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unicationUteroglobin-related protein 1 and Clara cell protein in induced sputum of

patients with asthma and rhinitis

Journal: CHEST

Manuscript ID: CHEST-06-0835.R1

Manuscript Type: Manuscript

Date Submitted by the Author:

30-Jun-2006

Complete List of Authors: Claire, de Burbure; Catholic University of Louvain, School of Public Health Patrizia, Pignatti; Fondazione Salvatore Maugeri, Allergy Corradi, Massimo; University of Parma, Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences Mario, Malerba; Universita of Brescia, department of Internal Medicine André, Clippe; Catholic University of Louvain, School of Public Health Xavier, Dumont; Catholic University of Louvain, School of Public Health Gianna, Moscato; Fondazione Salvatore Maugeri, Allergy Mutti, Antonio; University of Parma, Laboratorio di Tossicologie Industriale bernard, alfred; Catholic Universioty of Louvain, Public Health

Keywords: ALLERGY, ASTHMA, SPUTUM

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Uteroglobin-related protein 1 and Clara cell protein in induced sputum of patients with

asthma and rhinitis

de Burbure Claire1, Pignatti Patrizia2, Corradi Massimo3, Malerba Mario4, Clippe André1,

Dumont Xavier1, Moscato Gianna2, Mutti Antonio3, Bernard Alfred1

1Unit of Industrial Toxicology and Occupational Medicine, Faculty of Medicine, Université

Catholique de Louvain, Belgium

2Allergy and Immunology Unit, Fondazione Salvatore Maugeri, IRCCS, Pavia, Italy

3Laboratorio di Tossicologia Industriale, Universita degli Studi di Parma, Parma, Italy

4Department of Internal Medicine, University of Brescia, Spedali Civili, Brescia, Italy

Running title: Asthma/Rhinitis: New Sputum Proteins

Conflict of interest: none of the authors has a conflict of interest to disclose

Address correspondence to A. Bernard, Unit of Industrial Toxicology and Occupational

Medicine, Université Catholique de Louvain, Clos Chapelle-aux-Champs 30, bte 3054, B-

1200 Brussels Belgium Phone: 32-2-7643220, Fax: 32-2-7643228, e-mail:

bernard@toxi.ucl.ac.be

Total word count = 3,216

Key words: induced sputum, uteroglobin-related protein 1, Clara cell protein, asthma, rhinitis.

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LIST OF ABBREVIATIONS

Alb: albumin

CC16 : Clara cell protein

DTT : dithiothreitol

FEV1: forced expiratory volume in one second

PD20FEV1: methacholine dose causing a 20% fall in FEV1

UGRP1: uteroglobin-related protein 1

VC: vital capacity

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ABSTRACT

(Word count: 217)

Rationale: uteroglobin-related protein 1 (UGRP1) and Clara cell protein (CC16), members of

the secretoglobin family, increasingly appear to play a role in airway inflammatory response.

Objectives: to explore levels of UGRP1 and CC16 in induced sputum of patients with asthma

and rhinitis.

Methods: induced sputum samples of patients suffering from asthma or rhinitis (n = 32 each,

atopics 24/32 and 20/32 respectively) and from 19 non-smoking non-atopic controls were

analyzed for cytology and levels of UGRP1, CC16 and albumin.

Measurements and main results: sputum UGRP1 increased in both asthma and rhinitis,

most strikingly so in asthma, where changes were most significant in atopic individuals. By

contrast, sputum CC16 did not change significantly in either condition although it was

positively correlated with UGRP1 in patients and controls. Changes in sputum UGRP1 in

atopic asthma were not linked to permeability changes reflected by increased albumin levels,

but correlated positively with sputum macrophages and negatively with eosinophils. The

observed differences in UGRP1 and CC16 may be linked to different cell populations being

responsible for their secretion, UGRP1 being mainly secreted in larger conducting airways

whereas CC16 is mainly secreted by the nasal and peripheral airways epithelium.

Conclusions: the increase in UGRP1 but not of CC16 in asthma and rhinitis suggests that

UGRP1 may play a role in these inflammatory diseases.

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INTRODUCTION

(Word count: 450)

Non-invasive methods for assessing the airway inflammatory response, which is central to the

pathogenesis of asthma and rhinitis, have been developed in the last decade with the hope to

reliably and accurately monitor patient progress and response to therapy. One such newly

developed and evolving tool consists in the study of lung-specific proteins secreted into the

airways lining fluid by both the conducting and the respiratory airway epithelial cells1.

The Clara cell protein, small 16kD protein secreted throughout the airways but mainly by the

bronchiolar Clara cell (hence called CC16), has been studied in relation to asthma and rhinitis

in particular as its gene is localized on the p12-q13 region of human chromosome 11 involved

in inflammation regulation. CC16 expression is indeed decreased in asthma2 and in case of

respiratory tract inflammation in both man3 and animals4,5. A38G gene polymorphism has

also been linked to lower serum CC16 levels and a higher risk of being asthmatic in some

populations6,7. A recent gene profiling study in allergic rhinitis furthermore reported CC16 to

have the most decreased anti-inflammatory gene expression among over 44,927 genes8.

