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Peptides 63 (2015) 90–95

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

esearch paper

refoil factors (TFFs) are increased in bronchioalveolar lavage fluidrom patients with chronic obstructive lung disease (COPD)

iels-Erik Vibya,e,∗, Ebba Nexøb, Hannelouise Kissowa, Helle Andreassenc,aul Clementsenc, Lars Thimd, Steen Seier Poulsena

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, DenmarkDepartment of Clinical Biochemistry, Aarhus University Hospital, Aarhus, DenmarkDepartment of Pulmonology, Gentofte University Hospital, Hellerup, DenmarkDepartment of Protein Engineering, Novo Nordisk A/S, Maalov, DenmarkDepartment of Cardiothoracic Surgery, Copenhagen University Hospital, Denmark

r t i c l e i n f o

rticle history:eceived 3 July 2014eceived in revised form3 September 2014ccepted 23 September 2014vailable online 23 October 2014

eywords:

a b s t r a c t

Trefoil factors (TFFs) 1, 2 and 3 are small polypeptides that are co-secreted with mucin throughout thebody. They are up-regulated in cancer and inflammatory processes in the gastrointestinal system, wherethey are proposed to be involved in tissue regeneration, proliferation and protection. Our aim was toexplore their presence in pulmonary secretions and to investigate whether they are up-regulated inpulmonary diseases characterized by mucin hypersecretion. Bronchioalveolar lavage fluid was obtainedfrom 92 individuals referred to bronchoscopy. The patients were grouped according to diagnosis and pul-monary function. The concentrations of TFF1, TFF2 and TFF3 were measured by ELISA. All three peptides

ucinypersecretionsthmaFFronchoscopyung disease

were detected in bronchioalveolar lavage fluid. Patients with chronic obstructive pulmonary disease hadconcentrations two to three times above the levels in the healthy reference group, and patients withpulmonary malignancies had concentrations of TFF1 and TFF2 three times that of the reference group.The results suggest that TFFs are involved in tissue regeneration, proliferation and protection in lungdiseases.

© 2014 Elsevier Inc. All rights reserved.

ntroduction

Trefoil factors (TFFs) are small (12–22 kDa) protease-resistanteptides secreted by mucus-producing cells throughout the body15]. Three mammalian TFFs are known: TFF1, TFF2 and TFF3. Theyre characterized by the trefoil domain, a 42–43 amino acid chainith 3 disulfide bonds which create a characteristic three-looped

tructure. TFF2 contains two trefoil domains whereas TFF1 and TFF3ontain only one, but have a free cysteine residue that allows dimerormation [22,48]. Until recently TFF2 was described as a monomer,ut a dimeric form is found in the porcine pancreas [38]. All TFF pep-

ides has also been described as heteromers; TFF1 and TFF2 formsisulfide linked-heterodimers with gastrokine-2 [16], gastric TFF2

∗ Corresponding author at: Department of Cardiothoracic Surgery, Copenhagenniversity Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark.el.: +45 35451170.

E-mail address: njen0021@regionh.dk (N.-E. Viby).

ttp://dx.doi.org/10.1016/j.peptides.2014.09.026196-9781/© 2014 Elsevier Inc. All rights reserved.

is non-covalently linked to MUC6 [38] and TFF3 forms heterodimerswith Mucus Associated FCGBP Protein [2].

The TFFs are expressed in almost all mucus-secreting cells inmucosal surfaces and exocrine glands throughout the body [21].They have been investigated most extensively in the gastroin-testinal tract. Their expression is increased in inflammatory boweldisease (IBD) and peptic ulcer, both in the inflamed mucosa and inthe ulcer-associated cell-line (UACL) that can arise in an inflamedand ulcerated mucosa [15,42,43].

