Lung Collagen: More Than Scaffolding

Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

Thorax 1986;41:418-428

Review article Lung collagen: more than scaffolding

Collagens of various types are the major group of be given to its role in two disorders: pulmonary proteins in the lung. They are present in all major fibrosis and emphysema. structures, including airways, blood vessels, the interstitium of the lung parenchyma, and the basement Collagen structure and metabolism membranes of epithelial and endothelial cells. Any alterations in quantity, structure, or the geometry of This section offers a brief summary of what is cur- their distribution would be likely to alter lung func- rently known about the structure and metabolism of tion. Changes would have particularly dramatic collagen. Readers are referred elsewhere for more de- effects if they occurred in the interstitium, where the tailed reviews ofcollagen biochemistry.2 An appre- distance between air and blood may be as little as ciation of the pathways described here may be useful 50 nm.1 for an understanding of what follows, but some read- A collagen molecule typically has a rod like ers may wish to pass directly to the next section on structure, 300 nm in length and 3 nm in diameter, lung collagen. forming tightly packed fibrils with diameters up to Our perception of collagen has changed dra- about 100 nm. Any large deposits of collagen or col- matically over the last 10 years. One of the major lagen deposition with incorrect orientation within the breakthroughs has been the discovery of collagen interstitium may severely impede gas exchange; while isotypes and the recognition that there is a family of loss of collagen from the interstitium would lead to collagens-each having distinctive structural and disruption of the normal alveolar network, and loss metabolic characteristics, and in some cases different from basement membranes would compromise their functions. There are thought to be at least 11 different http://thorax.bmj.com/ role controlling entry ofmaterials to and from cells in collagens, coded by a group of about 20 genes6 (ta- the lung. Not surprisingly perhaps against this back- ble). Each of these so called collagen types comprises ground, lung diseases occur in which imbalances in three polypeptide chains (a chains), which intertwine collagen structure or metabolism appear to play a in a right handed triple helix. The individual chains part. This editorial review describes some of the de- typically contain about 1000 amino acids, and a char- velopments in our understanding oflung collagen and acteristic of all of them is a high proportion of gly- its role in pulmonary disease. Particular attention will cine, proline, and hydroxyproline residues. A large

of the on September 24, 2021 by guest. Protected copyright. Address for reprint requests: Dr Geoffrey J Laurent, Lung Biochem- proportion polypeptide chain contains repeat istry Unit, Department of Thoracic Medicine, Cardiothoracic Insti- units of the structure Gly-X-Y, where X and Y are tute, London SW3 6HP. either proline, alanine, or hydroxyproline. It is likely

Collagen types and their likely distribution in the lung Type Likely distribution in the lung Molecular structure Likely cells responsiblefor bulk ofreproduction Large bronchi, blood vessels, interstitium Fibroblasts I (trimer) Presence in lung uncertain [ai(I)] Fibroblasts II Bronchial and tracheal cartilage [ax(II)1 Chondrocytes III Large bronchi, blood vessels, interstitium [a;(III)b3 Fibroblasts IV Basement membranes Endothelial and epithelial cells V Basement membranes and interstitium [O(IV)12a (IV) Fibroblasts Structure uncertain, may be comprised of 3 distinct molecules VI Blood vessels and interstitium codistributing aO(VI)a2(VI)a3(VI) Fibroblasts with types I and III collagen VII Uncertain [al(VII)]3 Uncertain VIII Uncertain Uncertain Endothelial cells Ix Cartilage a1(IX)a2(IX)a3(IX) Chondrocytes x Cartilage [a21(X)13 Chondrocytes xI Cartilage Uncertain Chondrocytes 418 Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

Lung collagen: more than scaffolding 419 Chromatin RNA polymerase NUCLEUS Transcription (1)

