Isolation and Tissue Localization of Type AB2Collagen From Normal Lung Parenchyma

Joseph A. Madri, MD, PhD, and Heinz Furthmayr, MD

Type ABa collagen was isolated from normal lung parenchyma by pepsin extractionfollowed by differential salt extraction. This collagen comigrates with AB2 collagenisolated from placental membranes when run on 5% polvacrylamide gel elec-trophoresis; it has an aA and aB polypeptide chain ratio of 1: 2 and a cyanogen bromidepeptide profile similar to known AB2 collagen on 7.5% polvacrylamide gel elec-trophoresis. This AB2 collagen isolated from lung tissue specificalli inhibits passivehemagglutination of affinity-purified rabbit antibodies to AB2 collagen isolated fromamnionic and chorionic membranes. Bv indirect immunofluorescence microscopy, AB2collagen was found to be widely distributed throughout the lung and was foundpreferentially associated with cell surfaces (basement lamina) and basement mem-branes. (Am J Pathol 94:323-332, 1979)

THE CON-NECTIVE TISSL- E of the lung is morphologicallv- andbiochemically complex. Collagen has been found to compose up to 20 ofthe dry weight of the connectiv-e tissue matrix of the adult human lungand has been implicated to play an important role in differentiation anddevelopment as -ell as in the structure and function of this organ innormal and fibrotic disorders.' 5 Howvever. little is known about the impor-tance of the distinct types of collagen for these processes.There is ample evidence for the existence of at least five polymorphic

types of collagen. each composed of distinct a-chains. The chemicalstructure of Types I. II. and III collagen has been established.6 8 Thestructure of Type IV collagen (basement membrane) is still a matter ofcontroversy,9 and evidence of other collagen types (AB2 '0 and CP45 ")illustrates the fact that our knoxvledge of the collagen polymorphism maystill be incomplete.

Sev-eral investigators have noted multiple collagen types in the lung.' 5Using dissection techniques coupled with extraction and cvanogen bro-mide peptide analysis, they noted Types I and III collagen in the periph-ery and Type II collagen in tracheal and bronchial preparations.' 4.12These studies were completed before the discovery of Type AB2 collagen

Fro)m the Department of Pathologs Iale LUnix-ersitx School of Nledicine. Nemv Hasen. Con-necticut

Supported by Grant 1 1-204-77S from the Xmerican Heart Xssociation. Fairfield Count' Chapter.Inc Dr \ladri is a t-SPHS Felloss and recipient of Grant 1 F3_ HLO53.59-01 Dr Furthmavr is therecipient of a Facults- Research award from the American Cancer Societv. No 177.

Accepted for publication October 2. 1975.Address reprint requests to Joseph A. N\adri. \ID. PhD. Department of Pathology. Yale University

School of Medicine. 310 Cedar Street. Ness Hasven. CT 065100002-9440/79/0208-0323$01.00 323( 1979 American Association of Pathologists

324 MADRI AND FURTHMAYR American Journalof Pathology

by Burgeson et al 10 and Mayne et al 11; therefore, the presence of thiscollagen type in the lung was not sought or was not recognized (the maincyanogen bromide peptides of aA and aB chains elute with a1CB7 andalCB4,5,6, respectively, on CM-cellulose column chromatography 11).The present study reports the isolation of Type AB2 collagen from

human lung parenchyma and its tissue localization using specific anti-AB2collagen antibodies in an immunofluorescence study.

Materials and Methods

Differential Salt Precipitation of the Collagens

At autopsy, normal lungs were recovered intact from patients who had died of unrelatedcauses. Only ltungs without gross and microscopic evidence of disease were used in thisstudy. Tracheobronchial tissue and large vessels were removed by sharp dissection, andthe remaining peripheral lung was cut into 1-cm cubes, homogenized in a Waring blenderat 4 C, washed extensively with distilled water at 4 C, and lyophilized. Ten-gram samplesof dried, homogenized lung tissuie were rehomogenized in 0.5 M acetic acid, and pepsin(Worthington) was added at a concentration of 1 g/10 g dry weight of tissue. The mixturewas incul)ated for 24 houirs at 4 C.

