immunohistochemical study collagen types - thoraximmunohistochemical study ofcollagen types in...

Thorax 1981 ;36:645-653

Immunohistochemical study of collagen types inhuman foetal lung and fibrotic lung diseaseED BATEMAN, M TURNER-WARWICK, BC ADELMANN-GRILL

From the Department of Medicine, Cardiothoracic Institute, Brompton Hospital, London andMax-Planck-Institut fur Biochemie, Munchen, Germany

ABSTRACT Highly purified type-specific anti-collagen antibodies (prepared in animals to types I, II,III, and IV bovine collagen) were used in an indirect immunofluorescence method for the study ofhuman lung collagen. The tissue localisation of each collagen type, and the apparent type 1:111

collagen ratio was assessed in normal foetal and adult lung and in fibrotic lung lesions. In thelatter, the relationship of the findings to the natural history of the lesion was considered. Thismethod was compared with routine connective tissue stains. The following observations were made.(1) Foetal lung in the canalicular phase of development proved a useful substrate for validating andstandardising the procedure. (2) Collagen fluorescence was more sensitive than connective tissuestains in detecting collagen in foetal tissues and sites of early fibrosis. (3) On the basis of collagen-type fluorescence, two distinctive patterns of fibrosis were recognised. Areas of mature collagensurrounding vessels and bronchi and in established scar tissue, for example in asbestotic pleuralplaques, were virtually exclusively type I collagen. By contrast, areas of early active fibrosis likesarcoid nodules and organising pneumonia, which usually contained variable numbers of fibroblastsand chronic inflammatory cells, were characterised by an increased proportion of type III collagenand a greater intensity of both types I and III collagen fluorescence. The possible significance of thischange in type III: I collagen ratio is discussed. Determination of the stage of fibrotic lesions by thismethod might have applications in the prediction of disease progression, and influence managementof some conditions.

Human tissues have been found to contain at leastfive idiotypes of the collagen molecule, all of whichhave also been isolated from the lung.1-5 Type Icollagen, as in other organs, is the most plentiful,the majority being associated with the bronchialtree and blood vessels. A small amount is found inlung interstitium.1 Type I fibrils are packed tightlyand form fibres of great tensile strength.6 Type IIIcollagen constitutes a larger proportion of foetalthan of adult tissues7 and usually exists in fibrils ofsmaller diameter than type j.8 The presence of typeIII collagen is thought to permit multidirectionalflexibility, an important property in a compliantstructure like the lung. Type II collagen has beenfound only in cartilaginous tissue and in the vitreousbody of the eye.9 In the lung it is confined to trachealand bronchial cartilage.2 The collagens type IVand V are present in basement membranes, andtherefore lie adjacent to the endothelial, epithelial,Address for reprint requests: Dr ED Bateman, Departmentof Medicine, UCT Medical School, Observatory 7925,South Africa.

and smooth muscle cells from which they areproduced.3 810 They may provide support for thesecells and also contribute to the filtering function ofthe basement membranes.8

Biochemical analysis is the principal method ofdetermining the proportions of these variousmolecules from tissue samples. Quantitation of thecollagen types is difficult, however, since totalextraction is seldom achieved and their solubilitydepends on several properties including the degreeof cross-linking between collagen molecules.7 11Furthermore, in most tissues anatomical localisationof collagen types is not possible even when carefuldissection techniques are coupled with the extractionprocedure. On the other hand, the isolation, puri-fication, and immunochemical characterisation ofcollagen types has resulted in the development ofwell-defined antibodies to each collagen type, whichcan be used both for quantitation of extractedcollagens and for identification of these types byimmunohistological techniques. The latter methodspermit precise anatomical localisation of collagen

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Bateman, Turner- Warwick, Adelmann-Grill

types in tissue sections. The antibodies employedare raised in animals, and isolated from antisera byimmunoadsorption on immobilised antigens.12 13Their specificity and potency are characterised byradioimmunoassay. Although, for technical reasonsthese antibodies are usually raised to bovine col-lagens, there is a substantial cross-reactivity of bovineand human collagens and these antibodies have beenused successfully in the study of human tissues.12 13

In the present report we describe the use of type-specific antibodies in the immunofluorescence studyof collagen in several forms of pulmonary fibrosis anddemonstrate their value for determining the relativeamounts and distribution of collagen types in lungtissue at various stages of the fibrotic process.