CC16-deficient mice show increased sensitivity to hyperoxia or ozone damage, exaggerated

inflammatory responses1, with increased pulmonary eosinophilia when antigen-sensitized and

challenged9. CC16 has important immunosuppressive and antiinflammatory properties, which

include the inhibition of inflammatory mediators such as cytosolic phospholipase A2,

interferon-γ, tumor-necrosis factor α, as well as platelet-derived growth factor-induced

chemotaxis of fetal lung fibroblasts1. Measuring CC16 in bronchoalveolar lavage fluid, where

it reflects epithelial function, and in serum, bringing useful information on the permeability of

the alveolocapillary membrane, has therefore proven useful in assessing both acute and

chronic airway damage.

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Recently another small protein belonging to the same family as CC16, the secretoglobin

family10, has been discovered: uteroglobin-related protein 1 (UGRP1) is a 17 kDa

homodimeric lung-specific protein11,12,13 also called high in normal 2 protein (HIN2) or

secretoglobin SCGB3A212. As mouse UGRP1 has an overall aminoacid sequence identity of

25% to mouse Clara cell protein, including an area called antiflammin containing several

lysine residues, it is supposed to share some of its anti-inflammatory properties although its

precise functions are still unknown11. UGRP1 appears highly specific of the airways, and our

laboratory has been characterizing, sequencing and evaluating it along other pneumoproteins

as potential biomarker of lung damage since 1999 (GenBank deposit: UGRP2: AF436839-41;

UGRP1: AF439544-7)14. Expression of UGRP1 mRNA has been shown to decrease in mouse

lung after induction of inflammation by in situ hybridization11.

In this study, levels of CC16 and UGRP1 were analyzed for the first time in induced sputum

samples of patients with either asthma or rhinitis with a view to investigating the differential

diagnostic potential of the two pneumoproteins in these two conditions.

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MATERIALS AND METHODS

(Word count = 667 + online supplement!)

Population studied

Patients gave their signed informed consent to participate into the study, which was approved

by the Ethical Committees of the Salvatore Maugeri Foundation IRCCS and the University

of Brescia and was conducted according to the Declaration of Helsinki. Induced sputum

specimens and lung function tests were obtained from 32 patients with intermittent to

moderate-persistent bronchial asthma, 32 rhinitis patients and 19 healthy volunteers

(men/women: 21/11, 10/22 and 10/9 respectively). Patients came from Pavia (Allergy and

Immunology Unit, Fondazione Salvatore Maugeri) and controls from Brescia or Parma

University (Internal Medicine and Industrial Toxicology respectively). Atopy was defined as

at least one positive skin prick test for common inhalant allergens (with positive histamine

prick). Bronchial asthma was diagnosed according to NHI Guidelines15. Allergic rhinitis was

diagnosed according to ARIA Guidelines16. Patients with rhinitis symptoms and negative skin

tests, after consultation with an ear, nose and throat specialist, received the diagnosis of non-

allergic rhinitis.

Sputum induction and processing

Sputum induction was obtained as previously described17. Sputum processing was performed

with 0.1% dithiothreitol (DTT), according to International guidelines17,18. After separation

from supernatant by centrifugation, cell count and viability by trypan blue exclusion were

determined by light microscopy. Cytospins were stained with Diff-Quick (Dade Diagnostika

GmbH, Unterscheiβheim), analyzed for differential cell count by counting 500 nonsquamous

cells minimum, and reported as percentages of total nonsquamous cells. Only sputum samples

with less than 30% squamous cells were considered acceptable.

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Sample analysis

Sputum supernatants were stored at –80 °C until evaluation. Sputum CC16 and albumin were

quantified by semi-automated non-isotopic latex immunoassay as previously described19,20.

The procedure for quantifying UGRP1 was similar to the above, using a combination of two

in-house polyclonal anti-UGRP1 antibodies. The between and within-run coefficients of

variation of both immunoassays in biological fluids, including sputum, were between 5 and

10%. The effect of DTT treatment on the concentrations of UGRP1 and CC16 was assessed

by adding DTT (0.1%) to six sputum samples with UGRP1 and CC16 concentrations ranging

from 20 to 3,980 µg/l and 27 to 2,170 µg/l, respectively. The concentrations of UGRP1 in

treated and untreated samples were highly correlated (Pearson’s r = 0.98, p<0.001) with post-

treatment values averaging 70 % of pretreatment values. Concentrations of CC16 in treated

and untreated samples were also highly correlated (Pearson’s r = 0.94, p<0.001) with no

systematic difference (post-treatment values averaged 102 % of the pre-treatment values).