(2) CYTOPLASM I...... polypeptide with "signal peptide' Translation ,1% RER lumen g_ _ribosomes (movingakx-igmRNA) Rough endoplasmic reticulum (RER)L mRNA

pro collagen NH2 COOH a chain (3) 4 OH OH I hydroxylation (4) glycosylation (5) Golgi apparatus disulphide bond formation (6) H ;~O I OH OH procollagen molecule OH 4 OH http://thorax.bmj.com/ ,F ' OH StZ removal of N and Fibril £S> C terminal propeptides assembly site OH 3w ~~~~~~~~~(3) EXTRACELLAR SPACE )lecular nk on September 24, 2021 by guest. Protected copyright.

molecular slink (7)

Collagen fibril extracellular degradation (8)

Fig I Likely steps in the pathway oflung collagen synthesis and degradation.

that all the collagens that are currently well described major developments in our understanding of the' will be found in the lung, either in the parenchyma or pathways of collagen metabolism and the extensive in the higher airways. They are listed in the table and processing that occurs at intracellular and extra- discussed in more detail later. cellular sites. These include the following steps, many In addition to these advances in the appreciation of of which are illustrated in figure 1, with numbered the diversity of collagen structure, there have been cross references to this text. Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

420 Laurent 1 Extensive processing ofthe messenger RNAfor 5 Addition ofcarbohydrate moieties to the collagen polypeptide chains The gene for the a2(I) chain of type I collagen con- Carbohydrates, mostly glucose and galactose resi- tains many more bases than are required to code for dues, are covalantly bonded to hydroxylysine in reac- the polypeptide. The coding sequences (exons) are in- tions requiring the enzymes galactosyl transferase and terspersed throughout the gene, separated by about glucosyl transferase. The extent of glycosylation var- 50 intervening sequences (introns). During proces- ies greatly for the different collagens; it is particularly sing within the nucleus these sequences must high in type IV collagen, where these carbohydrate be specifically excised and the strands, subsequently moieties represent as much as 10% by weight of the ligated before transcription to the mRNA in the molecules. endoplasmic reticulum (fig 1). 6 Disulphide bonding between a chains For all collagens except type I disulphide bonds are 2 Synthesis with "signal" sequence formed between cysteine residues of adjacent chains, The initial transcript (pre-procollagen) has a short se- as well as interchain links between adjacent mole- quence of amino acids at one end of the molecule. cules. This sequence of predominantly hydrophobic amino acids functions to direct the molecule into the Golgi 7 Formation ofcovalant cross linked compounds apparatus, from which it is destined to be secreted between amino acids side chains from the cell. After the modifications described above, most of which take place in the Golgi apparatus, procollagen 3 Collagen synthesised in a precursorform molecules are secreted from cells via the secretory the poly- vacuoles (see fig 1). After removal of the propeptides, After removal of the "signal" sequence, collagens are assembled into fibrils in recesses of the peptide is still considerably longer than the chain that fibroblast plasma membrane. These fibrils are not sta- eventually forms fibrils in the extracellular matrix. chains There are peptide extensions at both ends of the mol- bilised until covalent cross links form between to as peptides), which do of adjacent molecules. The first step in this process is ecule (referred procollagen http://thorax.bmj.com/ not contain long repeat structures of the Gly-X-Y the modification of the lysine and hydroxylysine form and, apart from one short section, do not form residues in reactions requiring the copper dependent a triple helix. These extensions may have several func- enzyme lysyl oxidase. Cross linking then occurs tions: promoting formation of the triple helix, pre- via reactions between these products and unmodified venting premature fibril formation, and influencing lysine or hydroxylysine chains. have extracellular fibril formation. Possibly they also 8 Degradation ofprocollagen and collagen a role in the control of collagen production, with the Another recent development, discussed in detail later, N terminal propeptide exerting a negative feedback and is the finding that collagen turnover (synthesis on September 24, 2021 by guest. Protected copyright. on procollagen synthesis. The peptides are removed degradation) is more rapid in normal tissues than was by specific amino and carboxyproteases, which are originally believed. For this reason pathways of de- thought to act either at the membrane just before gradation, which are still relatively poorly under- secretion or some time after secretion from the cell, stood, are potentially important sites for regulation of depending on the collagen type. collagen homeostasis. The pathways of collagen degradation are complex and there are various sites, 4 Hydroxylation ofproline and lysine both intracellular and extracellular, where break- About one half of the proline residues in the pro- down may occur. Once it leaves the cell, the molecule collagen chain are hydroxylated after production of ages chemically with the formation of cross links that the molecule. There are specific enzymes (3- and render it less susceptible to degradation. In contrast, 4-prolyl hydroxylases) requiring as cofactors molecu- the procollagen molecule appears to be highly sus- lar oxygen, iron, ascorbic acid, and a ketoglutaric ceptible and a large proportion of newly synthesized acid. Another hydroxylase (lysyl hydroxylase) is res- molecules may be degraded within minutes of syn- ponsible for the hydroxylation of lysine residues. thesis, before secretion from the cell. Hydroxyproline is also found in some other The major pathway for degradation of extra- proteins (elastin, acelylcholinesterase, the Clq cellular collagen appears to depend on collagenases. components of complement, and a component of There are various collagenases that are capable of de- surfactant); but in quantitative terms this is grading one or more of the isotypes. The best charac- insignificant. This hydroxylation of proline is used as terised of these, first isolated from mammalian skin, is the basis for the quantitation of lung collagen. capable of degrading all the interstitial collagens Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