Methods

TISSUE SPECIMENSHuman foetal lung of 20 weeks gestational age froma therapeutic abortion and samples of normal adulthuman skin and kidney from surgical specimens wereobtained. Samples of normal and diseased adultlung and pleura were obtained from diagnosticopen lung biopsy, lobectomy, or pneumonectomyspecimens. All tissues were snap frozen fresh oncarbon dioxide ice, and stored at - 70°C beforesectioning. Four micron serial sections were cutfrom each on a Bright's cryostat, air dried, andstained by immunofluorescence techniques or fixedin formaldehyde for histochemical staining.

COLLAGEN ANTIBODIESAntibodies to native (triple-helical) bovine collagen,types I, II, and III were raised in guinea pigs andisolated by immunoadsorption on Sepharose-linkedcollagen of the corresponding type.14 Their speci-ficity was verified by radioimmunoassay.15 A prep-aration of purified antibodies to bovine type IVcollagen raised in rabbits was a generous gift fromProfessor AJ Bailey (Agricultural Research Council,Langford, Bristol).

TISSUE IMMUNOFLUORESCENCEThe type-specific collagen antibodies were used asthe first reactant in an indirect immunofluorescencetechnique. The second reactant was fluoresceinisothiocyanate-conjugated (FITC) immunoglobulinto guinea-pig or rabbit IgG (gamma-chain specific)prepared in rabbits or goats respectively (Miles-Yeda*Ltd, Rehovat, Israel). The former was used fortypes I, II, and III collagen fluorescence, and thelatter for type IV. The optimal dilutions of first andsecond reactants were determined in a chess-boardtitration on human adult kidney and skin, andfoetal lung, and used on all subsequent tissue

specimens.Serial sections of equal thickness from each tissue

were stained with one of four collagen antibodies.Comparisons were made on corresponding structuresand sites in consecutive sections. The semiquanti-tative assessment of the ratio of type I and type IIIcollagen was based on combined evaluation ofintensity of fluorescence and profusion of fluorescentfibres in each location, and scored on a four pointscale (absent or equivocal, +, 2+ or 3+). Oncontrol sections of each tissue anti-collagen anti-bodies were omitted, or substituted with normalguinea-pig serum (complement-preserved, WellcomeReagents Ltd, Beckenham, England) or rabbit serum.Microscopy was performed by one observer, anda single preparation of first and second layerreactants were used. Minimal interbatch variationand repeatability with this standardised techniquewas confirmed by examination of sections from asingle sample of foetal lung in each batch, and by thestudy of some pathological specimens on two ormore occasions.The sections were examined on a Reichert

"Zetopan" research microscope with ultravioletlight source for transmitted light, widefield im-mersion darkfield condenser, and a camera attach-ment for photography. Filters included a heatstopping filter (Schott and Genossen KG2/2 mm),a primary exciter filter (Schott and GenossenUG/1-5 mm) and secondary protective filters(Wratten 2B/3 mm and Schott GG 13/3 mm and1 mm). This combination permitted better histo-logical orientation and contrast between specificapple-green fluorescence and non-specific auto-fluorescence than that obtained with incident lightsystems, because tissues not stained by FITC-conjugate appeared as various shades of blue(depending on their refractile properties), insteadof black or dull yellow-green as occurred with in-cident light. Orientation and adequate contrastbetween true and false fluorescence was found to beimportant in the study of tissues like diseased lungbecause confusing distortion of architecture iscommon.

HISTOCHEMICAL STAINSSerial sections of each tissue were also stained witheach of the following: haematoxylin and eosin,modified Glees' "reticulin" silver impregnation,'6Miller's elastin van Giesen and Masson's trichromestains17 and examined on a Nikon Apophot (NipponKogaku KK) light microscope with AFM photo-micrographic attachment.The clinical history and course of each patient

was only revealed when the tissue assessment hadbeen completed.