Additional detail on the method for making UGRP1 measurements is provided in an online

data supplement. Due to the small volumes of some samples, all biological parameters could

not be determined in all subjects, exact numbers are indicated in the tables.

Lung function tests

Spirometry was performed according to European Respiratory Society guidelines21 with a

computerized water-sealed spirometer (BIOMEDIN, Padova). Measurements were expressed

as percentage of predicted normal values based on race, age, sex, and height. Methacholine

challenge testing was performed with a nebulizer (MEFAR, Brescia) connected to a dosimeter

as previously reported22. The methacholine dose causing a 20% fall in FEV1 (PD20FEV1)

was considered positive if PD20 was <1000 µg.

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Statistical analysis

Results are presented as means ± SEM. For statistical analysis, UGRP1, CC16, albumin and

absolute macrophage or eosinophil counts in sputum were normalized by log transformation,

and their results are presented as geometric means. Group means were compared by two-sided

unpaired Student’s t test or by ANOVA followed by Dunnett’s post hoc test. Associations

between sputum UGRP1, CC16, albumin and sputum cell counts were evaluated by simple

regression analysis and calculating Pearson’s correlation coefficient. Influence of age, gender

(male/female), diagnosis (asthma, 0/1; rhinitis, 0/1), smoking status (never-smoker/current

smoker) and atopy (0/1) was studied by stepwise multiple regression (entry: p = 0.25, staying

in model: p = 0.05). Three past-smokers were grouped with never-smokers. All statistical

analyses were performed using StatView® (StatView 5, SAS 2000-2001 © SAS Institute

Inc.). Figures were drawn with StatView or GraphPad Prism (GraphPad Prism 3.03, 2002, ©

GrapPad Prism Inc.). Statistical significance was set at 0.05.

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RESULTS

(Word count = 713)

The general characteristics, lung function and sputum cytology of controls, rhinitis and

asthmatic patients are given in Table 1, including disease duration, atopy and smoking status,

steroid treatment, methacholine sensitivity, sputum cytology and some lung function tests.

Whereas all 19 controls were non-smokers and non-atopic, about four in ten asthmatic and

rhinitis patients were smokers, and two thirds atopic. There were no significant differences in

lung function between patients and controls, indicating overall good control of the disease.

However, in comparison with rhinitis patients, asthmatics had significantly lower mean values

of FEV1, Tiffeneau index and vital capacity. Only one rhinitis patient (1/32, 3.1%) was

methacholine sensitive compared to 12/32 asthmatics (37.5%). The proportions of asthmatics

with a positive methacholine test were similar between those receiving inhaled corticosteroids

(7/19, 36.8%; low dose, 1/6; medium dose 4/9 and high dose, 1/6) and those who were not

under steroid medication (5/13, 38.5%). As expected, asthmatics had significantly raised

sputum eosinophil numbers but reduced macrophages compared to both controls and rhinitis

patients. Eosinophils were also raised in rhinitis compared to controls, though much less

dramatically so than in asthma. Epithelial cell numbers were increased in both groups of

patients compared to controls.

Concentrations of sputum UGRP1, CC16 and albumin in control, rhinitis and asthma subjects

are compared in Figure 1. Whereas variations in CC16 levels were not significantly different

between the three groups, UGRP1 was elevated in both rhinitis and asthma patients compared

to controls, particularly so in asthma. Sputum albumin levels only rose significantly in

asthma. Stepwise multiple regression analysis on the whole population taking age, gender,

smoking status, asthma and rhinitis into consideration confirmed the only significant

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determinants of sputum UGRP1 to be both asthma (partial r = 0.35, p = 0.013) and rhinitis

(partial r = 0.27, p = 0.011), while none of the tested predictors influenced sputum CC16.

Albumin levels were positively associated with asthma (partial r = 0.48, p < 0.0001), whereas

smoking status was a negative determinant (partial r = -0.26, p = 0.0112). Division by atopy

did not change results concerning CC16 and albumin. By contrast, this splitting revealed that

rhinitis determined UGRP1 levels in non-atopics only (partial r = 0.354, p = 0.029) while

asthma remained sole significant determinant in atopics (partial r = 0.415, p = 0.005). The

influence of atopy on sputum UGRP1 levels is illustrated in Figure 2.

Methacholine sensitivity did not appear to affect either CC16 or UGRP1 sputum levels

globally. Among atopics, however, methacholine positive subjects had significantly increased

protein levels (CC16 and UGRP1: p = 0.045 and 0.009 respectively between 10 positive vs 28

negative subjects). Sputum albumin levels also increased with positive methacholine

challenge (12 positive vs 40 negative subjects: p = 0.024), but again this increase only

concerned atopics (9 positive vs 26 negative subjects: p = 0.008, p not significant for non-

atopic methacholine groups). Comparing the various inhaled steroid regimens in our small

number of treated subjects did not show any statistically significant differences in sputum

UGRP1 and CC16 (results not shown).

