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Induced sputum a window to lung pathology


Induced sputum a window to lung pathology

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									                               868   Biochemical Society Transactions (2009) Volume 37, part 4

                                     Induced sputum: a window to lung pathology
                                     Ben Nicholas1 and Ratko Djukanovi´
                                     Division of Infection, Inflammation and Immunity, University of Southampton School of Medicine, Tremona Road, Southampton SO16 6YD, U.K.

                                                      Sputum is recognized as a sampling method for the monitoring and assessment of chronic lung diseases such
                                                      as asthma, COPD (chronic obstructive pulmonary disease) and cystic fibrosis. Sputum samples the central
                                                      airways and its protein components (e.g. mucins and cytokines), cellular components (e.g. eosinophils and
                                                      neutrophils) and microbiological components (e.g. viruses and bacteria) can be used as markers of disease
                                                      severity, exacerbation, susceptibility or progression. This paper describes the basic constituents of induced

                                                      sputum and how these influence the quantification and identification of novel biomarkers of chronic lung
                                                      diseases using techniques such as proteomics.

                                     Clinical utility of sputum                                                                   Collection of induced sputum
                                     Induced sputum is recognized as a very useful sampling                                       The method of sputum sampling to some extent influences
                                     method for both research and clinical use aiding both the                                    its final composition, since the balance of tracheobronchial
                                     diagnosis and monitoring of disease status, particularly in                                  secretions to other biofluids may be altered through conta-
                                     relation to chronic lung diseases such as asthma, COPD                                       mination. Thus it is generally accepted that, through travel
                                     (chronic obstructive pulmonary disease) and, increasingly,                                   via the buccal cavity to the sample dish, sputum will be
                                     interstitial lung disease [1,2]. A great advantage of the                                    contaminated by saliva, and if its collection requires signi-
                                     technique is that it enables sampling of the airways in a                                    ficant expectoration (often the case in healthy non-smoking
                                     non-invasive manner [3], in contrast with other methods                                      volunteers), may also be contaminated by plasma proteins
                                     such as bronchial biopsy, bronchial brushing and BAL                                         through increased exudate induced by the expectoration
                                     (bronchoalveolar lavage), all of which require bronchoscopy                                  process. Therefore one might expect differences between
                                     and the discomfort and risk that it entails. This is particularly                            subjects with healthy or irritated airways independent of any
                                     important in the examination of patients with severe airways                                 disease process, simply reflecting differences in the ease of
                                     disease where endoscopy poses significant risk to oxygen                                     expectoration [8]. In addition, repeated sputum sampling
                                     saturation levels. While sputum induction is not completely                                  can itself influence sputum composition through induction
                                     without risk in such patients [4], steps such as concomitant                                 of a localized inflammatory response [9]. Therefore, a
                                     application of bronchodilators and/or stepwise increase in                                   standardized protocol of sputum induction applied to all
                                     saline application can serve to reduce risk of the procedure [5].                            patients, using nebulized saline to loosen phlegm and aid
Biochemical Society Transactions

                                        A further advantage of induced sputum as a sampling                                       expectoration, is critical for sputum sampling, especially
                                     method is the large body of knowledge now available on                                       where comparisons in biomarkers of disease are to be made.
                                     its characteristics in both normal subjects and patients with
                                     disease, particularly in relation to its inflammatory cell                                   Composition of sputum
                                     content, but also relating to other physical properties such                                 Contaminants apart, sputum consists of constituents derived
                                     as purulence and bacterial load [6,7].                                                       from the respiratory epithelium, mainly sampling the
                                        A greater future potential for induced sputum has also                                    central airways [10]. Additional components are provided
                                     been recognized within the last 10 years: since it samples the                               by transepithelial exudate from tissue plasma, salt, lipids,
                                     airways, sputum may contain protein/peptide components                                       inflammatory cell components from cells resident within
                                     that could act as biomarkers of disease presence or severity.                                either the tissue or the airways lumen, externally derived
                                     One caveat, however, is that, despite well-defined protocols                                 particulate/inhaled matter from the environment, microbial
                                     for its collection and processing, sputum sampling is                                        products from any colonizing bacteria or viruses, and also
                                     known to be variable, although stable enough for repeat                                      cellular debris from all of these compartments.
                                     samplings [2], and its viscosity requires further steps in                                      Thus it is composed of a mixture of mucins (approx. 2–4%
                                     processing, which may add to this variability. The present                                   of the final weight) and other exudates with physiological
                                     paper describes the current state of knowledge regarding                                     relevance to the airways. The gel forming mucins MUC5B
                                     the utility of induced sputum for biomarker discovery and                                    and MUC5AC are the predominant mucin forms in the
                                     quantification in the examination of pulmonary disease.                                      airways [11] and are secreted in the submucosal glands of the
                                                                                                                                  epithelium and the goblet cells of the epithelium respectively.
                                     Key words: biomarker, chronic lung disease, mucin, proteomics, sputum.
                                                                                                                                     Mucins are large glycoproteins made up of tandemly
                                     Abbreviations used: BAL, bronchoalveolar lavage; BALF, BAL fluid; COPD, chronic obstructive
                                     pulmonary disease; DTT, dithiothreitol.                                                      repeating amino acids (serine- and threonine-rich), which
                                       To whom correspondence should be addressed (email bln1@soton.ac.uk).                       are O-linked glycosylation sites [12]. The high carbohydrate

