Adam, M.

Deyl, Z.

Dostal, C.

Clinical Chemistry and Laboratory Medicine (CCLM)

English

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Collagen Heterogeneity in Systemic Scleroderma and Other Diseases

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Adam, Dostal and Deyi: Collagen heterogeneity In systemic scleroderma and other diseases 495J. Clin. Chem. Clin. Biochem.Vol. 17,1979, pp. 495-498Collagen Heterogeneity in Systemic Scleroderma and Other DiseasesBy M. Adam, C DostalResearch Institute for Rheumatic Diseases, Prague, CSSR,andZ. DeylInstitute of Physiology, Czechoslovak Academy of Sciences, Prague, CSSRSummary: Proportions of collagen type I and type III were investigated in different tissues (kidney, liver, heartmuscle, esophagus, lung and skin) in a single case of malignant systemic scleroderma. The proportion of collagentype HI was higher than in healthy controls. Similarly, an increase in the proportion of collagen type III was shownin tissues affected by other inflammatory diseases. Further the authors suggest that the increase in total kidney col-lagen, especially type III, has important consequences for kidney function; in the described case it had the criticalinfluence on the fatal course of the disease, and the patient died from uremia.Kollagen-Heterogenität bei systemischer Sklerodermie und anderen ErkrankungenZusammenfassung: Die Anteile von Kollagen Typ I und Typ III wurden in verschiedenen Geweben (Niere, Leber,Myokard, Oesophagus, Lunge und Haut) bei einem Fäll von maligner systemischer Sklerodermie untersucht. Generellwurde im Vergleich zu Gesunden eine Zunahme des Anteils von Kollagen Typ III festgestellt. Gleichermaßen eine Zu-nahme des Anteils von Kollagen Typ III findet in Geweben bei anderen entzündlichen Erkrankungen statt. DieAutoren weisen daraufhin, daß die Zunahme des Gesamt-Kollagen, insbesondere des Kollagen Typ III, in der Nierevon Bedeutung für die Funktion der Niere ist und daß dies im beschriebenen Fall einen kritischen Einfluß auf denVerlauf der Erkrankung hatte, da der Patient an Uraemie verstarb.IntroductionCurrent knowledge of systemic scleroderma extendsmainly to the descriptive phase, and our understandingof the disease remains superficial. The skin and sub-cutaneous elements are not exclusively affected, butthe process may be widespread and involve virtually allthe tissues of the body. The prognosis of life in thepatient with systemic scleroderma depends on thepresence and degree of visceral involvement. The mostcritical organ involvement^ which can be fatal in ämatter of weeks or several months, are renal orcardiopulmpnary. On the other hand esophageal hypo-mobility is the most common visceral manifestation ofscleroderma, and it often appears early. Histologieexaminations of systemic scleroderma lesions show alter-ation of collagen fibers (1). On the other hand the resultsof different authors (2,3) suggest increased collagen bio-synthesis in a number of patients with systemic sclero-derma.Recent advances in collagen chemistry have madepossible the detailed characterization of the insolubleifraction. These studies have led to the discovery ofcollagen heterogeneity. The more abundant type Icollagen of molecular composition ( ( ))2 2 ispresent, together with another genetically distinctcollagen type III of molecular composition (a1(III))3in interstitial tissues. Other genetically distinctcollagens are: collagens type IV of composition(ai(IV))3, and the newly discovered type V from bloodvessels, composed of chains oA and aB (4). The occur-rence of collagen polymorphism has been identified indifferent tissues such as skin, aorta, liver, lung, kidney,synovial membrane, peripheral nerve. The content ofcollagen type II in the loose connective tissue changes incertain physiological and pathological states.In this report we present data suggesting an increase in.the quantity of collagen type III in different tissues0340-076X/79/0017-0495S02.00© by Walter de Gruyter & Co. · Berlin · New York496 Adam, Dostal and Deyl: Collagen heterogeneity in systemic scleroderma and other diseasesinvolved with systemic scleroderma, as well as othersources of inflammatory diseases.Material and MethodsSamples of tissuesOne case of malignant systemic scleroderma was studied. Thiswas a 42 year-old female, in which only six months elapsedbetween the onset of symptoms and death. In spite of highdoses of corticosteroids, the situation of the patient rapidlybecame worse; urea nitrogen in the blood increased to119 mg/dl, severe chest pains appeared, extrasystolae wereobserved in ECG and finally anuria occured before exitus.Ischemic necroses of renal cortex, serofibrinous pericarditisand esophagal erosions, besides others, were