Undigested residue was separated by centrifugation at 10,000g for 1 hour at 4 C. Theextracted collagens were precipitated by the addition of solid NaCl to a final concentrationof 1.0 M NaCI. The precipitate was solubilized in 1.0 M NaCl, 50 mM Tris-HCl, pH 7.5,at 4 C. Type III collagen was precipitated by dialysis against 1.7 M NaCl, 50 mM Tris-HCO, pH 7.5, at 4 C. Type I collagen was precipitated by dialysis of the Type IIIsupernatatnt fluiid against 2.6 NI NaCl, 50 mM Tris-HCl, pH 7.5, at 4 C. Type AB2 collagenwas precipitated by dialysis of the Type I supernatant fluid against distilled water at 4 C.The collagens were then dissolved in and dialyzed against 0.1 M acetic acid and werelyophilized.Type I, III, and AB2 human collagens were prepared from human amnionic and

chorionic membranes according to the method of Burgeson et al,'0 with the exception thatType AB2 collagen was precipiated by dialysis against distilled water. Type II collagen wasprepared from calf nasal septums according to the method of Trelstad et al.'3 Type IVcollagein was prepared from murine EHS sarcoma matrix according to the method ofTimpl et al."4

SDS-Acrylamide Gel Electrophoresis of Collagen ChainsSDS-polvacrylamide gel electrophoresis of collagen chains and cyanogen bromide

peptides was conducted on 5% and 7.5% polyacrylamide gels (36 hours, 6 mA/gel for 5%gels, and 12 hours, 6 mA/gel for 7.5% gels) using the procedure of Furthmayr and Timpl.'5

Amino acid hydrolyses of the collagens were conducted according to the method ofMiller. "'

Antibodies against Type AB2 collagen isolated from human amnionic and chorionicmembranes were prepared in rabbits according to the methods of Nowack et al."7 Theantibodies were purified by cross-absorption on affinity columns composed of Type I andType III collagens conjuigated to CNBr-activated Sepharose 4B,"8 followed by immuno-absorption on a Tvpe AB2-sepharose affinity column and elution with 1.0 M acetic acid,0.15 M NaCI. After adjusting the pH to approximately 7, the affinity-purified antibodieswere dialyzed against phosphate-buffered saline and concentrated by Amicon ultrafiltra-tion. Titers and specificity were determined by the hemagglutination inhibition methodsdescribed by Beil et al."9

Vol. 94, No. 2 LOCALIZATION OF AB2 COLLAGEN IN LUNG 325February 1979

TLssue Immunofluorescence

Sheep antirabbit IgG %vas prepared using purified rabbit IgG as immunogen and rabbitIgG (Miles) conjugated Sepharose 4B as an immunoabsorbent."' Fluorescein-conjugatedsheep antirabbit IgG wsas prepared using standard techniques.20 Frozen sections of lungtissue and subsequent immunofluorescence staining and microscopy %vere done by stan-dard techniques 21 and using a Leitz WTetzlar Ortholux Research microscope equipped w itha fullv automatic Orthomat microscope camera and a fluorescence vertical illuminator.

Results

Chemical Studies

The differential salt precipitation of collagen Types I. III. and AB2yielded a ratio of Type I: Type III: Type AB2 of 35 :58 :7 by w-eight of thelyophilized collagens. The total collagen extracted wvas approximately 830mg/25 g dry wveight of tissue. The identification and purity of the Types 1,III, AB2 collagen wvere assessed by 5% polvacrylamide electrophoresis ofthe a-chains and cyanogen bromide peptide pattern on 7.5%c poly-acrylamide gel electrophoresis (Text-figures 1 and 2). Type I collagen hadan al a2 chain ratio of 2: 1; Type III collagen wvas composed of only alchains; Type AB2 collagen isolated from lung tissue had an aA to aB chainratio of 1 :2. The polvpeptide chains of Types I III, and AB2 extractedfrom lung comigrate wvith those isolated from human amnion and cho-rionic membranes (arrows, Text-figure 1). A faint unidentified band wasnoted. migrating betwveen the ,B components and a chains in the Types Iand III collagen fractions. T-wo other faint unidentified bands were alsonoted migrating below the a chains in the Types I and III collagenfractions (arrowheads. Text-figure 2). No bands comigrating wvith Type INcollagen isolated from the EHS sarcoma wvere noted.