                                     C   The Authors Journal compilation C 2009 Biochemical Society                                              Biochem. Soc. Trans. (2009) 37, 868–872; doi:10.1042/BST0370868
                                                                                                             Biochemical Basis of Respiratory Disease   869

content of the mucin structure is thought to stiffen the protein   transporting them to the external environment. A further
filaments, increasing the volume domain of the molecule and        biological effect of mucins occurs through their unique bio-
accounting for some of its gel forming properties. Many            chemical properties. Mucins are capable of binding a number
bacteria also bind to certain sugar moieties, and thus bacterial   of molecules in biofluids, and recent work categorizing the
binding and clearance may be the primary function of mucins        proteome of airway epithelia has identified proteins that
[11]. The mucin monomers or subunits are approx. 2–3 MDa,          bind with particular affinity to mucins, inferring potential
but are capable of forming an oligomerized meshwork                importance in disease progression [20]. Such molecules
through a mixture of different molecular interactions              include secretory IgA and its transporter molecule in the
(including hydrogen bonds, van der Waals forces and ionic          epithelium, the polyIg receptor, proline-rich proteins,
bonds), which tangles to form a low-viscosity network [13];        transferrins, lysozyme and defensins [11]. Such molecules
furthermore, disulfide linkages can result in polymer gels         are part of the structural cell innate immunity to microbial
of much greater size. The gel forming mucins provide a             infections and, in the case of IgA, provide a link between
protective coating to the airways epithelium. They are diverse     the acquired and innate immune cell defences also. Other
in molecular mass, ranging from 5 to 50 MDa [14], and appear       proteins unique to induced sputum mucin-rich fractions, and
filamentous when viewed using electron microscopy.                 not present in apical secretions of differentiated epithelial cell
    The normal movements of sputum in the mucocilliary es-         secretions, are in the majority derived from innate immune
calator ensure that there is constant movement of these com-       cells such as neutrophils [20].
ponents to prevent epithelial cell damage from its compo-              A number of natural modulators of mucus gels have
nents. Effective clearance of mucus requires a balance of          been found. These include substances that act as mucolytics,
sputum volume and viscoelasticity along with an effective          including gelsolin, a substance that reduces F-actin
cilliary apparatus and pericilliary liquid volume.                 (filamentous actin) to its soluble form [18], thioredoxin [21]
    Mucins are responsible for most of the physical character-     and bacterial chitinases [22], all natural secretory products
istics of mucus, conferring elasticity and other cohesive/         of either healthy or diseased airways. Other substances can
adhesive properties. Mucus is a gel that acts in a non-            act upstream to prevent epithelial cell damage in response to
Newtonian liquid manner (i.e. has liquid and solid                 oxidant stress such as smoking and consequent goblet cell
behaviour) such that in normal secretions it can enable cilia      hyperplasia, such as the antioxidant haem oxygenase 1 [23].
to convert kinetic energy into mucus movement. In models,
it has been shown that increasing viscosity will improve
mucus clearance [15].                                              Factors affecting mediator measurement
    A change in sputum volume, of necessity accompanied            in sputum
by changes in physical characteristics, is a feature of airways    As previously mentioned, sputum is a complex biofluid
disease. One consequence of airway epithelial damage is            and has several properties which make measurement of
replacement of ciliated cells by goblet cells, a process that is   its components more difficult: (i) The non-mucin protein
largely under the control of the EGFR (epidermal growth            component is only a small proportion of the total sputum
factor receptor) signalling cascade [16]. This is thought to       contents. (ii) Liquefaction agents such as DTT (dithiothreitol)
occur as a physiological response to increase airways irrit-       or NAC (N-acetylcysteine) are chemically active and may
ation, resulting in improved clearance of pathogens and            modify the components such that they are no longer
irritants, and demonstrates the dynamic nature of mucus            measurable [24]. (iii) Akin to BAL, there is no endogenous
control, alongside the physical barrier element of mucus.          component useful for normalization of quantitative data.
    Where sputum volume increases sufficiently, or where its       (iv) Sputum is a salty biofluid and this ionic component
physical properties are altered, preventing adequate clearance,    can adversely affect biochemical analyses (e.g. isoelectric
resultant coughing occurs to aid clearance of the airways          focusing, see Figure 1). (v) Sputum is prone to contamination
[17]. Other constituents of mucus are known to affect these        by plasma, cells, saliva, microbes etc., all of which make the
properties. For example, there are significant quantities of       interpretation of results more challenging.
DNA and actin present in the mucus of patients with various           To overcome these difficulties, several modifications to
airways diseases derived from necrotic inflammatory cells in       the collection protocol can be made, including: selection of
the lumen, and these can act to reduce elasticity or prevent       ‘mucoid’ components, which reduces salivary contamination,
clearance by changing sputum physical properties [18]. In          centrifugation or filtration to remove cell/microbial products,
addition to changes in the total quantity of mucin forms,          renaturation of sputum by dialysis to refold proteins and
changes in the relative proportions of their glycosylated          reduce salt content after DTT treatment [25] and weighing
isoforms may well contribute to airway physiology through          of selected sputum to normalize for sputum weight. Without
knock-on effects on gel forming properties [19].                   such modifications, quantification of biomarkers in sputum
                                                                   has seen a great deal of variability or significant numbers of
Mucins and protein binding effects                                 samples with zero data values. In an effort to redress this
As previously indicated, the primary role of mucus appears         issue, several criteria for quantification of markers in sputum
to be an innate defence mechanism by the epithelium. Mucus         have been proposed, including the use of sample ‘spikes’ with
forms a protective barrier, trapping airways particulates and      recombinant proteins to determine percentage recoveries of