Sputum UGRP1 was significantly correlated to CC16 in all three subject groups whereas

correlations of these two proteins with albumin were weak and even non-existent in the case

of asthma (Figure 3). In atopic asthmatics, sputum UGRP1 and CC16 correlated positively

with macrophages and negatively with eosinophils expressed as total cell percentage, whereas

albumin indicated opposite correlations (Figure 4). These correlations were confirmed when

using absolute cell numbers per milligram and more pertinently per milliliter of sputum, unit

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used for proteins. In particular, positive correlations with sputum macrophages were then also

observed for the whole population (UGRP1 (n=64): R2=0.165, p=0.0009 and R2=0.187,

p=0.0004; CC16 (n=64): R2=0.21, p=0.0001 and R2=0.234, p<0.0001 with macrophage

numbers per ml and mg of sputum, respectively). It is important to realize that correlations

emerging between URGP1 and CC16 as well as between these proteins and cells counts, were

calculated over very wide ranges of values, requiring logarithmic scales to be displayed.

Although statistically very significant, most of these correlations reflect associations that are

relatively weak. UGRP1 concentration in sputum, for instance, can show variations of more

than one order of magnitude for a given value of CC16 (Figure 3) while less than 25% of

UGRP1 or CC16 variations are related to changes in sputum eosinophils (Figure 4).

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DISCUSSION

(word count = 961)

To our knowledge this is the first report of UGRP1 protein being assayed in any human

biological sample, and the first time CC16 is measured in induced sputum. Most interestingly,

UGRP1 levels were significantly increased in both asthma and rhinitis compared to controls,

the rise in atopic asthma being the most remarkable. CC16 levels, although correlated to

UGRP1, were by contrast not observed to change significantly in either asthma or rhinitis.

These increases in UGRP1 cannot be explained by epithelial permeability changes, known to

occur in both asthma and rhinitis23. UGRP1 levels were indeed only weakly or not at all

correlated with sputum albumin. Furthermore, as UGRP1was undetectable in serum samples

of this study (see materials and methods) transepithelial leakage from blood is unlikely to be

the cause of the high sputum UGRP1 levels. The only plausible mechanism for increased

UGRP1 in sputum appears therefore to be an increased synthesis and secretion of the protein

in those areas contributing to sputum collection.

Our findings concerning CC16 in sputum were rather unexpected from several points of view.

According to the literature, a decrease in CC16 could have been expected both in asthma and

in rhinitis. Indeed, patients who suffered from asthma for longer than 10 years have decreased

serum CC1624, in correlation with decreased populations of Clara cells in their airways2.

Furthermore, children suffering from allergic rhinitis were recently shown to have decreased

CC16 levels in nasal fluid25 and highly reduced CC16 gene expression8. The lack of changes

of sputum CC16 in asthma or rhinitis probably stems from the fact that induced sputum

mainly originates from central airways, with little or no recovery from the peripheral

respiratory system where CC16 is predominantly produced26. By contrast, serum levels of

CC16 mainly originate from distal and peripheral airways, which offer a vast surface area for

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transepithelial leakage into the bloodstream1. In comparison with sputum, serum might

therefore be a more reliable indicator of the amount of CC16 secreted in the lower respiratory

tract. Even though sputum CC16 levels in atopic asthmatics were overall unchanged, they

were found to correlate positively with macrophage and negatively with eosinophil

percentages. The correlation with macrophages might simply reflect their role in the

respiratory tract defense mechanisms, and maybe also tie up with the known role of

macrophages in phagocytosis and recycling of surfactant and other lung secretory proteins27.

The negative correlation with eosinophils is likely linked to the anti-inflammatory role of

CC168,25.

With regard to UGRP1, similarly to CC16, genetic studies predicted lower uteroglobin-related

protein 1 levels in some asthmatics, following observations of reduced transcriptional activity

linked to a G-A point mutation at –112bp in the human UGRP1 gene promoter13. However,

studies in CC16-null mice demonstrated compensatory increases in UGRP1 and UGRP2

mRNA expression28. Assuming altered CC16 gene expression in asthma and rhinitis, as

suggested by the above-mentioned studies8,24, could therefore in part explain a compensatory

rise in UGRP1 as observed in our studies. Individual genetic differences linked to known

CC16 and UGRP1 gene polymorphism could further explain the differences observed,

notably the significant rise in UGRP1 in atopic asthmatics only29,13. Interestingly, although

weak and not specific of URGP1 (also found with CC16) the negative correlation in atopic

asthmatics between UGRP1 and eosinophil percentage is consistent with the downregulation

of UGRP1 expression observed in ovalbumin-challenged mice with high tissue eosinophilia

linked to high interleukin-5 levels30, as well as the very recent study by Chiba et al. (2006,

epub ahead of print)31 reporting that intranasal administration of adeno-UGRP1 markedly

reduced the number of infiltrating inflammatory cells, particularly eosinophils. As with CC16,

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correlations of UGRP1 with sputum macrophages may be linked to their role in defense

mechanisms, UGRP1 being a known ligand of the MARCO receptor (macrophage scavenger

receptor with collagenous structure) of alveolar macrophages32. Interestingly, although cell

numbers were overall in agreement with induced sputum reference values33,34, total cell

numbers were increased in our asthmatic patients, whereas macrophage percentages were

significantly reduced. Further studies would be required to fully comprehend the interactions

between UGRP1 and macrophages, particularly in asthma where macrophage function

alterations feature high in the recent literature35,36.