I III AB2

TEXT-FIGLFRE 1-gDizel electrophoresis of the a -chains of the collagens extracted from puilmonary Ivparenchyma The extracted pulmonary collagens are -compared with Types I. III. and AB2,collagen isolated aBfrom htiman amnionic and chorionic membranes and aATvpe IV collagen isolated from murine EHS sarcoma a _arrottus ULnidentified bands are designated by ar- W 22 Irou heads !

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326 MADRI AND FURTHMAYR American Journalof Pathology

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TEXT-FIGURE 2-7.5% gel electro-phoresis of the cyanogen bromide pep-tides of the collagens isolated from pul-monary parenchyma. The labeled peakscorrespond to those described by Bur-geson et al 10 and are identical to thoseisolated from placental membranes.

The cyanogen bromide peptide patterns for Types I, III, and AB2collagens extracted from lung were identical with Types I, III, and AB2isolated from human placental membranes. The major peptides for eachof the collagens are labeled in Text-figure 2 according to previouslypublished results.10Amino acid compositions of Types I, III, and Alt collagens extracted

from lung were in agreement with compositions of Types I, III, and AB2isolated from placental membranes and previously published values 10

(data not shown).

Immunochemical and Tissue Immunofluorescent Analysis

The hyperimmune serums obtained after a series of four subcutaneousinjections of 1 mg of AB2 collagen in complete Freund's adjuvant, each 2weeks apart, had a titer of 1: 2560 by passive hemagglutination. Thehemagglutination reaction was only weakly inhibited by the addition of

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Vd. 94, No. 2 LOCALIZATION OF AB2 COLLAGEN IN LUNG 327February 1979

Types I, II, III, and IV collagen and was completely inhibited by theaddition of 2.5 ,g of AB2 collagen (data not shown). The affinity-purifiedanti-AB2 collagen antibodv had a final titer of 1: 100,000 in the passivehemagglutination assay. The hemagglutination reaction is only weakl,inhibited bv the addition of Type I, II, III, or IV collagen and is com-pletelv inhibited by the addition of 2.5 ,g of AB2 collagen. The hemagglu-tination is also completely inhibited of 2.5 jg AB2 collagen extracted fromthe lung tissue and purified byr differential salt precipitation (Text-figure3). Neither the antiserums nor the purified antibody showed any cross-reaction with denatured AB2 collagen (aA and aB chains) by hemaggluti-nation-inhibition or bv hemagglutination with human red cells con-jugated with aA or aB chains.

Frozen sections of normal human lung tissue stained with affinity-purified anti-AB2 collagen antibodv and a secondary, fluorescein-labeledantibody reveal intense fluorescent staining of alveolar septums (Figures1A and 1B). Intense fluorescence is noted (arrowheads) on capillary base-ment membranes and on the alveolar wall aspects of the epithelial liningcells of the alveoli in linear basement-membrane-like pattems (arrows).This pattem is consistent with a basement membrane localization and issimilar to that observed in Goodpasture's syndrome. 2223

Intense fluorescent staining was also noted on bronchiolar basementmembranes (arrowheads) and on bronchial epithelial lining cell mem-branes in a delicate irregular pattem (arrows) (Figure 2). This pattem offluorescence is also consistent with a basement membrane (basement

TEXT-FICGRE 3-Hemagglutination inhibition studv °0of the specificity of anti-A%B collagen antibodv. Initial <dilution of antibod!- was 1: 100. Concentration of thecollagens used as inhibitors was 2.3 Mg. I = human 6placental pepsin-extractable T-pe I collagen. II = calf °nasal septal pepsin-extractable Type II collagen. III =human placental pepsin-extractable T%pe III collagen. 4IN = murine chondrosarcoma acid-soluble Type IV |collagen. AB, = human placental pepsin-extractable oAB2 collagen. 4B2L = human lung pepsin-extractable 4 2AB2 collagen. 0 = no inhibitor.