                                                                                       C   The Authors Journal compilation C 2009 Biochemical Society
870   Biochemical Society Transactions (2009) Volume 37, part 4

      Figure 1 High salt and mucin content adversely affects isoelectric focusing of DTE (dithioerythritol)-processed sputum samples,
                      resulting in poor resolution
                      Removal of these contaminants by size-exclusion filtration and subsequent dialysis restores spot resolution on
                      two-dimensional gels (B. Nicholas, unpublished work).

      each analyte as a measure of the effect of changes in sputum            fibrosis disease pathology, and many have examined the
      quality in disease on the marker quantification itself [24,26].         change in bacterial phenotype with colonization and anti-
                                                                              biotic resistance [36,37]. The most interesting evidence in
                                                                              disease, however, lies in the reduction of intact mucins
      Quantification of markers of airways
                                                                              in the sputum of cystic fibrosis patients, which belies the
      disease in sputum samples                                               common belief that cystic fibrosis results in a straightforward
      A number of publications exist that describe quantification
                                                                              increase in sputum volume, which is detrimental to cough
      of markers in sputum and their change with disease. These
                                                                              clearance. The decrease in intact mucin in cystic fibrosis
      studies have varied widely in their processing, normalization
                                                                              sputum may be due to increased degradation and dilution
      and assay validation protocols. Altered levels of a number
                                                                              with other cellular products [38]. Such information has been
      of markers in sputum have been observed in stable COPD,
                                                                              discovered by altering sputum analysis methods to include
      including those related to remodelling [27], chemoattraction
                                                                              the high-molecular-mass material, which would normally be
      of inflammatory cells related to disease pathology [28,29]
                                                                              excluded using gel-based fractionation methods [20].
      and co-morbidities such as metabolic dysfunction reflected
                                                                                  An increasing number of proteomic studies are investigat-
      in leptin levels [30]. Changes have also been observed during
                                                                              ing the effects of smoking and chronic disease in the pulmon-
      exacerbations of COPD [31,32] and in comparisons of
                                                                              ary compartment. Benchmark proteomic studies in the form
      COPD with other airways diseases such as cystic fibrosis [33].
                                                                              of so-called ‘shotgun’ analyses have been used to generate
      However, a disappointing element of studies using known
                                                                              proteome lists for many compartments of the airways,
      biomarkers is that a number of candidate biomarkers appear
                                                                              including a number of biofluids, including saliva [39,40],
      to be universally altered in airways disease, irrespective
                                                                              BALF (BAL fluid) [41,42], nasal lavage fluid/mucus [43,44],
      of disease aetiology. These studies are also limited by the
                                                                              epithelial lining fluid [45] and sputum [46]. Such studies
      literature on biomarkers, which may have a bias towards
                                                                              are beginning to enable comparisons of proteomes between
      systemic rather than locally produced biomarkers due to a
                                                                              different airways compartments and different diseases
      historical reliance on blood-derived samples for analyses.
                                                                              (Figure 2).
      Therefore there has been increasing interest in identifying
                                                                                  A wide variety of unbiased proteomic studies comparing
      novel biomarkers of disease, and sputum, through its non-
                                                                              health with disease in the pulmonary compartment have iden-
      invasiveness and direct sampling of the airways, provides an
                                                                              tified mechanisms of disease or surrogate biomarkers of dis-
      excellent potential sample in which to find such biomarkers.
                                                                              ease [47]. Comparative unbiased studies of sputum involving
                                                                              large numbers of patients or independent cohorts have not
      Proteomics of induced sputum                                            been reported; however, pilot studies have identified candid-
      There have been relatively few proteomic studies of induced             ate biomarkers for diseases such as COPD [48], cystic fibrosis
      sputum. It would be fair to say that cystic fibrosis has                [35] and bronchiolitis [49]. Interesting data are emerging from
      occupied the highest profile in research using sputum, both             these types of studies. In common with proteomic studies
      in terms of bacteriology and mucolytic therapy; however,                from other mucosal sites, the proteomic signature from the
      due to the recognized mechanisms of this disease, there is              pulmonary compartment in response to any disease/stimulus
      possibly a lower motivation for performing expensive and                appears to be made up of several categories of proteins, in-
      time-consuming unbiased proteomic studies for surrogate                 cluding those arising from the systemic compartment, prob-
      markers for the disease. A few studies have, nevertheless,              ably present through increased tissue leakage such as albumin,
      been performed, looking at biomarkers in BAL [34]                       immunoglobulins and α 1 -antitrypsin, those of the innate
      and sputum [35] with the aim of understanding cystic                    structural cell defences such as PLUNC (palate, lung and

      C   The Authors Journal compilation C 2009 Biochemical Society
                                                                                                                                Biochemical Basis of Respiratory Disease   871