Morphological studies offer possible mechanistic clues for the observed differences between

UGRP1 and CC16: human UGRP1-secreting cells are located not only primarily in the larger

upper airways but in sub-mucosal glands in particular, whereas CC16 secretion is

predominant in the lower conducting airways although it also occurs in sub-mucosal acinar

and duct cells early in life11,28 and in some goblet cells37. Since asthma is known to lead to

airway remodeling, not only with goblet cell hyperplasia but with hypertrophy and

hyperplasia of the submucosal glands in particular38,39, the rise in UGRP1 observed in

asthmatic patients could reflect this hypersecretory state and therefore become an indirect

biomarker of airway remodeling. The significant rise in UGRP1 observed in rhinitis, albeit

smaller than in asthma, corroborates recent reports that remodeling occurs not only in asthma

but also in rhinitis and other conditions inducing chronic cough40,41. It would furthermore be

worth investigating how CC16 and UGRP1 levels would respond to corticosteroid treatment

recently shown to reverse airway remodeling in mice43, as the small number of patients in

each treatment category did not allow us to make conclusive observations.

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Finally, it appears from our study in asthma and rhinitis that induced sputum, clearly less

invasive than bronchial lavage, could prove a most helpful tool for monitoring inflammatory

cells and anti-inflammatory proteins such as UGRP1 and CC16. This could be practical for

clinicians keen to monitor treatment effect on remodeling in individual patients44. As this

study is the first report of UGRP1 and CC16 levels in induced sputum, further studies

conducted in larger numbers of atopic and non-atopic patients would be required to confirm

the differential levels of these two new biomarkers in asthma and rhinitis.

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ACKNOWLEDGMENTS

(word count = 35 words)

The authors wish to thank the staff members of the Internal Medicine and Industrial

Toxicology Departments of Brescia and Parma Universities respectively for volunteering to

provide the induced sputum control samples used in this study.

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REFERENCES

1. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential

applications as markers. Am J Respir Crit Care Med. 1999 Feb;159(2):646-78.

2. Shijubo N, Itoh Y, Yamaguchi T et al. Clara cell protein-positive epithelial cells are

reduced in small airways of asthmatics. Am J Respir Crit Care Med. 1999

Sep;160(3):930-3.

3. Lensmar C, Nord M, Gudmundsson GH et al. Decreased pulmonary levels of the anti-

inflammatory Clara cell 16 kDa protein after induction of airway inflammation in

asthmatics. Cell Mol Life Sci. 2000 Jun;57(6):976-81.

4. Arsalane K, Broeckaert F, Knoops B, Wiedig M, Toubeau G, Bernard A. Clara cell

specific protein (CC16) expression after acute lung inflammation induced by intratracheal

lipopolysaccharide administration. Am J Respir Crit Care Med. 2000 May;161(5):1624-

30.

5. Hayashida S, Harrod KS, Whitsett JA. Regulation and function of CCSP during

pulmonary Pseudomonas aeruginosa infection in vivo. Am J Physiol Lung Cell Mol

Physiol. 2000 Sep;279(3):L452-9.

6. Laing IA, Hermans C, Bernard A, Burton PR, Goldblatt J, Le Souef PN. Association

between plasma CC16 levels, the A38G polymorphism, and asthma. Am J Respir Crit

Care Med. 2000 Jan;161(1):124-7.

7. Candelaria PV, Backer V, Laing IA et al. Association between asthma-related phenotypes

and the CC16 A38G polymorphism in an unselected population of young adult Danes.

Immunogenetics. 2005 Apr;57(1-2):25-32. Epub 2005 Mar 3.

8. Benson M, Jansson L, Adner M, Luts A, Uddman R, Cardell LO. Gene profiling reveals

decreased expression of uteroglobin and other anti-inflammatory genes in nasal fluid cells

from patients with intermittent allergic rhinitis. Clin Exp Allergy. 2005 Apr;35(4):473-8.

Page 17 of 32

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unication

18

9. Chen LC, Zhang Z, Myers AC, Huang SK. Cutting edge: altered pulmonary eosinophilic

inflammation in mice deficient for Clara cell secretory 10-kDa protein. J Immunol. 2001

Sep 15;167(6):3025-8.