Lung collagen: more than scaffolding 421 (types I, II, and III) but not, so far as we know, the of breakdown for extracellular collagen is certainly other types. Degradation occurs in a site specific way, much slower, and possibly highly cross linked col- the protease cleaving the chain of the mature triple lagen does not turn over under normal circumstances. helix at a peptide bond about three quarters of the The implications of these studies are that way from one end. Once this occurs the molecule be- fibroblasts are not quiescent cells that are stimulated comes susceptible to various other proteases, which to produce collagen only in disease; rather, they are may act extracellularly (at neutral pH), or alterna- continually synthesising collagen and may be res- tively the denatured chains may be taken up by cells ponsible for its degradation intracellularly and ex- during endocytosis and the chain degraded intra- tracellularly. Furthermore, changes in either synthesis cellularly (probably in lysosomes). Collagenase is or degradation could potentially play a major part in produced as an inactive proenzyme and is converted the regulation of collagen mass in lung disease (fig 2). to the active form by several proteases, including plas- The ways in which these processes are regulated are min and trypsin. Several agents are capable of ini- still poorly understood; clearly, however, there are tiating this conversion (including prostaglandins and many potential points for regulation (figs 1 and 2) and cyclic AMP) and at least two antiproteases can act as recently a host of mediators have been described collagenase inhibitors-they are fJ, anticollagenase that can, for cells in culture at least, influence rates and a2 macroglobulin. Both these compounds are at which collagen is synthesised and degraded. Many found in plasma and would normally bind any col- of these mediators are derived from the cells of the lagenase in serum. The collagenase described above is immune and reticuloendothelial system and for that known to be produced by fibroblasts and is probably reason there has been much conjecture about their prominent in normal collagen turnover. Other col- role in diseases where rates of connective tissue pro- lagenases may be important during disease. Macro- tein metabolism are believed to alter. phages can produce a metalloprotease that is capable of degrading types IV and V collagen. Neutrophils can produce two proteases; one is specific for type IV collagen and the other degrades type III collagen as well as elastin. a) NORMAL ADULT LUNG Translation Post- translational http://thorax.bmj.com/ Collagen in the normal lung Replication Transcription (initiation, modifications or cell death (RNA production elongation & (hydroxylation, Collagens of the various types constitute about 15% (cell number) & processing) termination) cross linking) of the dry weight of human lung tissue and are the major protein group. Several groups of workers have \~~~~\\4 0/ / synthesis CONNECTIVE isolated collagens from lung7 -9 and examined their AMINO ACIDS TISSUE distribution with immunohistochemical tech- degradation PROTEINS niques.9'13 Types I and III are the most abundant of the lung collagens and are present in a ratio of about on September 24, 2021 by guest. Protected copyright. 2:1,14-16 codistributing in airways and blood vessels Proteases Antiproteases Active oxygen Post-translational Substrate as well as in species modifications modifications the interstitium. The molecular structure (i e hydroxyl of proteases and distribution of these and other collagen types in radicals) (i e activation of lung are summarised in the table. proenzymes) Comparatively little is known of the rates of collagen metabolism in normal lung. Formerly the prevailing view was that lung collagen, like collagen b) FIBROSIS a) - - increased synthesis in other tissues, was either inert or turned over (net deposition) extremely slowly.'7 18 More recent studies, however, or b) - decreased degradation _. increased synthesis using improved techniques have suggested that syn- or o-rcc) decreased degradation& thesis and degradation rates may be quite rapid. In adult rats and rabbits the turnover rate is about 10% a day, implying that amounts of collagen equivalent c) EMPHYSEMA a) - - decreased synthesis to one tenth of the total pool are being synthesised (net loss) and degraded each day. Furthermore, studies of the or b) increased degradation kinetics of decreased synthesis & this process in vivo'9 and in vitro20 have o indicated that about 30% of lung collagen is being or c - increased degradation degraded intracellularly within minutes of its syn- Fig 2 Collagen metabolism in the normal lung andpossible thesis, before secretion from cells. In contrast, the rate changes during pulmonaryfibrosis and emphysema. Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