Figure 2 Comparison of the proteomes of sputum BAL and saliva                     5 Delvaux, M., Henket, M., Lau, L., Kange, P., Bartsch, P., Djukanovic, R.
and their degree of overlap                                                         and Louis, R. (2004) Nebulised salbutamol administered during sputum
                                                                                    induction improves bronchoprotection in patients with asthma. Thorax
Reproduced with permission from B. Nicholas, P. Skipp, R. Mould, S.                 59, 111–115
Rennard, D.E. Davies, C.D. O’Connor and R. Djukanovic, Shotgun pro-               6 Nair, B., Stapp, J., Stapp, L., Bugni, L., Van Dalfsen, J. and Burns, J.L.
teomic analysis of human-induced sputum, Proteomics, 2006, vol. 6,                  (2002) Utility of gram staining for evaluation of the quality of cystic
                                                                                    fibrosis sputum samples. J. Clin. Microbiol. 40, 2791–2794
pp. 4390–4401. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.                        7 Brusse-Keizer, M.G., Grotenhuis, A.J., Kerstjens, H.A., Telgen, M.C.,
                                                                                    van der Palen, J., Hendrix, M.G. and van der Valk, P.D. (2008) Relation
                                                                                    of sputum colour to bacterial load in acute exacerbations of COPD.
                                                                                    Respir. Med. 103, 601–606
                                                                                  8 Schoonbrood, D.F., Out, T.A., Lutter, R., Reimert, C.M., van Overveld, F.J.
                                                                                    and Jansen, H.M. (1995) Plasma protein leakage and local secretion of
                                                                                    proteins assessed in sputum in asthma and COPD: the effect of inhaled
                                                                                    corticosteroids. Clin. Chim. Acta 240, 163–178
                                                                                  9 van der Vaart, H., Postma, D.S., Timens, W., Kauffman, H.F., Hylkema,
                                                                                    M.N. and Ten Hacken, N.H. (2006) Repeated sputum inductions induce a
                                                                                    transient neutrophilic and eosinophilic response. Chest 130,
                                                                                 10 Alexis, N.E., Hu, S.C., Zeman, K., Alter, T. and Bennett, W.D. (2001)
                                                                                    Induced sputum derives from the central airways: confirmation using a
                                                                                    radiolabeled aerosol bolus delivery technique. Am. J. Respir. Crit.
                                                                                    Care Med. 164, 1964–1970
                                                                                 11 Thornton, D.J. and Sheehan, J.K. (2004) From mucins to mucus: toward a
nasal epithelium carcinoma-associated protein), and those
                                                                                    more coherent understanding of this essential barrier. Proc. Am.
of the innate immune defence system, such as neutrophil-                            Thorac. Soc. 1, 54–61
and macrophage-derived proteins, including lipocalin-2 and                       12 Rose, M.C. and Voynow, J.A. (2006) Respiratory tract mucin genes and
                                                                                    mucin glycoproteins in health and disease. Physiol. Rev. 86,
myeloperoxidase, and other epithelial cell-derived secretory
products, which may be the more ideal candidates for                             13 Dasgupta, B., Tomkiewicz, R.P., Boyd, W.A., Brown, N.E. and King, M.
novel disease biomarkers. One cannot forget also that                               (1995) Effects of combined treatment with rhDNase and airflow
local modulation of systemic and locally produced proteins                          oscillations on spinnability of cystic fibrosis sputum in vitro.