10. Klug J, Beier HM, Bernard A et al. Uteroglobin/Clara cell 10-kDa family of proteins:

nomenclature committee report. Ann N Y Acad Sci. 2000;923:348-54.

11. Niimi T, Keck-Waggoner CL, Popescu NC, Zhou Y, Levitt RC, Kimura S. UGRP1, a

uteroglobin/Clara cell secretory protein-related protein, is a novel lung-enriched

downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Mol

Endocrinol. 2001 Nov;15(11):2021 -36.

12. Niimi T, Copeland NG, Gilbert DJ et al. Cloning, expression, and chromosomal

localization of the mouse gene (Scgb3a1, alias Ugrp2) that encodes a member of the novel

uteroglobin-related protein gene family. Cytogenet Genome Res. 2002a;97(1-2):120-7.

13. Niimi T, Munakata M, Keck-Waggoner CL et al. A polymorphism in the human UGRP1

gene promoter that regulates transcription is associated with an increased risk of asthma.

Am J Hum Genet. 2002b;70(3):718-25. Epub 2002 Jan 25.

14. HELIOS Project Final Report, Research and Technological Development Programme

“Quality of Life and Management of Living Resources” Key action 4 “Environment and

Health” QLK4-CT-1999-01308, 2003; pp 1-63; http://airnet.iras.uu.nl/products/

reports_and_annexes/HELIOS/HELIOS_final_report_Part_A.pdf

15. National Asthma Education and Prevention Program, Guidelines for the diagnosis and

management of Asthma, Expert Panel Report 2, National Institute of Health, National

Heart, Lung, and Blood Institute, 1997.

16. Bousquet J, Van Cauwenberge P, Khaltaev N; Aria Workshop Group; World Health

Organization. Allergic rhinitis and its impact on asthma J Allergy Clin Immunol. 2001 (5

Suppl):S147-334.

Page 18 of 32

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19

17. Djukanovic R, Sterk P, Fahy JV, Hargreave FE. Standardized methodology of sputum

induction and processing. Eur Resp J 2002;20 supp.37:1s-55s.

18. Pignatti P, Delmastro M, Perfetti L et al. Is dithiothreitol affecting cells and soluble

mediators during sputum processing? A modified methodology to process sputum. J

Allergy Clin Immunol 2002;110:667-8.

19. Bernard AM, Lauwerys RR. Continuous-flow system for automation of latex

immunoassay by particle counting. Clin Chem. 1983;29:1007-11.

20. Bernard A, Marchandise FX, Depelchin S, Lauserys R, Sibille Y. Clara cell protein in

serum and bronchoalveolar lavage. Eur Respir J. 1992;5:1231-8.

21. Quanjer PH. Standardization of lung function testing. Bull Europ Physiopathol Resp

1983;19 (Suppl 5):45-51.

22. Crapo RO, Casaburi R, Enright PL et al. Guidelines for methacholine and exercise

challenge testing-1999. Am J Respir Crit Care Med 2000;161:309-29.

23. Svensson C, Andersson M, Greiff L, Alkner U, Persson CG. Exudative

hyperresponsiveness of the airway microcirculation in seasonal allergic rhinitis. Clin Exp

Allergy. 1995 Oct;25(10):942-50.

24. Shijubo N, Itoh Y, Yamaguchi T et al. Serum levels of Clara cell 10-kDa protein are

decreased in patients with asthma. Lung. 1999;177(1):45-52.

25. Johansson S, Keen C, Stahl A, Wennergren G, Benson M. Low levels of CC16 in nasal

fluid of children with birch pollen-induced rhinitis. Allergy. 2005 May;60(5):638-42.

26. Alexis NE, Hu SC, Zeman K, Alter T, Bennett WD. Induced sputum derives from the

central airways: confirmation using a radiolabeled aerosol bolus delivery technique. Am J

Respir Crit Care Med. 2001 Nov 15;164(10 Pt 1):1964-70.

27. Wright JR. Clearance and recycling of pulmonary surfactant. Am J Physiol. 1990

Aug;259(2 Pt 1):L1-12.

Page 19 of 32

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123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

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20

28. Reynolds SD, Reynolds PR, Pryhuber GS, Finder JD, Stripp BR. Secretoglobins

SCGB3A1 and SCGB3A2 define secretory cell subsets in mouse and human airways. Am

J Respir Crit Care Med. 2002 Dec 1;166(11):1498-509. Epub 2002 Aug 01.

29. Sengler C, Heinzmann A, Jerkic SP et al. Clara cell protein 16 (CC16) gene

polymorphism influences the degree of airway responsiveness in asthmatic children. J

Allergy Clin Immunol. 2003 Mar;111(3):515-9.

30. Chiba Y, Srisodsai A, Supavilai P, Kimura S. Interleukin- 5 reduces the expression of

uteroglobin-related protein (UGRP) 1 gene in allergic airway inflammation. Immunol Lett.