TFF2 contains two trefoil domains which cross-link mucinmolecules, and produce a highly viscous gel and the dimeric formsof TFF1 and TFF3 form smaller mucin complexes with lower viscos-ity [5,40,41]. The single trefoil domain on monomeric TFF1 and TFF3is able to block the binding sites in the mucin molecules, therebyinhibiting polymerization. The TFF peptides are also present inthe respiratory tract. Immunohistochemical studies have local-

ized TFF1 and TFF3 to goblet cells, Clara cells and submucosalglands with TFF3 being most abundant [7,47]. TFF2 mRNA has beendetected only in one study in the lungs of asthma patients but notin healthy individuals [18]. TFF3 has shown to be implicated in

N.-E. Viby et al. / Peptides 63 (2015) 90–95 91

Table 1Characteristics of all patients referred to bronchoscopy. The reference group included patients with no pathology seen in bronchoscopy and FEV1 ≥ 70%. The group of patientswith pulmonary cancer and metastasis were diagnosed by biopsies and subsequent pathological analysis. The group of patients with normal bronchoscopy had either aFEV1/FVC < 70% or no pulmonary function test performed and could therefore not be included in the reference group. The group of patients with other diagnosis includedpatients with pulmonary infection, heart failure, asthma and bronchiectasis. The group of patients with unknown diagnosis included patients who could not be diagnosedby bronchoscopy and/or patients who wished no further examinations.

All Referencegroup

Pulmonarycancer

Metastasis Normal bron-choscopy

Otherdiagnosis

Unknowndiagnosis

All 92 12 34 5 14 21 6

GenderMale 45 8 14 4 7 8 4Female 47 4 20 1 7 13 2

Smoking statusNever smoker 15 1 2 3 2 6 1Former smoker 29 4 11 1 3 9 1Current smoker 43 7 21 1 9 4 1Unknown 5 0 0 0 0 2 3

Pulmonary function

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FEV1/FVC < 70% 27 0 13

FEV1/FVC ≥ 70% 35 12 8

Pulmonary function test not available 30 0 13

ucosal protection and repair processes, stimulating cell migrationia chemotaxis [6].

Airway mucins are hypersecreted by patients with pulmonaryiseases such as asthma and chronic obstructive pulmonary diseaseCOPD) [30] and the hypersecretion contributes to airway obstruc-ion and to patient morbidity and mortality [29,31].

Because the TFF peptides are secreted with mucin and becausehe amount of mucus is increased in lung diseases, we hypothe-ized that the amount of TFF would also be increased in pulmonaryiseases.

In the present study, we establish the presence of all three TFFsn pulmonary secretions. We report an increase in the levels of allhree peptides in bronchioalveolar lavage (BAL) fluid from patientsith COPD and a negative correlation between the levels of both

FF1 and TFF3 and pulmonary function. In addition we observedncreased levels of TFF1 and TFF2 in patients with malignant lesionsn the lungs.

aterials and methods

articipants

Ninety-two consecutive patients (31–85 years of age, 45en, 47 women), undergoing diagnostic bronchoscopy at theepartment of Pulmonology, Gentofte University Hospital, Copen-agen Denmark, were included in the study. The indications

or bronchoscopy were radiological findings in the form ofumor-like infiltrates, atelectasis, bronchiektasiae and abscess andymptoms such as hemoptysis, prolonged cough and recurringneumonias. Spirometry was retrievable in 62 patients beforeronchoscopy as a part of the routine examination program ofhe department. Forced expiratory volume in 1 second (FEV1)nd forced vital capacity (FVC) was used. Information aboutulmonary symptoms, medication, smoking, pulmonary functionnd final diagnosis were retrieved from the patients’ recordsetrospectively.

The group of individuals included was very heterogeneous butith a high prevalence of pulmonary cancer and COPD. The indi-

iduals were firstly divided according to the diagnosis stated in theronchoscopy report and the pathology report (Table 1) and sec-

ndly according to pulmonary function (Table 2). As a consequencef this there is an overlap between the COPD diagnosis and the otheriagnoses, for instance, 13 individuals with pulmonary cancer alsoad COPD.

1 9 3 11 0 13 13 5 5 4

Twelve individuals had normal pulmonary function, no pul-monary symptoms at the time of bronchoscopy and no pathologicalchanges found at the bronchoscopy or other examinations. Theywere typically individuals who had experienced a single haemopt-ysis during an otherwise uncomplicated respiratory tract infection.These twelve individuals were used as a reference group.

