article. REFERENCES 1. Barnes PJ. The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest. 2008;118:3546–3556. 2. Boitano S, Flynn AN, Sherwood CL, et al. Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2. Am J Physiol Lung Cell Mol Physiol. 2011;300:L605–L614. 3. Day SB, Ledford JR, Zhou P, et al. German cockroach proteases and protease-activated receptor-2 regulate chemokine production and dendritic cell recruitment. J Innate Immun. 2012;4:100–110. 4. Day SB, Zhou P, Ledford JR, et al. German cockroach frass proteases modulate the innate immune response via activation of protease-activated receptor-2. J Innate Immun. 2010;2:495–504. 5. Ito T, Wang YH, Duramad O, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med. 2005;202:1213–1223. r




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