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Lung collagen immunofluorescence 647

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-X h S:: ~~Fig 1 Photomicrographs of type-specific collagen fluorescence in oblique serial sections ofhumanfoetal bronchus(20 weeks gestation), obtained with an indirect immunofluorescence technique. In original preparations, transmittedultraviolet light gave complete colour contrast between apple-green specific collagen fluorescence and blueautofluorescence ofunstained tissues (seen here as ill-defined "fluffy" white areas). (a) Type I collagen fluorescence inbronchial wall and entire developing cartilage plate (P). Bronchial lumen marked L. (b) Type III collagenfluorescence in bronchial wall but not in cartilage plate. The chondrocytes shown appeared blue. (c) Type IV collagenfluorescence delineating folds in bronchial mucosal basement membrane. No fluorescence in cartilage (a, b, coriginal magnification x 400). (d) Type II collagen fluorescence confined to cartilage plate (original magnificationx 800).

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Results

FOETAL LUNGThis tissue at the canalicular phase of development(sixteenth to twenty-fourth week) served as a refer-ence tissue for evaluation of the anti-collagenantibodies. The bronchial walls contain carti-laginous plates which stained for type I and type JIcollagen (fig 1). Whereas type IL was confined tothis site, type I was plentiful in all other layers ofthe developing bronchus, except the mucosa. TypeIll collagen was not found in cartilage but in allother sites it occurred in association with type I, insmaller quantities. The apparent ratio of type IIIto type I collagen in foetal lung was similar in allsites and higher than in adult lung. The fluorescentsemiquantitation for both types I and III collagenin the walls of different structures decreased in thefollowing order: arteries, bronchi, pleura, veins,and budding alveolar sacs. Although the microscopyused did not provide sufficient resolution fordetailed study of differences in fibrillar forms of thesecollagen types, in alveolar walls the type III collagenappeared to have a finer, more reticular pattern. Adistinctive linear fluorescent appearance liningalveoli, bronchi, blood vessels, and pleura wasobtained with type IV collagen antibodies (figs 1and 2). In bronchi, the characteristic convolutionof the mucosal basement membrane was evident(fig 1). Outlining basement membrane in this wayhelped orientation of tissue sections and definitionof anatomical structures in foetal lung.

ADULT LUNGThe relative amounts of type I and type III collagenin different sites in normal and diseased lungs areshown in the table. Normal alveolar walls containedminimal amounts of type I collagen and barelydetectable amounts of type III collagen. Pleural,peribronchial, and perivascular fibrous tissue con-tained only small amounts of type III collagen, andeven type I collagen fluorescence was weaker than inthe same sites in foetal lung. An exception to thisratio of type I to III fluorescence was found inbronchial submucosa and subintima of vessels,where similar amounts of type I and HI collagenfluorescence were observed.As demonstrated in foetal lung, type IL collagen

was confined to bronchial cartilage in both normaland diseased tissues studied. Type IV collagen formeda continuous lining in alveolar walls, capillaries,bronchi, vessels, and pleura, and served as a usefuladditional means of defining these structures,particularly in diseased tissues where distortion ordestruction had occurred. The basement membraneof new vessels within scar tissue and surrounding

smooth muscle bundles in vascular and bronchialwalls were readily defined.

Organising pneumonia A biopsy specimen from alocalised area of right basal pulmonary consolidationof three months' duration in a patient with dermato-myositis showed large amounts of immunofluores-cent type I and type III collagen (of approximatelyequal intensity) in areas of intra-alveolar and alveolarwall fibrosis.

Sarcoid nodules A similar increase in both collagentypes was observed in established sarcoid nodulesin lung from a patient with asymptomatic sarcoidosisand 4-8 mm diameter diffuse nodularity on chestradiograph. This ratio was also present in areas ofmild alveolar wall thickening and cellularity,whereas intervening alveolar walls were normal in allrespects. Giant-cell-containing islands of mono-nuclear cells were free of collagen.