Of all the patients 27 individuals were diagnosed with COPDaccording to the National Clinical Guideline Center (NICE) guide-lines based on the results of their pulmonary function test(FEV1/FVC < 70%) [23]. The criteria of the Global initiative forChronic Obstructive Lung Disease (GOLD) was used to fur-ther divide the patients in severity of COPD (stage I: mildCOPD = FEV1 ≥ 80% predicted, stage II: moderate = 50% ≤ FEV1 < 80%predicted, stage III: severe = 30% ≤ FEV1 < 50% predicted) [1]. Clini-cal symptoms such as cough, mucus production and shortness ofbreath were also obtained but the COPD status was determined bythe result of the pulmonary function test alone.

Thirty-nine individuals were diagnosed with pulmonary malig-nancies. They were divided into two groups, primary lung cancer(n = 34) and patients with metastases to the lungs from cancers inother organs (n = 5). In the primary lung cancer group, 14 individ-uals were further typed into either small cell lung cancer (SCLC)(n = 3), adenocarcinomas (n = 5) or squamous cell carcinoma (n = 6),based on pathology reports on biopsies; the rest were not fur-ther typed and could therefore only be categorized as non smallcell lung cancer (NSCLC) (n = 20). TNM classification of malignanttumors was used to describe the extent of the patients cancer [36].Dividing into groups was performed when the spirometry data wasavailable, before anay measurements were performed in the BALfluid.

The local scientific ethics committee approved all procedures(permit number KA 04008 m). All participants gave oral and writtenconsent.

BAL fluid sampling

The BAL fluid was collected according to the ERS statementGuidelines for measurement of acellular components and standard-ization of BAL [10]. After inspection of the airways through thebronchoscope, and before any brushings or biopsies were taken,

the bronchoscope was introduced into a segmental bronchus. Themiddle lobe or the lingula was used if no macroscopic lesionwhere found elsewhere in the lung. The instrument was wedgedto occlude the airway. Saline was introduced in aliquots of 50 ml

92 N.-E. Viby et al. / Peptides 63 (2015) 90–95

Table 2Characteristics of reference group and COPD group and determination of GOLD stage. GOLD stage assesses the severity of COPD by FEV1% predicted. As seen the COPD groupalso involved 13 patients with malignancies, most of them having GOLD II stage COPD. The COPD group did not involve patients with other or unknown diagnoses.

All Reference group COPD GOLD I GOLD II GOLD III

All 35 12 23 4 16 3GenderMale 21 8 13 3 8 2Female 14 4 10 1 8 1

Smoking statusNever smoker 5 1 4 2 2 1Former smoker 11 4 7 1 6 0Current smoker 19 7 12 2 8 2

Medication�-2 agonist 4 0 4 1 3 0Steroid 0 0 0 0 0 0Anticholinergic 0 0 0 0 0 0�-2 agonists + steroid 4 0 4 1 2 1�-2 agonist + anticholinergic 1 0 1 0 1 0�-2 agonist +steroid +anticholinergic 2 0 2 1 0 0No medication 24 12 12 1 10 2

ars

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MalignanciesPrimary pulmonary cancer 13 0

Matastasis 1 0

nd subsequently aspirated under gentle suction. The first ten mletracted were collected and frozen at −20 ◦C for later TFF mea-urement.

easurement of TFF by ELISA

The concentrations of the TFFs where measured by ELISAechniques as previously described [43,44]. The main concern mea-uring TFF in BAL fluid was that it had a very inhomogeneousonsistency with a watery component containing semi-solid andiscous mucus fractions in which TFF could be undetectable by theLISA antibodies. Another concern was that the relatively high vis-osity of the BAL fluid would inhibit the diffusion of antigens in theamples during measurement. In order to obtain a uniform sample,he BAL fluid underwent ultrasound treatment three times (10 sach time with 5 s intervals). Samples of 250 �l were then cen-rifuged through a 0.22 �m microspin filter (Milipore) for 10 min at2000 G. The filters were flushed through with 250 �l ELISA buffer10 mmol/l phosphate, 145 mmol/l NaCl, pH 7.4) again for 10 mint 12000G. The two obtained filtrates were pooled and this resultedn fluid samples free of solid components to be analyzed.