I II III IV AS,AlO0Anti AB2 Antibody

328 MADRI AND FURTHMAYR American Journalof Pathology

lamina) localization of the antibody. The fluorescence was completelyinhibited by the addition of lung or placental membrane-extracted AB2collagen to the primary antibody before application to the tissue section.No fluorescence was observed when normal rabbit serums were used inplace of the anti-AB2 collagen antibody or when only the secondaryfluorescein-labeled sheep antirabbit IgG antibody was used (data notshown).

DiscussionSeveral investigators have noted collagen heterogeneity in the lung

using salt, acid, and pepsin extraction; cyanogen bromide tissue hydroly-sis; and biosynthesis utilizing lung tissue explants.15 These studies havedemonstrated Types I and III collagen in pulmonary parenchyma andType II collagen apparently localized to the tracheobronchial tree. TypeIV (basement membrane) collagen has been demonstrated indirectly inpatients with Goodpasture's syndrome, whose serums exhibit basementmembrane deposition in lung tissue and renal glomeruli when studied byindirect immunofluorescence.

In the differential salt precipitation studies done previously, AB2 colla-gen was not found because it is soluble in 2.5 M NaCl, 50 mM Tris-HCI,pH 7.4, and the 2.5 M NaCl supernatant fluid was not further analyzed. Incyanogen bromide tissue hydrolysis studies and by CM-cellulose columnchromatography coupled with cyanogen bromide peptide analysis, AB2collagen could have easily gone unrecognized because of the very similarcyanogen bromide peptide elution profileQ of aICB7,8 and aACBa,b anda1CB6 on CM-cellulose column chromatography and aBCBc.'0 "

In the present study, we find AB2 collagen to make up approximately7% of the pepsin-extractable collagens of the lung. We realize that thisfigure may not reflect the actual content of AB2 collagen in the lungbecause of possible incomplete recovery due to cross-linking and in-solubility, different solubilities of the extracted collagens, and probablepepsin sensitivity of Type IV collagen influencing the total collagenextracted.The immunofluorescence study reveals that AB2 collagen has a wide-

spread distribution in the lung. The finding of specific fluorescence inbronchial basement membranes, on bronchial epithelial cell membranes,and on alveolar and capillary basement membranes suggests that AB2collagen may be a cell-surface (basement lamina)-associated collagen anda component of certain basement membranes. The lack of cross-reactivitybetween anti-AB2 antibody and Type IV collagen isolated from the mu-rine EHS sarcoma suggests that different basement membranes are most

Va.94, No.2 LOCALIZATION OF ABS COLLAGEN IN LUNG 329February 1979

likely composed of different collagenous proteins as well as other non-collagenous components. Antibodies to the Type IV collagen used in thisstudy have been shown to react with basement membranes.1'The presence of AB2 collagen in the lung warrants reinvestigation of the

changes in collagen type previously noted in pulmonary fibrosis. Animmunofluorescence study of pulmonary fibrosis utilizing specific anti-types I, II, III, IV, and AR, collagen antibodies, coupled with immuno-chemical quantitation is in progress and may lead to a better understand-ing of the changes in clinical parameters noted in pulmonary fibrosis.Studies of the interaction and anatomic location of the different collagensin fibrotic disorders compared with those in the normal lung are possiblyas important as investigations of changes in collagen types for a betterunderstanding of the pathophysiology of pulmonary fibrosis.

Refemces1. Bradlev K, McConnell-Breul S, Crystal RG: Lung collagen heterogeneity: Protein

synthesis. Proc Natl Acad Sci USA 71:2828-2832, 19742. Bradley KH, McConnell SD, Crystal RG: Lung collagen composition and svn-

thesis: Characterization and synthesis with age. J Biol Chem 249:2674-2683, 19743. Bradley K, McConnell-Breul S, Crvstal RG: Collagen in the human lung. J Clin

Invest 55:543-50, 19754. Hance AJ, Bradley K, Crystal RG: Lung collagen heterogeneitv: Synthesis of type

I and type II collagen by rabbit and human lung cells in culture. J Clin Invest 57:102-111, 1976

5. Seyer JM, Hutcheson ET, Kang AH: Collagen polysmorphism in idiopathic chronicpulmonary fibrosis. J Clin Invest 57:1498-1507, 1976