2005 Feb 15;97(1):123-9.

31. Chiba Y, Kurotani R, Kusakabe T, Miura T, Link BW, Misawa M, Kimura S.

Uteroglobin-related Protein 1 Expression Suppresses Allergic Airway Inflammation in

Mice. Am J Respir Crit Care Med. 2006 Feb 2; [Epub ahead of print]

32. Bin LH, Nielson LD, Liu X, Mason RJ, Shu HB. Identification of uteroglobin-related

protein 1 and macrophage scavenger receptor with collagenous structure as a lung-specific

ligand-receptor pair. J Immunol. 2003 Jul 15;171(2):924-30.

33. Belda J, Leigh R, Parameswaran K, O'Byrne PM, Sears MR, Hargreave FE. Induced

sputum cell counts in healthy adults. Am J Respir Crit Care Med. 2000 Feb;161(2 Pt

1):475-8.

34. Spanevello A, Confalonieri M, Sulotto F et al. Induced sputum cellularity. Reference

values and distribution in normal volunteers. Am J Respir Crit Care Med. 2000 Sep;162(3

Pt 1):1172-4.

35. Alexis NE, Soukup J, Nierkens S, Becker S. Association between airway hyperreactivity

and bronchial macrophage dysfunction in individuals with mild asthma. Am J Physiol

Lung Cell Mol Physiol. 2001 Feb;280(2):L369-75.

Page 20 of 32

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123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

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unication

21

36. Peters-Golden M. The alveolar macrophage: the forgotten cell in asthma. Am J Respir

Cell Mol Biol. 2004 Jul;31(1):3-7.

37. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of Clara cells in

normal human airway epithelium. Am J Respir Crit Care Med. 1999 May;159(5 Pt

1):1585-91.

38. Morcillo EJ, Cortijo J. Mucus and MUC in asthma. Curr Opin Pulm Med. 2006

Jan;12(1):1-6.

39. Boser SR, Park H, Perry SF, Menache MG, Green FH. Fractal geometry of airway

remodeling in human asthma. Am J Respir Crit Care Med. 2005 Oct 1;172(7):817-23.

Epub 2005 Jun 23.

40. Frieri M. Inflammatory issues in allergic rhinitis and asthma. Allergy Asthma Proc. 2005

May-Jun;26(3):163-9.

41. Niimi A, Torrego A, Nicholson AG, Cosio BG, Oates TB, Chung KF. Nature of airway

inflammation and remodeling in chronic cough. J Allergy Clin Immunol. 2005

Sep;116(3):565-70.

42. Bernard AM, Gonzalez-Lorenzo JM, Siles E, Trujillano G, Lauwerys R. Early decrease of

serum Clara cell protein in silica-exposed workers. Eur Respir J. 1994 Nov;7(11):1932-7.

43. Cho JY, Miller M, McElwain K, McElwain S, Broide DH. Combination of corticosteroid

therapy and allergen avoidance reverses allergen-induced airway remodeling in mice. J

Allergy Clin Immunol. 2005 Nov;116(5):1116-22. Epub 2005 Oct 10.

44. Woodruff PG, Fahy JV. Airway remodeling in asthma. Semin Respir Crit Care Med. 2002

Aug;23(4):361-7.

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FIGURE LEGENDS

Figure 1: Observed concentrations of uteroglobin-related protein 1 (UGRP1), Clara cell

protein (CC16) and albumin (Alb) in sputum samples of controls, rhinitis and asthma patients

(geometric means and relevant significant ANOVA results are indicated).

Figure 2: Levels of uteroglobin-related protein 1 (UGRP1) in sputum samples of controls,

rhinitis and asthma patients divided according to atopic status (geometric means and relevant

significant Dunnett test results are indicated).

Figure 3: Simple correlations of uteroglobin-related protein 1 (UGRP1) with Clara cell

protein (CC16) then those of UGRP1 and CC16 with albumin in induced sputum samples of

controls, rhinitis and asthma patients. Note the very strong correlations between UGRP1 and

CC16 and the weaker or absent correlations of both UGRP1 and CC16 with albumin, notably

in asthma.

Figure 4: Correlations between uteroglobin-related protein 1 (UGRP1), Clara cell protein

(CC16) and albumin (Alb) with macrophage and eosinophil percentages in induced sputum of

atopic asthmatics.

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Table 1: Population characteristics, sputum parameters and lung function tests according to

diagnosis (exact numbers are indicated between brackets if different from total).