Silicosis In pulmonary silicotic nodules, the overallcollagen fluorescence was less than that in sarcoidgranulomata, particularly in the central hyalinisedzones and in the whorled bundles of densely packedcollagen (fig 3). In these zones, type I was exclusivelypresent, whereas in the outer zones, containingdust-laden macrophages and numerous fibroblasts,both types I and III were present in almost equalproportions. This feature was also observed through-out smaller cellular nodules that lacked the acellularcentral zone. New vessels were identified in the outerzones by their basement membrane fluorescence.A similar distribution of types I and III collagenwas found in the fibrosis compressing and invadingvessles.

Pleural plaques Plaques from a patient withasbestosis and pleural mesothelioma contained somefibroblasts and gave strong uniform fluorescencewith type I antibodies, but negligible fluorescencewith type LII.

Fibrous pleural thickening The fibrous pleura fromthree patients with systemic lupus erythematosis(SLE) and two patients with cryptogenic fibrosingalveolitis (CFA) with pleuritis were examined. Inall cases the major collagen in thickened pleura wastype I, but in two patients with SLE and one withCFA, moderate amounts of type III collagen werealso present. The patients in whom type III wasfound had a short disease history (four, eight and12 months) and progressive clinical deteriora-tion. The two with SLE showed a striking improve-ment in symptoms, lung function tests and chestradiography when treated with corticosteroids,

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Lung collagen immunofluorescence

whereas the third did not respond. Patients withoutdemonstrable type III collagen had a longerhistory (12 months and four years) and a stableclinical course without treatment.

COMPARISON OF HISTOLOGICAL STAINS AND

COLLAGEN IMMUNOFLUORESCENCE

In foetal lung, the fluorescence method using either

Fig 2 Photomicrogr-aphs of type-specific collagenfluorescen7ce in walls of alveolat duicts froom human foetalIluzng (20 wseeks gestation). Purified type-specific anttibodieswere uised in az inidir-ect immunofluorescence mlethod. Airspaces miarked A. Photomtiicrographs a anid b showv serialsectionis stainiedfor types I antd III collagen respectively.(c) Basetent memwbrane type IV collagen fluorescence inan adjacent area (original magnification x 1000).

type I or type II antibodies appeared to be moresensitive for detecting collagen in tissue sectionsthan conventional histological stains. The same wastrue in adult tissues in areas with increased type IIIcontent. In both these circumstances the Masson'strichrome and Miller elastin van Giesen failed todetect some of the smaller deposits of collagen,whereas the pattern of collagen obtained with the

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650 Bateman, Turner- Warwick, Adelmann-Grill

Table Scores oftype I and type III collagen content in human lunig

Tissue Type I collagen Type III collagen

Foetal: Bronchial cartilage +++ ± (Type II + + +)Alveolar wall ++ +Bronchi and vessels +++ + +Pleura and lung septa +++ + +

Normal adult alveolar wall +Adult pleura. peribronchial, and perivascular connective

tissue +:+Bronchial submucosa and subintima + + + +Organising pneumonia +++ ++ - ++Sarcoid nodule: all layers +++ ++ -+++Silicotic nodule: outer zone ++ - +++ + - ++

centre + _ ++Asbestotic pleural plaque: all layers + + + ±Fibrous pleural thickening:

in CFA ++ - +++in SLE +++ ++

Tissues were stained by an indirect immunofluorescence technique using type-specific collagen antibodies on serial sections.Semiquantitative scores were based on combined assessment of distribution and intensity of fluorescence in each location, and scored as ±(equivocal or absent), +, + +, or + + +.

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silver impregnation method coincided most closelywith that of collagen immunofluorescence. Finally,in normal adult lung, and in fibrous tissue withminimal type IIH collagen, all three stains comparedfavourably with fluorescence.

Discussion

Immunofluorescence techniques have been used forthe identification of collagen types in specificanatomical sites in several tissues,1213 1819 but wehave found no reports of their application to thestudy of fibrotic lung disease.