found atnecropsy.For comparison skin biopsy was taken from a patient (48years old) with systemic scleroderma, which was taking anormal course. Cases of bronchopneumonia and a case ofliver cirrhosis were also analyzed, in order to study thedifferences in the respective organs. Further, the compositionof collagen was investigated in rheumatoid nodule and inrheumatoid synovial tissue.Samples of organs under investigation were obtained atnecropsy from 3 healthy individuals (46, 53 and 62 yearsold) that died during traffic accidents. The diagnoses ofscleroderma and of rheumatoid arthritis were done accordingto the A.R.A. criteria (5).Collagen preparationsCollagens type I and III were isolated by pepsin digestionaccording to Fujji & Kühn (6) and the values were calculatedfrom the hydroxyproline content using correction factorswith respect to different hydroxyproline concentration in bothcollagen types (7.46 for type I and 5.5 for type III).Separation techniquesPolyacrylamide gel electrophoresis was performed by themodified method of Stark & Kühn (7), using 0-alanine bufferpH 4.3. The gels were stained with Amido Black 10 B andthen evaluated at 680 nm in a Perkin Elmer UV Spectrophoto-meter Model 402 equipped with a gel scanning accessory.CM cellulose chromatography (CM cellulose 52 Whatman) wasby the original method ofPiez et al. (8). The column was1.6 X 6 cm thermostated at 40°C with a flow rate 72 ml/h.A linear gradient was prepared from two 120 ml quantities ofappropriate buffers. The effluent was monitored on a UVspectrophotometer (Perkin Elmer Model 402) using an ultramicroflow-through cell at 230 nm.Other analysesHydroxyproline was assayed according to Stegeman (9).Results and DiscussionSolubility criteria have served for years äs the onlyindicator of differences in collagen from differentsources. This included differences between normaltissues, and differences caused by pathologicalconditions. With the discovery of different collagentypes, the question of the relative proportions of thesebecame pertinent.The main tissues involved in malignant systemicscleroderma were therefore investigated with respectto the content of collagen type III. Normal tissues aswell as tissues affected with various diseases served ascontrols. The identity and purity of isolated collagentype III were proved in all samples by carboxymethylcellulose chromatography as well as by polyaciylamidegel electrophoresis without and with dithiothreitolpretreatment (fig. 1, 2). Electrophoretic runs revealed theexpected increase of the -band after dithiothreitol treat-ment. The Chromatographie and electrophoretic profileswere in all cases rather similar, and in the figurescollagen type III from systemic scleroderma malignantskin is shown as an example.The data reported here are valid of course for the pepsinsolubilized fraction of insoluble collagen, as this solubili-zatiori is incomplete in old tissues. According to theresults of the methods used the possibility cannot beexcluded that small amounts of collagen type I may bepresent in the fraction precipitated with 1.7 mol/1NaCl; this would be unlikely, however, to have apronounced effect upon the results. On the other handbasement membrane collagen (type IV) has a precipi-tation profile similar to that of type III (10), and thismight influence the presented data to a greater extentin liver and kidney samples. In healthy tissues the0.100 -I§0.050 -70 o 140Eff luen t volume [ml]210Fig. 1. CM cellulose chromatography of collagen type I ( ) and collagen type III (- ) Column 1.6 X 6 cm therinostated at40 °C, flow rate 72 ml/h; linear gradient from two 120 ml quantities of appropriate buffers (8).J. Clin. Chem. din. Biochem. / Vol. 17,1979 / No. ?Adam, Dostal and Deyl: Collagen heterogeneity in systemic scleroderma and other diseases 497α1 16 5 4 3 2 1 0 - 6 5 4 3 2 1 0Distance lern]Fig. 2. Densitometric tracing of polyacrylamide gel electro-phoresis.a) without dithiothreitol;b) after dithiothreitol pretreatment of collagen type 111.lowest amounts of collagen type HI occurred in synovialtissue and skin (about 20%), while parenchymatousorgans, heart muscle and esophagus constitute aboutone third of pepsin solubilized collagen (tab. 1). It isto be expected that under normal conditions the amountof collagen type III will vary with age, as shown for skinby Epstein (11) and Deyl & Adam (12). Furthermore,from the table, it is also evident that collagen type III isincreased in inflammed tissues, i.e. in tissues involvedwith systemic sclerosis as well as with rheumatoidarthritis and bronchopneumonia.Organ function, especially in parenchymatous organs,is affected not only by the concentration of singlecollagen types, but also by the total collagen