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through, for example, proteolytic degradation, may account                       14 Kirkham, S., Kolsum, U., Rousseau, K., Singh, D., Vestbo, J. and Thornton,
for some of the biomarkers found using proteomic studies in                         D.J. (2008) MUC5B is the major mucin in the gel phase of sputum in
biofluids, although often the modulator itself remains cryptic.                     chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med.
                                                                                    178, 1033–1039
   In conclusion, sputum is of increasing importance as a                        15 Rubin, B.K. (2002) Physiology of airway mucus clearance. Respir. Care
diagnostic tool for the diagnosis and monitoring of disease,                        47, 761–768
the study of disease pathogenesis and its treatment, through                     16 Takeyama, K., Dabbagh, K., Lee, H.M., Agusti, C., Lausier, J.A., Ueki, I.F.,
                                                                                    Grattan, K.M. and Nadel, J.A. (1999) Epidermal growth factor system
its inflammatory cell, bacterial, volatiles, mucin and protein                      regulates mucin production in airways. Proc. Natl. Acad. Sci. U.S.A. 96,
content. Measurement of these components is increasingly                            3081–3086
sophisticated and quantifiable, and the search for novel                         17 Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W.
                                                                                    and Boucher, R.C. (1998) Evidence for periciliary liquid layer depletion,
biomarkers of airways disease in this biofluid is under way.                        not abnormal ion composition, in the pathogenesis of cystic fibrosis
However, proteomic analyses to date have not provided                               airways disease. Cell 95, 1005–1015
any clinically proven biomarkers of lung disease. With the                       18 Kater, A., Henke, M.O. and Rubin, B.K. (2007) The role of DNA and actin
                                                                                    polymers on the polymer structure and rheology of cystic fibrosis
rapid pace of development of MS methods, with improved                              sputum and depolymerization by gelsolin or thymosin β4. Ann. N. Y.
accuracy and throughput, and avoiding traditional gel-based                         Acad. Sci. 1112, 140–153
applications, sputum will be increasingly utilized for such                      19 Schulz, B.L., Sloane, A.J., Robinson, L.J., Prasad, S.S., Lindner, R.A.,
                                                                                    Robinson, M., Bye, P.T., Nielson, D.W., Harry, J.L., Packer, N.H. et al.
proteomic studies.                                                                  (2007) Glycosylation of sputum mucins is altered in cystic fibrosis
                                                                                    patients. Glycobiology 17, 698–712
Funding                                                                          20 Kesimer, M., Kirkham, S., Pickles, R.J., Henderson, A.G., Alexis, N.E.,
                                                                                    Demaria, G., Knight, D., Thornton, D.J. and Sheehan, J.K. (2009)
                                                                                    Tracheobronchial air–liquid interface cell culture: a model for innate
Work is supported by the National Institutes of Health [grant                       mucosal defense of the upper airways? Am. J. Physiol. Lung Cell.
number RO1-HL72356-01].                                                             Mol. Physiol. 296, L92–L100
                                                                                 21 Rancourt, R.C., Lee, R.L., O’Neill, H., Accurso, F.J. and White, C.W. (2007)
                                                                                    Reduced thioredoxin increases proinflammatory cytokines and neutrophil
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      C   The Authors Journal compilation C 2009 Biochemical Society

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