6. Rauterberg J, Kuhn K: Acid soluble calf skin collagen. Eur J Biochem 19:398-407,1971

7. Miller EJ, Lunde LG: Isolation and characterization of the cyanogen bromidepeptides from the al (II) chain of bovine and human cartilage collagen. Biochemistr,12:3153-3159, 1973

8. Chung E, Keele EM, Miller EJ: Isolation and characterization of the cvanogenbromide peptides from the al (III) chain of human collagen. Biochemistrv 13:3459-3464, 1974

9. Orkin RW, Gehron P, McGoodwin EB, Martin GR, Valentine T, Swarm R: Amurine tumor producing a matrix of basement membrane. J Exp Med 145:204-220,197d

10. Burgeson RE. El Adli FA, Kaitila II. Hollister DW: Fetal membrane collagens:Identification of two new collagen a chains. Proc Natl Acad Sci USA 73:2579-2583,1976

11. Mavne R. Vail MS, Miller EJ: Characterization of the collagen chains synthesizedby cultured smooth muscle cells derived from Rhesus monkev thoracic aorta. Bio-chemistry 17:446-452, 1978

12. McLees BD, Schleiter G, Pinnell S: Isolation of type III collagen from human adultparenchymal lung tissue. Biochemistry 16:185-190, 1977

13. Trelstad RL, Kang AH, Toole BP, Gross J: Collagen heterogeneity. J Biol Chem247:6469-6473, 1972

14. Timpl R, Martin GR. Bruckner P, Wick G, Wiedemann H: Nature of the collag-enous protein in a tumor basement membrane. Eur J Biochem 84:43-52, 1978

330 MADRI AND FURTHMAYR American Journalof Pathology

15. Furthmayr H, Timpl R: Characterization of collagen peptides by sodium dodecyl-sulfate-polyacrylamide electrophoresis. Anal Biochem 41:510-516, 1971

16. Miller EJ: Structural studies on cartilage collagen employing limited cleavage andsolubilization with pepsin. Biochemistry 11:4903-4909, 1972

17. Nowack H, Gay S, Wick G, Becker U, Timpl R: Preparation and use in immuno-histology of antibodies specific for type I and type III collagen and procollagen. JImmunol Methods 12:117-124, 1976

18. Parikh I, Marsh S, Cuatrecasas P: Topics in the methodology of substitutionreactions with agarose. Methods in Enzymology, Vol 34. Edited by WB Jacoby, MWilchek. New York, Academic Press, Inc., 1977, p 77

19. Beil W, Furthmayr H, Timpl R: Chicken antibodies to soluble rat collagen. I.Characterization of the immune response by precipitation and agglutination meth-ods. Immunochemistry 9:779-788, 1972

20. Wells AF, Miller CE, Nadel MK: Rapid fluorescein and protein assay method forfluorescent-antibody conjugates. Appl Microbiol 14:271-275, 1966

21. Goodman M: Fluorescent Antibody Methods. New York, Academic Press, Inc.,1968

22. Beirne, GJ, Octaviano GN, Kopp WL, Burns RO: Immunohistology of the lung inGoodpasture's syndrome. Ann Intern Med 69:1207-1212, 1968

23. Lerner RA, Glassock RJ, Dixon FJ: The role of anti-glomerular basement mem-brane antibody in the pathogenesis of human glomerulonephritis. J Exp Med126:989-1004, 1967

AcknowledgmentsWe thank Mr. John Albert and Mr. John Braslin for excellent photographic assistance and Mrs.

Maria Silberberg for typing the manuscript.

Fiure IA-Indirect immunofluoresence pattern of pulmonary pa-renchyma stained with anti-AB2 collagen antibody at 1 :1000 dilution. Thefluorescence is noted on alveolar septum basement membranes (arrows)as well as surrounding capillary walls (arrowheads). B-High-powerfield exhibiting a linear basement-membrane-like immunofluorescencepattern of pulmonary parenchyma stained with anti-AB2 collagen antibodyat 1:1000 dilution. (A, x 250; B, x 500) Figure 2-Indirect immuno-fluorescence pattern of bronchi stained with anti-AB2 collagen antibody.Intense linear fluorescence is noted on the bronchial basement mem-brane (arrowheads). A weak irregular delicate fluorescence is also notedon bronchial epithelial lining cells (arrows). (x 500)