The samples were further diluted with ELISA buffer prior to anal-ses to a final dilution of: TFF1: 100 and 1000 fold, TFF2: 4 and 10old, TFF3: 1000 and 5000 fold.

tatistical analysis

As the data did not follow a normal distribution, non-parametrictatistics were used. Results are expressed as medians with range.he Mann–Whitney-U test was used to compare the COPD and ref-rence groups, the Kruskal–Wallis tests to compare groups withifferent diagnoses, COPD stages, and type of cancer and if signif-

cant a post analysis with Dunnetts comparison was performed.pearman correlation test was used to investigate correlationetween the TFF levels and the pulmonary function, and to test the

mpact of possible confounders. All statistical analyses were donen SAS 9.13 (SAS Institute Inc. Cary, NC, USA) and Graph Pad Prism

for windows (Graph Pad Software Inc. La Jolla, CA, USA).

esults

The individuals included in this study ended up with a varietyf diagnoses after bronchoscopy and were divided into: A group

13 2 10 11 0 1 0

with no apparent lung disease, designated as the reference group(n = 12), a group of patients with malignant lung disease (n = 39),either primary lung cancer (n = 34) or metastatic cancer (n = 5), agroup of patients with normal bronchoscopy but FEV1/FVC <70%or no available pulmonary function test, and a group of other dis-eases such as pulmonary infection, bronchiectasis and heart failure(n = 21) or unknown diagnoses (n = 6).

In all the patients TFF3 was the most abundant of the pep-tides with a median (range) of 6962 (<5-91085) pmol/l, followedby TFF1, 986 (25-31810) pmol/l, and TFF2, <5 (<5-740). In the refer-ence group TFF3 was also the most abundant of the peptides witha median (range) of 3580 (313-13280) pmol/l, followed by TFF1,518 (25-2338) pmol/l, and TFF2, <5 (<5-147) pmol/l (Supplemen-tary Table 1). The TFF levels were compared to age, gender, smoking,medication, or clinical symptoms, but no impact of these factorswas found.

Due to the heterogeneity of diagnoses and lack of patient infor-mation the results from the groups of other and unknown diagnoseswere excluded from further analysis.

Twenty-three patients with COPD were compared to the refer-ence group. The concentrations of all TFFs were increased in BALfluid, the median concentration was increased three-fold for TFF1(p < 0.01) and TFF2 (p < 0.05) and two-fold for TFF3 (p < 0.05) (Fig. 1,Table 3).

When we staged the patients in the COPD group, we found thatpatients with stage II and III COPD had a higher concentration ofTFF1 and TFF3 in BAL fluid than patients with stage I COPD indicat-ing an increase in TFF with the severity of COPD (Table 3). Whenall the included patients with an available pulmonary function testwere correlated to the concentrations of TFF1, TFF2 and TFF3 inBAL fluid, we found a correlation between TFF1 and TFF3 concen-trations and FEV1 measurements, i.e. individuals with low FEV1tended to have higher concentrations of TFF1 (Spearman r = 0.169,p < 0.01) and TFF3 (spearman r = 0.090, p < 0.05) in the BAL- fluid(Fig. 2).

Patients diagnosed with primary lung cancer and lung metas-tases from other organs had a three-fold higher level ofTFF1compared to the reference group (p < 0.05). Patients with lungmetastases also had a significant higher level of TFF2 (p < 0.05)

(Fig. 3). Cancer stage (by TNM classification) or cancer type (ade-nocarcinoma, squamous cell carcinoma, SCLC or NSCLC) had noinfluence on the level of any of the peptides (Supplementary Table2).

N.-E. Viby et al. / Peptides 63 (2015) 90–95 93

Fig. 1. TFF levels in BAL fluid obtained from a reference group and patients with COPD. Results are shown as median and range. (A) TFF1, (B) TFF2 and (C) TFF3. Levels of thethree peptides are significant higher in the COPD group (**p < 0.01, *p < 0.05, Mann–Whitney-U test).