This study confirms that type I is themajor collagentype of established fibrous scar tissue in the lung,as it is in other organs.6 In addition, on the basis ofcollagen type fluorescence two distinctive patterns offibrosis have been identified, the first in sites ofestablished mature fibrous tissue, the second inareas of early active fibrosis. In the former situations,for example in asbestotic pleural plaques, thewhorled central zone of silicotic nodules and inperibronchial and perivascular connective tissue,collagen forms large fibre bundles. These are morerefractile than loosely arranged collagen and thusdisplay a greater degree of light-blue non-specificautofluorescence in ultraviolet light. Specific fluores-cence is obtained with antibodies to type I collagenand individual fibres are coated, giving a "tubular"rather than uniform fluorescent appearance. Inaddition, the overall intensity of fluorescence isdiminished in these sites. The reduced fluorescenceand tubular effect is probably the result of feweraccessible antigenic sites on the fibres, because ofmore cross-linkages,"l 20 21 fibril packing, or changesin connective tissue matrix. Type m collagen isseldom detected in these locations. By contrast, insites of early or active fibrosis, the content of bothtype I and type III is increased, and quantitatedfluorescence of the latter often approaches that oftype I. Other features include a more intense overallfluorescence of both collagen types as a result ofuniform coating of collagen fibres with fluorescentantibody complexes, and less non-specific auto-fluorescence. These sites are usually more cellularand contain variable numbers of fibroblasts andchronic inflammatory cells.The two patterns frequently coexist in a single

lesion, as seen in the silicotic nodule where thecentral zone is "mature", and the outer cellularzone has features of active fibrogenesis. However insome biopsies, for example from acute organisingpneumonia, active sarcoidosis, and fibrous pleurisyof short duration, the fluorescence appearances aresufficiently uniform to permit an overall assessmentof early active fibrosis to be made.

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The finding in this study that type III collagenincreases in sites of early active fibrosis in the lungaccords with observations in other organs and tissues.It has been found in immunofluorescence studies ofwound healing in children,'8 fresh atheroscleroticintimal fibrosis,22 human and experimentally inducedliver cirrhosis,19 23 24 and in biochemical studies ofdermal collagen synthesis in response to acute orchronic inflammatory stimuli.25 Human embryonicskin fibroblasts have been shown to be capable ofsimultaneous synthesis of both type I and type Mcollagen,26 and it is probable that in response toinflammation and other stimuli, "invading" orproliferating fibroblasts or possibly myofibroblasts27increase type HI collagen synthesis. Bailey et al25found that the proportion of type m collagen inmature rat skin increased from about 10% to 40%in acute inflammatory fibrosis.The physiological implications of changes in

collagen ratios are being increasingly recognised.Besides the structural differences between collagentypes which can influence mechanical properties oftissues, the various types have different biochemicalproperties. For example, the cell attachment protein,fibronectin, binds more avidly to type III collagenthan to type 1, II and basement membrane col-lagens,28 and provides the mechanism by which typeIII collagen induces more platelet aggregation thanthe other types.29 30 This may have importantconsequences in early atherosclerosis, as well as inother lesions. Furthermore, the binding site offibronectin on collagen corresponds to the specificbinding site for mammalian collagenase, suggestingthat the binding of fibronectin might play a role inthe regulation of different rates of degradation ofcollagen types.31The comparison of connective tissue stains in this

study showed that silver impregnation is the stain ofchoice for detecting collagen in foetal lung and inearly fibrotic lesions. This supports the findings ofHuang32 in studies of alveolar wall collagen. Con-troversy exists as to whether argyrophilic connectivetissue is all collagenous,3233 but its profusion inimmature and embryonic tissues is well recognised,and is not explained by collagen type, fibril size,or glycosaminoglycan content, although it mightbe related to fibre organisation.8 The relativelyhigh type III collagen content in these locations, asshown by immunofluorescence, suggests that alarge proportion of argyrophilic fibres are type mcollagen. There is, however, as yet no certain in-formation on the relative silver staining propertiesof types I andm collagen fibres.There are several potential,sources7of error in the

methods used in this study, and precise standardisa-tion is impossible. The chief difficulties are the sub-