422 Laurent Collagen in lung disease the notion that collagen deposition is progressive and increases with advancement of the disease. The human lung may be exposed to many toxins- Studies of collagen metabolism have also been con- particularly those derived from cigarette smoke, but troversial. There is widespread agreement that syn- also many other environmental agents. Minor epi- thesis rates are increased in experimentally induced sodes of lung damage are likely to occur con- fibrosis, but in man this has not yet been demon- tinuously, and to be met by the "repair cascade," strated biochemically. One early study in vitro, which eventually leading to collagen deposition in scar tis- examined uptake of radiolabelled proline into the sue. Once the causative agent is no longer present this hydroxyproline of biopsy samples obtained from deposited collagen is likely to be broken down in the patients with pulmonary fibrosis, suggested that "resolution pathway". The important point, how- synthesis was no different from the synthesis in con- ever, is that there has to be a tight balance between trol material.2' A similar result has been reported by synthesis and degradation of connective tissue pro- Selman and coworkers,28 who studied a quite large teins. Two common disorders of the lung where an group of patients. Again the major problem relates to imbalance of connective tissue protein metabolism the way synthesis is expressed. In both the above occurs are pulmonary fibrosis and emphysema. studies rates have been expressed with respect to concentration of DNA and therefore an influx of inflammatory cells may mask increases in collagen Collagen content and metabolism in pulmonaryfibrosis synthesis rates. This seems likely since studies in ex- Pulmonary fibrosis is a general term to describe those perimental animals have shown increased synthesis in disorders for which there is histological evidence of terms of fractional rate (that is, with respect to the diffuse thickening of alveolar walls. Characteristically total collagen pool) but no change when synthesis was fibrous tissue, believed to consist mostly of collagen, related to DNA concentration.29 The inherent lim- is localised predominantly in the interstitium, where itations of in vitro studies have also been questioned gas exchange occurs. Histologists have for some time since the rates of synthesis obtained for sliced tissue reported an increase in collagen deposition, although preparations appear to be an order of magnitude biochemical evidence for this has been controversial. lower than those obtained in vivo.30 Indirect evidence An initial study of lung biopsy samples from patients suggesting an increased synthesis rate in man has http://thorax.bmj.com/ with idiopathic pulmonary fibrosis (cryptogenic come from studies of the enzymes of collagen syn- fibrosing alveolitis) suggested no change in collagen thesis and the production of procollagen peptide (see concentration or synthesis rates.2' The severe lim- previous section on collagen structure and metabo- itations inherent in studies of biopsy samples are, lism). Increased levels of type III procollagen peptide however, now well recognised. Briefly, studies of bi- have been reported in serum3" and lavage fluid,32 and opsy material demand that collagen content should increased serum levels of glucosyl transferase have be expressed with respect to some other constituent, also been observed for patients with fibrosing lung and DNA content has been used most commonly. disorders.3 on September 24, 2021 by guest. Protected copyright. Histological studies have established that there is a In contrast, there are few studies of collagen de- considerable cellular infiltrate during fibrosis, which gradation during fibrosis. This pathway may be im- increases the wet and dry weight as well as the DNA portant in the light of recent observations suggesting content of the tissue. The histological appearance and that collagen turnover in the adult lung is more rapid collagen concentrations also vary considerably with than was traditionally believed.2930 On the basis of the site of the biopsy, and the degree of fibrosis (as- the appearance of labelled hydroxyproline after ad- sessed histologically or biochemically) is known to ministration of labelled proline in vivo, a decreased vary with the stage of the disease (see ref 22 for full degradation of newly synthesised collagen has been discussion). suggested for rabbits with bleomycin induced Studies of acute lung disease in man23 24 and of fibrosis.29 There are less data on this in man, al- experimentally induced fibrosis'1 25 - 27 have shown though one study of collagenase activity in biopsy appreciable and extremely rapid increases in total samples from patients with pulmonary fibrosis sug- lung collagen, although changes in collagen concen- gested decreased collagenase mediated degradation.28 trations were not always apparent. In addition, a This contrasts with a report of increased collagenase recent study of a larger series of patients with activity in lavage fluid derived from patients with pul- pulmonary fibrosis showed a considerable increase in monary fibrosis,34 although whether this reflects total lung collagen with appreciable increases in col- changes in the interstitium is uncertain. Studies such lagen concentration.222 Furthermore, the collagen as these highlight the importance of considering de- concentration was shown to be greater in postmortem gradation as well as synthesis in understanding the lung samples than in biopsy samples,22 supporting mechanisms leading to pulmonary fibrosis (fig 2). Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