Fig. 2. Correlations between the levels of TFF measured in BAL fluid and pulmonary function in patients with an available pulmonary function test. Patients with unknown orno diagnoses were not included. (A) TFF1, (B) TFF2 and (C) TFF3. There is significant correlation between TFF1 and FEV1% of predicted value (p < 0.01, r = 0.169) and betweenTFF3 and FEV1% predicted value (p < 0.05, r = 0.090) (Spearmann correlation test).

Fig. 3. TFF levels in BAL fluid obtained from a reference group and patients with primary lung cancer and lung metastases. Grey dots represent patients diagnosed withCOPD, black dots represent patients with either normal or no spirometry data. (A) TFF1, (B) TFF2 and (C) TFF3. Levels of TFF1 are significantly increased in patients with lungcancer and in patients with metastases and levels of TFF2 are significantly increased in patients with metastases compared to the reference group (*p < 0.05, Kruskal–Wallistests and post analysis with Dunnetts comparison to reference group).

Table 3TFF levels in BAL fluid obtained from COPD patients. Results are given as median (range). Reference group had no pathology found in bronchoscopy and a normal FEV1/FVC.Patients in the COPD group had FEV1/FVC < 70%, and were staged by GOLD criteria using FEV1 according to the Global initiative for chronic Obstructive Lung Disease guidelines.Comparison between reference group and COPD is done by non-parametric test (Mann–Whitney U), comparison between GOLD stages is made by nonparametric one wayANOVA (Kruskal–Wallis followed by Dunnett comparison to GOLD stage I).

Group n TFF1 pmol/l p TFF2 pmol/l p TFF3 pmol/l p

Reference 12 518 (25-2337) – <5 (<5-147) – 3580 (313-13280) –COPD 23 1284 (193-10379) <0.01 21 (<5-733) <0.05 7044 (2812-54992) <0.05GOLD stage TFF1 pmol/l p TFF2 pmol/l p TFF3 pmol/l pGOLD I 4 701 (405-885) ns <5 (<5-85) ns 4093 (2812-5500) nsGOLD II 16 1983 (193-10379) <0.05 26 (<5-733) ns 7756 (3941-54992) <0.05GOLD III 3 4401 (1207-9015) <0.05 44 (<5-64) ns 16314 (4892-17168) ns

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iscussion

Previous data have suggested increased expression of TFFeptides in relation to mucosal inflammation primarily in the gas-rointestinal tract. Here we report the presence of TFF peptides inulmonary secretions and we report an increased level of TFF pep-ides in BAL fluids from patients with COPD and malignant lungiseases.

We found TFF3 to be the most abundant in BAL fluid, followed byFF1 and with TFF2 occurring in the lowest concentration. Identifi-ation of the TFF peptides in BAL fluid is in agreement with previousndings of TFF1 and TFF3 peptides in lung tissue by immunohisto-hemistry and by mRNA determination [7,21,47].

Interestingly we found all of the TFF peptides in increasedmounts in patients with COPD, and increased levels of TFF1 andFF2 in patients with pulmonary malignancies. Goblet cell metapla-ia and hyperplasia, together with peribronchiolar inflammationnd fibrosis is characteristic in the pathobiology of COPD [13] andhis appears to be preceded by an increase in TFF production.he increased TFF secretion could also be a consequence of thencreased amounts of mucin secretion in combination with theresence of several inflammatory cytokines. Inappropriate mucusroduction due to metaplasia in the bronchioli, has several demen-rial effects, including a reduction in bronchiolar antiprotease,eading to epithelial cell destruction [13]. The exact stimulus forhe goblet cell to increase the expression of the peptide is still notlucidated, but the epithelial damage could be a mediator, sinceFFs are shown to be involved in protection and regeneration inther tissues [23]. The results are in accordance with our previousndings; in a limited number of subjects we examined TFF pep-ides in sputum and serum of COPD (stage III) patients and asthmaatients. We found an increase in TFF1 and TFF3 in sputum, only inOPD patients not in asthmatic patients [45].