content,which is also increased in diseased tissues. We suggestthat this fact is very important for the function of kidney.In the case reported here, collagen exerted an indis-putably critical influence on the fatal course of thedisease, and the patient died from uremia. As expectedfrom the clinical course and pathological picture,collagen metabolism was also affected in other visceralorgans (liver, lung, heart muscle, esophagus).Of particular interest are collagen changes in liver, wherein cirrhosis the amount of collagen type III was lower thanin healthy controls. Fleischmajer et al. (13) reported thatin sclerodermous skin collagen type III is increased mainlyin the lower dermis and in subcutaneous tissue.In general, synthesis of collagen type III predominates innewly formed tissues. Gay et al. (14) showed that a singlecell is capable of synthesizing both collagen types I andIII. In other words it appears that there is a certaincategory of pathological situations in which themechanisms determining the type of collagen to besynthesized by a certain population of fibroblasts, or themechanisms of collagen degradation, are altered. Withrespect to scleroderma, the protein synthesizingmechanisms must be considered, since it has beenshown by Brady (15) that in a number of organs in thisdisease, there is little or no collagenolytic activity.The actual proportions between individual collagentypes, as well as the total amount of collagen, are, ofcourse, influenced by the course of the pathologicalprocess. Variations are found in the solubility of skincollagen in scleroderma, and increased as well asdecreased collagen concentrations can be found in thesoluble fractions (16, 17, 18). In the active stage ofscleroderma an increase of collagen biosynthesis hasbeen definitely proved (19), while decreased values havebeen described in extreme fibrosis.It is now apparent, that in addition to being the majorstructural protein, stabilizing body organs by the mecha-nical strength of its fibers, collagen is also essential forcytodifferentiation, in which it plays a key role. Colla-gen metabolism and its disturbances are, from this pointof view, of crucial importance.Tab. 1. Collagen type III content from insoluble pepsin-treated collagen in various tissues (in mg/g of total solubilized collagen).Investigated tissueSkinEsophagusHeart muscleLiverLungKidneySynovial tissueNormal4622134232539130235124 years old53 62years old208332337379288340191331301382294312204DiseasedSSM+Systemic scleroderma (48 years old)SSMSSMSSMCirrhosis, late stage (62 years old)SSMBronchopneumonia 1 st case (68 years old)2nd case (54 years old)SSMRheumatoid arthritis (35 years old)Rheumatoid nodule (44 years old)411362444408458302418467380430318438+SSM - systemic scleroderma malignant (42 years old)J. CUn. Chem. Clin. Biochem. / Vol. 17,1979 / No. 7498 Adam, Dostal and Deyl: Collagen heterpgeneity in systemic scleroderma and other diseasesReferences1. Beigelman, P. M., Goldner, F. Jr. & Bayles T. B., (1953), N.Engl. J. Med. 249, 45-54.2. Le Roy,E. C, (1972), J. Exp. Med. 135,1351-1362.3. Krieg, T., Müller, P. K. & Goertz, G., (1977), Arch.Dermatol. Forsch. 259,105-107.4. Burgeson, R. E., El Adly, F. A., Kaitila, E. E. & Hpllister,D. W., (1976), Proc. Natl. Acad. Sei. U.S.A. 73,2579-2583.5. Bennet, P. H. & Burch, T. A., (1967), Bull. Rheum. Dis.77,453-462.6. Fujii, T. & Kühn, K., (1975), Hoppe Seyler's Z. Physipl.Chem.JJtf, 1793-1801.7. Stark, M. & Kühn, K., (1968), European J. Biochem. 6,534-541.8. Piez, K. A., Eigner, E. A. & Lewis, M. S., (1963), Biochemistry2, 58-66.9. Stegemann, H., (1958), Hoppe Seyler's Z. Physiol. Chem.311,41-51.10. Trelstad, R. L., Catanese, V. M. & Rubin, D. Fs, (1976), Anal.Biochem. 77,114-118.11. Epstein, J. H. Jr., (1974), L Biol. Chem. 249, 3225-3231.12. beyi, Z. & Adam, M., (1977), Mechanisms Aging Develop.6, 25-33.13. Fleischmajer, R., Gay, S., Mejgel, W. N. & Perlish, J. S.,(1978), Arthr. Rheum. 27, 418-428.14. Gay, H. S., Martin, G. R., MüUer, P. K., Timpl, R. & Kühn,K., (1976), Proc. Natl. Acad. Sei. U.S.A. 739 4037-4040.15. Brady, A. H., (1975), J. Clin. Invest. 56,1175-1180.16. Adam, M. & Deyl, Z., (1978), Reumatplogia 16, 75-81.17. Bashey, R. L, Halpern, S., Stephens, R. E.", Perlish, J. S. &Fleischmajer, R., (1975), Biochem. Biophys. Res. Commun.62, 303-307.18. Zimmermann, B. K. & Balda, B. R., (1972), Arch.Dermätol. Forsch. 243, 357-363.19. Le Roy, E. C., (1974), J. Clin. Invest. 54, 880-889.Milan Adam, M.D.., Sc. D.,NaSlupi4,CS-128 50 Praha 2CSSRJ. Clin. Chem. Clin. Biochem. / Vol. 17,1979 / No. 7

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Collagen Heterogeneity in Systemic Scleroderma and Other Diseases

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