Lung collagen: more than scaffolding 423 Collagen types in pulmonaryfibrosis other data in the assessment of prognosis and res- There has been considerable interest in the role of ponse to treatment is more encouraging and should changes in collagen types in fibrosis. It has been sug- be pursued. gested that shifts to excessive type I collagen with a more fibrillar, rigid structure may lead to impaired Pathways leading to changes in collagen metabolism in gas exchange and some of the physiological charac- pulmonaryfibrosis teristics of fibrosis. An early study of collagen types The deposition of collagen is a relatively late event in reported an increase in the amount of type I collagen the sequence of steps broadly described as the "repair relative to type III in postmortem samples from cascade," a process central to normal body homeo- patients with idiopathic pulmonary fibrosis,14 and stasis. To see it in isolation is misleading and likely to this has been confirmed in both acute35 and chronic36 produce a distorted vision of the pathogenesis of forms of this disease. fibrosing lung disorders. What is important here is A somewhat different picture has emerged from that in normal scar formation the collagen deposition studies of biopsy samples. Bateman et al,'0 using is limited and apparently reversible, whereas in vari- immunohistochemical methods, suggested a re- ous pathological states, including some forms of lationship between the presence of type III collagen pulmonary fibrosis, it seems to be progressive and and disease activity. Kirk et a136 developed biochem- does not appear to be reversible. ical procedures for examining biopsy samples and, The early pathways leading to lung damage and although a wide range of values was seen, several pa- eventual collagen deposition are now beginning to be tients had type III to type I ratios greater than were understood, although the pathways are likely to be seen in either controls or postmortem samples. In ad- different for different forms of pulmonary fibrosis. dition, patients in the earlier stages of disease also For example, in asbestosis and pneumoconiosis the tended to have a greater proportion of type III col- nature of the causative agents is known in some detail lagen, as was suggested by immunohistochemical and the early pathways may be very different from studies.'3 3 Further, there was a relationship be- those where pulmonary fibrosis occurs as a side effect tween the relative proportions of these collagen types of bleomycin treatment or in cryptogenic fibrosing al- and response to treatment, implying that biochemical veolitis. In the case of dust induced diseases inter- measurements of collagen types might be useful in action with alveolar cells may be the primary event, http://thorax.bmj.com/ staging fibrosis.36 To examine these issues further whereas for bleomycin there is evidence that inter- Kirk and colleagues measured the concentration of action of bleomycin with DNA (in the presence of the type III procollagen peptide (fig 1) in serum of iron and oxygen), to form an active oxygen species, is patients with pulmonary fibrosis.3' The concen- probably the important primary event (see ref 38 for tration of this peptide in the serum was higher in pa- review). In cryptogenic fibrosing alveolitis (or idio- tients than in controls and there was a correlation pathic pulmonary fibrosis) in man the initial cause is between serum concentrations and the results of type unknown but the presence of immune complexes de- III measurement in lung biopsy samples. Further- posited on endothelial surfaces may be an important more, patients with the highest concentration of pep- initiating step.39 Whatever the initial cause, an influx on September 24, 2021 by guest. Protected copyright. tide tended to show a better response to steroid of inflammatory cells into the lung is common to all treatment, and this response was associated with a these disorders. The nature of these cells may in part decrease in serum peptide concentration. From what depend on the initiating agent but large numbers of we know of the pathway ofcollagen metabolism these neutrophils, macrophages, and lymphocytes are often results suggest that synthesis may be increased in seen. The persistent presence of these cells and other fibrosis and that treatment with corticosteroids di- plasma elements (that is, platelets, fibronectin, plasma minishes the rate. derived growth factors) is almost certainly important In the light of these studies ofcollagen types we can not only in the injury itself but also for the repair make some critical appraisal of their importance in mechanism (fig 3). fibrotic lung disease. The changes reported have in There is now strong evidence that cells of the im- general been quite small and the overlap between pa- mune and reticuloendothelial system may play a part tients and control subjects means that measurements in the regulation of collagen metabolism in fibrotic in individual patients are of little value. Furthermore, lung disorders (see refs 40 and 41 for review). Interest the shifts to excessive type I collagen are quite a late in these cells in pulmonary fibrosis was initially event and unlikely to constitute an important step in prompted by studies suggesting that peritoneal mac- the pathology of the disease. The possibility, how- rophages exposed to mineral fibres release a factor ever, of obtaining serial measurements of type III that stimulates collagen production by fibroblasts.42 procollagen peptides (or other indices of collagen More recent studies of alveolar macrophages derived metabolism) in the same patients and using these with from man and experimental animals have tended to Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