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jectivity of histological assessments, particularlythose involving immunofluorescence, and the inter-batch differences in specificity and affinity ofanticollagen and fluorescein-labelled antibodies.Provided that highly purified anticollagen antibodiesare used, with certain measures a degree of standard-isation can be achieved, which permits at least avalid comparison within a single series. The measuresemployed in this study included (a) the use of asingle batch of anticollagen antibodies and secondlayer reactants in predetermined dilutions, (b) theuse of the same second layer reactant for types I, ITand III collagen antibodies, and (c) the assessment ofrepeatability and interbatch variation on a standardsubstrate, and by several examinations of somepathological specimens. In addition, fluorescencemicroscopy was performed by a single observerwho had no previous knowledge of the histo-pathology or clinical details of each case.Human foetal lung in the canalicular phase of

development proved a valuable substrate for valida-tion of type-specific antibodies to collagen andstandardisation of the technique. These measures arean absolute requirement before pathological tissuesamples are studied.2 34 Human foetal epiphysealcartilage growth plates have also been used for thispurpose.'8 The value of foetal lung is based on thepresence of types I, II, III, and IV collagen atdifferent easily defined locations in the same section,the immaturity of the collagen fibres, and the relativeabsence of elastic fibres at this stage.The different locations in which the collagens

occur in foetal lung are: types I and II withindeveloping cartilage plates, type III in the samedistribution as type I with the exception of cartilage,and type IV collagen exclusively in sites containingbasement membranes. The simultaneous presenceof types I and II collagen in maturing cartilageplates is of interest since adult cartilage in othersites contains only type II collagen.35 There are,however, no reports of detailed biochemistry ofadult or foetal bronchial cartilage freed of itsfibrous perichondrium. In a study of limb develop-ment in chicks, Linsenmayer et a136 showed that atransition in collagen type occurred: from type Iin the limb bud mesenchyme, to solely type II intibial diaphysis by day 8. Later type I reappeared asosteogenesis began. Cartilage plates are found inproximal bronchi from the tenth week of intrauterinedevelopment, and their appearance proceeds distallyuntil the end of the canalicular phase.37 Thematuration of the cartilage matrix takes severalweeks longer.37 Cartilage development during thecanalicular phase might therefore correspond tothe transitional phase in chick limb buds describedby Linsenmayer et a136 when both type I and type II

collagen are present.A second advantage of using foetal lung in the

canalicular phase as a standard substrate is itsminimal elastic tissue content. At this stage a smallamount is visualised in arterial walls and major air-ways, but not in alveolar walls.38 Elastic fibres arehighly refractile and display characteristic ice-blueautofluorescence in ultraviolet light, the intensityof which may interfere with evaluation of the specificfluorescence of adjacent collagen. Third, the collagenin foetal tissue is less mature, has fewer cross-links,and is less tightly packed than in adults.' 11 It there-fore stains with predictable intensity. Finally, theavailability of foetal lung also commends it for useas a standard substrate, and a single sample can beincluded in many batches.

In conclusion, the immunohistochemical study ofcollagen is a useful sensitive method for detectingsmall overall increases in lung collagen, possibly atstages at which they are not detected with otherconnective tissue stains. It also has an importantapplication for defining areas of active or earlyfibrosis, whether widespread or focal. This is gaugedfrom the increased proportion of type III collagen,the intense uniform fluorescence of both types Iand III fibres, and the relative absence of non-specific collagen autofluorescence. In contrast, latelesions or mature scar tissues are identified byweaker specific fluorescence and more pronouncedautofluorescence of the type I collagen fibre bundles,the absence of type III fluorescence, and the relativeacellularity of these connective tissue sites. Thismethod is usefully combined with routine histologicalassessment which has the advantage of defining thecell types present. In some conditions, this informa-tion will assist with decisions about treatment, andthis application has recently been assessed in agroup of patients with cryptogenic fibrosing alveo-litis (unpublished observations).

This work was supported in part by grants fromICI South Africa (Pharmaceuticals) Ltd, LillyIndustries Ltd, and Dista Pharmaceuticals, England,to whom we are very grateful.

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