Controls Rhinitis Asthma

Population studied

Total number 19 32 32

Men/Women 10/9 10/22 21/11

Age (years) 39.4 ± 3.4 35.2 ± 2.1 43.9 ± 2.5°

Disease length (months) - 51.7 ± 12.3 128.2 ± 30.0°

Inhaled steroids 0 0 6 high, 9 medium,

4 low doses

Atopics 0 20 24

Smokers 0 13 13

Pack-years 0 13.1 ± 4.3 15.8 ± 3.8

Lung function tests

Methacholine test not evaluated 1/32 12/32

PD20FEV1 (µg) - 584 538 ± 92.3

FEV1 % 100.4 ± 1.9 (13) 107.4 ± 1.8 (31) 95.3 ± 4.1 (31)°

VC % 105.4 ± 1.8 (13) 109.9 ± 2.4 (29) 102.6 ± 3.6 (30)°

Tiffeneau Index - 102.4 ± 1.2 (31) 93.1 ± 2.2 (31)°

Sputum parameters

Cells/mg 5,356 ± 581 (12) 5,301 ± 905 10,320 ± 2,320

Vitality % - 77.2 ± 2.2 74.7 ± 2.5

Macrophages % 45.3 ± 3.6 (12) 43.5 ± 4.4 26.6 ± 3.3*°

Neutrophils % 51.2 ± 3.6 (12) 44.1 ± 4.8 46.8 ± 5.1

Eosinophils % 0.08 ± 0.08 (12) 5.3 ± 2.5* 19.4 ± 4.7*°

Lymphocytes % 2.4 ± 0.4 (12) 2.9 ± 0.6 1.6 ± 0.3

Epithelial cells % 1.1 ± 0.4 (12) 4.3 ± 1.2* 5.4 ± 1.1*

*: p < 0.05 between patients and controls

°: p < 0.05 between asthma and rhinitis patients

Arithmetic means ± SEM. PD20FEV1: methacholine dose causing a 20% fall in FEV1;

FEV1: forced expiratory volume in 1 second; VC: vital capacity.

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Figure 1

p = NS

10

1

0.1

2.33.33.8

100

CC

16 (m

g/l)

p=0.0047

p<0.0001

1

0.1

0.01

10

0.12

0.780.39

UG

RP

1 (m

g/l)

1

10

100

1000p<0.0001

p=0.0001

23.7

117.5

36.5

Controls Rhinitis Asthma

Alb

(mg

/l)

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Figure 2

Contro

ls

Rhinitis

- Ato

py

Rhinitis

+ A

topy

Asthm

a - A

topy

Asthm

a + A

topy

p < 0.0110

1

0.1

0.01

0.001

p < 0.05

0.13

0.970.58

0.31 0.35

UG

RP

1 (m

g/l)

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Figure 3

Controls

0.001

0.01

0.1

1

0.1 1 10

UG

RP

1 (m

g/l)

CC16 (mg/l)

n = 19R² = 0.439p = 0.002

Rhinitis

0.01

0.1

1

10

0.1 1 10 100CC16 (mg/l)

n = 32R² = 0.602p < 0.0001

Asthma

0.1

1

10

0.1 10 100CC16 (mg/l)

n = 32R² = 0.372p = 0.0002

1

0.001

0.1

1

1 10 100Alb (mg/l)

n = 13R² = 0.338p = 0.037

UG

RP

1 (m

g/l)

0.01

0.01

0.1

1

10

10 100 1000Alb (mg/l)

n = 30R² = 0.181p = 0.019

0.1

1

10

10 100 1000Alb (mg/l)

n = 29R² = 1.31E-5

p = 0.985

0.1

1

10

1 10 100

Alb (mg/l)

n = 13R² = 0.026p = 0.597

CC

16 (

mg

/l)

0.1

1

10

100

10 100 1000

Alb (mg/l)

n = 29R² = 0.002p = 0.839

0.1

1

10

100

10 100 1000Alb (mg/l)

n = 30R² = 0.23p = 0.0073

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Figure 4

0 20 40 60 80

n = 25R² = 0.1377p = 0.0676

Atopic Asthmatics10

1

0.1U

GR

P1

(mg/

l)

0 20 40 60 80

n = 25R² = 0.2458p = 0.0117

100

1

0.1

10

CC

16 (

mg/

l)

0 20 40 60 80

n = 25R² = 0.4145p = 0.0005

100

1

0.1

10

CC

16 (

mg/

l)

0 20 40 60 80

n = 25R² = 0.2215p = 0.0176

Atopic Asthmatics10

1

0.1

UG

RP

1 (m

g/l)

0 20 40 60 80

n = 23R² = 0.1765p = 0.0461

1000

10

100

Sputum Macrophages (%)

Alb

(m

g/l)

0 20 40 60 80

n = 23R² = 0.1843p = 0.0409

1000

10

100

Sputum Eosinophils (%)

Alb

(m

g/l)

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unicationFigure 1

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Figure 2

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Figure 3

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Figure 4

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unicationLIST OF ABBREVIATIONS

Alb: albumin

CC16 : Clara cell protein

DDT : dithiothreitol

FEV1: forced expiratory volume in one second

PD20FEV1: methacholine dose causing a 20% fall in FEV1

UGRP1: uteroglobin-related protein 1

VC: vital capacity

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