424 Laurent

INITIATING AGENT RESIDENT LUNG CELLS

) Pulmonary macrophages

Fibroblasts

(. Mast cells INFLUX OF CELLS OF IMMUNE & RETICULO- ENDOTHELIAL SYSTEM (E Endothelial cells cell damage (g) Monocytes Type I epithelial cells permeability changes ( Neutrophils Type 1I epithelial cells chemoattractants () Eosinophils 4 1 Lymphoid tissue 0Al (lymphocytes) Lymphocytes

MEDIATORS (i e growth factors, chemotactic agents, active oxygen species, proteases) http://thorax.bmj.com/

Elastin Fibroblasts

Proteoglycans

Effects on synthesis and degradation of proteins in the connective tissue matrix

|net deposition | | net loss FIBROSIS EMPHYSEMA

Fig 3 Possible cellular interactions leading to changes in synthesis and degradation of connective tissue proteins.

support this view, indicating that the macrophage is capable of releasing a neutrophil chemotactic factor47 capable of releasing growth factors that lead to and fibronectin, which is a chemoattractant for fibroblast replication.43 45 Here an important obser- fibroblasts.48 It can also produce active oxygen spe- vation is that alveolar macrophages from patients cies that are capable of degrading a wide range of with idiopathic pulmonary fibrosis tend to secrete a macromolecules, leading to eventual cell death. Thus growth factor spontaneously.4e The alveolar macro- mechanisms exist whereby the alveolar macrophage, phage may have a role for other reasons. This cell is by releasing various mediators, is capable not only of Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