The increased levels of TFFs in patients with COPD were relatedo the pulmonary function: low FEV1 was related to high concentra-ions of both TFF1 and TFF3. Since the cause of COPD developments extensive and persistent toxic damage, this seems to result inn increased production and hypersecretion of the peptides. Sev-ral functions of TFF peptides have been suggested in relation toancer pathogenesis including enhanced cell migration [37,39], cellifferentiation, angiogenesis [28,37,39], and anti-apoptotic effects4,8], all traits that are associated with cancer development, growthnd invasion [26]. The hyperproduction and secretion of the pep-ides after long lasting damage could be involved in the pathwayrom COPD/heavy smoking to cancer, but this hypothesis need fur-her investigation. We measured the peptides with a well validatedLISA, using recombinant peptides as standards and antibodiesaised against human TFFs, the different in-house and commercialLISAs have recently been reviewed by Samson [34]. The peptidesre shown to be found in different forms both monomeric, dimericnd hetreo-dimeric in secretions from the gastro intestinal system,ut it is still unknown if this is also the case in secretions from theespiratory tract. However it is a challenge measuring the exact con-entrations of the peptides, since some of the forms could escapehe binding sites of the ELISA assay. We assume this failure to beystematic and therefore used the ELISA to compare the differentroups.

TFFs have been proposed as promising targets to treat airwayiseases based on experimental data. Kouznetsova et al. found thathen murine Clara cells were trans-differentiating into a mucoushenotype after allergen exposure, TFF1 expression was increased17] and LeSimple et al. showed that exogenous TFF3 added to

n vivo humanized airway zenograft, increased the number of cellsositive for the ciliated cell marker �-tubulin [19]. Together theseesults support our results showing that TFF1 and TFF3 increasehen pulmonary epithelia are damaged and that the peptides play

s 63 (2015) 90–95

a role in cell-differentiation. A recent study from 2013 showed thatexogenous TFF2 was directly protective against epithelial damagein a naphthalene induced murine model of airway injury [32].

The TFF peptide expression is altered in several cancers includ-ing lung, mammary, gastro intestinal cancers and prostate cancers[3,9,14,20,24,25,27,33,35]. Primarily TFF1 and to some extent TFF2and TFF3 are over-expressed in the cancers. Pulmonary adenocar-cinomas of the subtypes acinar and bronchoalveolar carcinomas,and squamous cell carcinomas express high levels of TFF1, shownby immunohistochemistry and radio-immuno assays [33]. Fortypercent of small cell lung cancers and carcinoid lung tumors havepositive TFF1 immunostaining [46]. Expression of TFF1 by a pul-monary tumor is associated with a poor prognosis, possibly linkedto increased expression of CA125 and decreased expression of theEGF-receptor [11]. TFF1 is elevated in serum in advanced stagesof adenocarcinomas and bronchoalveolar carcinomas and it isnormalized after removal of the tumor [12]. Our results are in accor-dance with these findings in the literature. We found elevated levelsof TFFs (though TFF2 and TFF3 not significantly) in patients with pri-mary pulmonary cancer. We had a very limited number of patientsand this may be the reason why we found no significant differencein levels of the peptides among the different cancer stage groupsor cancer type groups.

Five patients were diagnosed with metastasis, and thesepatients showed an increase in TFF1 and TFF2, compared to thereference group. We do not know if this increased secretion relatesfrom the tumor itself or is a consequence of the pathological tis-sue present in the lung. The linear regression analysis did not findany confounding effect of gender, age, symptoms, medication orsmoking, but very few of the cancer patients were actually “neversmokers”, meaning that the toxic damages to the lung had beenpersistent.

In conclusion we report the presence of TFF peptides, especiallyTFF3, in pulmonary secretions. The concentrations of TFF in theBAL fluid are highly increased in patients with COPD and show aninverse correlation to lung function. Furthermore we found a highlevel of TFF1 and TFF 2 in patients with pulmonary cancer.

Disclosure statement

Lars Thim is employed at Novo Nordisk A/S. Niels-Erik Viby, EbbaNexø, Hannelouise Kissow, Helle Andreassen, Paul Clementsen andSteen Seier Poulsen has nothing to declare.

Acknowledgments

We warmly acknowledge the expert technical assistance ofInger Marie Jensen and the assistance in the patient chart archivesof Heidi Marie Paulsen.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.peptides.2014.09.026.

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