Lung collagen: more than scaffolding 425 expanding the fibroblast population and attracting important in the pathways leading to pulmonary fibroblasts to the site of the injury but also of attrac- fibrosis constitutes a major challenge for the future. ting inflammatory cells and thereby causing further cell damage. Pulmonary emphysema: is collagen important? Another possibility is that cells, cell fragments, or Advances in emphysema research have on the face of mediator are derived from the circulation. It has been it outstripped those in fibrosis. Some workers now recognised for many years that in the earliest events believe that the pathways underlying the development leading to pulmonary fibrosis there is an exudative of emphysema are sufficiently well understood to per- phase with appreciable accumulation of fluid derived mit evaluation of treatment directed at the proposed from the blood. It has also been suggested that ex- molecular defect. Excessive elastin breakdown is udation follows from capillary endothelial damage thought to be crucial. Antielastase treatment has been and that this may underlie the primary events in the proposed and the use of oa antitrypsin replacement pathways leading to pulmonary fibrosis.49 Consid- therapy is currently being evaluated in clinical trials. erable protein exudation has been demonstrated Its rationale is not the subject of this editorial, and during bleomycin induced pulmonary fibrosis by this approach and the pathways ofelastin metabolism measurement of radioisotopically labelled albumin in in relation to emphysema are described else- the pulmonary extravascular space.50 The influx of where.58 59 Rather, the aim here is to examine crit- various agents from the circulation could have appre- ically the hypothesis that emphysema is a disorder ciable effects on collagen metabolism in the lung, exclusively of elastin metabolism. overwhelming any effects associated with resident Pulmonary emphysema and fibrosis has tradi- cells (that is, alveolar macrophages). This could be tionally been thought of as opposites. Emphysema is due to agents free in plasma or those derived indi- characterised histologically by enlarged air spaces rectly from cells or platelets that enter the lung. and physiologically by increased compliance and di- Platelets contain an extremely potent mitogen for minished recoil. This contrasts with pulmonary fibroblasts (platelet derived growth factor), which is fibrosis, where there is a decreased compliance and a also a chemoattractant for fibroblasts.5' reduction in lung volume. They have also by and large Lymphocytes and neutrophils are both present in been thought of as diseases of elastin and collagen increased numbers, particularly in the early stages respectively. The reasons for this are not

clear and http://thorax.bmj.com/ of pulmonary fibrosis. Lymphocytes are known to should be reappraised. In biochemical studies where release a host of factors capable of stimulating elastin and collagen have been examined the changes increased collagen secretion by fibroblasts, causing in elastin have been similar to those in colla- fibroblast proliferation and migration (see ref 52 for gen.'5 6061 Studies on emphysema are controversial, review). Furthermore, studies of bleomycin induced with evidence for no change in elastin, expressed fibrosis in T cell depleted mice and thymectomised either as total lung content6263 or as a concen- rats suggested that these animals develop a less in- tration,62-64 although a more recent study suggested tense lesion,53 54 which gives in vivo credence to their a decreased concentration as a proportion of con- role. Such evidence has not yet been obtained for nective tissue components.65 on September 24, 2021 by guest. Protected copyright. macrophages. Differences also exist in the way researchers in the There is also a large influx of neutrophils in pul- two areas have directed their efforts. In pulmonary monary fibrosis, probably stimulated by release of fibrosis they have concentrated almost exclusively on various chemoattractants. Fibronectin has already the synthetic pathways, whereas in emphysema effort been mentioned and products ofcellular damage may has been directed toward degradation. This polar- also be important. Breakdown products of collagen isation has not been entirely useful and it may have are known to be chemoattractants for neutro- been more productive to examine the balance between philS55 56 as well as fibroblasts.57 In this way a cas- synthesis and degradation of connective tissue com- cade effect develops, bringing more neutrophils to the ponents in these disorders, since both may be im- site of injury and leading to further damage as well as portant (fig 2). repair by fibrosis. This may explain the progressive The current concept of emphysema is that it is a nature of the damage in many of the fibrotic lung disease which depends on changes in elastin de- disorders. Clearly there are many mediators, derived gradation, a view based on two observations. Firstly, from various sources, that are capable of inducing instillation of pancreatic elastase into lungs of experi- changes in collagen metabolism consistent with the mental animals induced a lesion typical of em- responses occurring during pulmonary fibrosis. This physema. Later neutrophil elastase was shown to information comes from in vitro studies, however; produce a similar response. The role of collagen was there is so far little in vivo evidence that they have a thought to be unimportant because instillation of role. Determining which, if any, of these factors is bacterial collagenase did not produce such a response Thorax: first published as 10.1136/thx.41.6.418 on 1 June 1986. Downloaded from

426 Laurent (see ref 66 for review). The second observation was 4 Bailey AJ, Etherington DJ. Metabolism of collagen and the discovery of the genetic disorder of a, antitrypsin elastin. In: Florkin M, Neuberger A, van Deenen LLM, deficiency, in which individuals were found to be sus- eds. Comprehensive biochemistry. Vol 19B, part 1: Pro- tein metabolism. Amsterdam: Elsevier, 1980:299-460. ceptible to early onset of emphysema.67 This led to 5 Nimni ME. Collagen: structure, function, and metabo- the so called protease-antiprotease theory of em- lism in normal and fibrotic tissues. Sem Arth Rheum physema, in which elastolytic enzymes were normally 1983;13: 1-86. met and controlled by a screen of a, antitrypsin. Elas- 6 Cheah KS. Collagen genes and inherited connective tin degradation and emphysema occurred when the tissue disease. Biochem J 1985;229:287-303. inhibitory screen. 7 Bradley KH, McConnell SD, Crystal RG. Lung collagen enzyme load exceeded the composition and synthesis. Characterization and These observations alone fall short of proving that changes with age. J Biol Chem 1974;249:2674-83. only elastin has a role. The lack of effect by col- 8 Laurent GJ. Collagen in normal lung and during. pul- lagenase depends on the nature of the enzyme used. monary fibrosis. In: Cumming G, Bonsignore G, eds. There are now known to be various collagenases, Cellular biology of the lung. New York: Plenum Press, which have different specificities for the different col- 1982:311-20. structure 9 Madri JA, Furthmayr H. Collagen polymorphism in the lagen types (see previous section on collagen lung. An immunohistochemical study of pulmonary and metabolism). Here it is important that neutrophil fibrosis. Hum Pathol 1980;11:355-65. elastase is capable of degrading types III and IV col- 10 Bateman ED, Haslam PL, Adelman-Grill BC, Turner- lagens as well as elastin.68 - 69 The results of studies of Warwick M. Cryptogenic fibrosing alveolitis: prediction metabolism in experimental animals are somewhat of fibrogenic activity from immunochemical studies of equivocal. They indicate that there is increased tum- collagen types in lung biopsy specimens. Thorax 1983;38:1 19-28. over (that is, synthesis and degradation) of collagen 11 Takiya C, Peyrol S, Cordier J-F, Grimaud J-A. Con- and proteoglycans as well as elastin. Furthermore, at nective matrix organization in human pulmonary a time when concentrations of collagen and elastin fibrosis. Virchows Arch [Cell PatholJ 1983;44:223-40. are normal there is still histological evidence of 12 Gibson MA, Cleary EG. CL Glycoprotein is the tissue emphysema.70 This is consistent with the results of form of type VI collagen. J Biol Chem 1985; some studies in man, in which total lung elastin 260:11149-59. 13 Raghu G, Striker LJ, Hudson LD, Striker GE. Extra- contents were not observed to alter in patients with cellular matrix in normal and fibrotic human lungs. Am emphysema.59 60 Rev Respir Dis 1985;131:281-9. http://thorax.bmj.com/ Further, even if elastin breakdown does constitute 14 Seyer JM, Hutcheson ET, Kang AH. Collagen poly- an initial step in the pathways leading to emphysema morphism in idiopathic chronic pulmonary fibrosis. J elastin breakdown products, or damage caused by Clin Invest 1976;57:1498-507. 15 Laurent GJ, McAnulty RJ, Corrin B, Cockerill P. Bio- elastase, are known to lead to an inflammatory cell chemical and histological changes in pulmonary fibrosis influx. These, as discussed above, are capable of re- induced in rabbits with intratracheal bleomycin. Eur J leasing enzymes or oxygen radicals that can lead to Clin Invest 1981;11:441-8. subsequent collagen breakdown. Perhaps therefore 16 Reiser KM, Last JA. 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