Collagen Synthesis and Degradation by Systemic Sclerosis Lung Fibroblasts

chondroitin sulfate, and heparan sulfate and low concentrations of desulfated chondroitin sulfate and dermatan sulfate were below control levels (1-3%). High concentrations of 6bronectin, chondroitin sulfate, and heparan sulfate produced labeling indices at or near those of untreated matrices. Low concentrations of type IV collagen and high concentrations of desulfated chondroitin sulfate and dennatan sulfate produced labeling indices above control levels (7, 5.5, and 6.5%, respectively). At 96 h in culture, cells cultured on untreated plastic had labeling indices of 4.5-5.5%. High concentrations of nbronectin and heparan sulfate produced labeling indices near control levels., while the labeling indices for all other matrices were well above control levels. Most notably, high concentrations of laminin produced an index of 11 .5%. All other matrices were associated with labeling indices in the 7.5-9.0% range. At 144 h, nearly aU cultures were confluent and some had commenced dome formation, in which the only dividing cells were confined to the dome structure. Because of this regional activity, these cultures were not further evaluated. The studies on matrix biosynthesis indicated that the isolated type 11 cells produced a similar panel of ECM components at 72 h in culture, regardless of the matrix substrate. Fibronectin, laminin, and type IV collagen were all produced by cultured type II cells. DISCUSSION

All of the matrix substrates studied generally supported isolated type II cell attachment and growth, most showing a depressed mitotic index at the 48-h time point compared with controls (uncoat.e d tissue culture plastic). Notably, low concentrations of type IV collagen and high concentrations of desulfated chondroitin sulfate or dermatan sulfate were associated with labeling indices above control values at 48 h . At 96 h , the mitotic indices for the type II cells on the various matrices were nearly all in the range of 50-100% above control levels. The exception was with high-concentration laminin substrates., which yielded indices nearly 2.50% above control values. These data indicate that matrix substrate influences the proliferative characteristics of isolated type 11 cells in culture. For the 6rst time, to our knowledge, sulfated glycosaminoglycans and their desulfated forms have been demonstrated to support type II cell proliferation. These effects., along with those of more commonly used matrix substrates., appear to be at least partially concentration dependent. The patterns of biosynthesis of ECM components were found to be independent of substrate and concentration. It may be that cells "traumatized" by isolation procedures synthesize ECM molecules in a genetically programmed fashion, independent of the cell's new e nvironment. This study establishes a baseline for further investigations on the possible role of sulfated ECM in influencing type II cell phenotypic characteristics and proliferative and functional capacities in vitro and ultimately in vivo. REFERENC ES

1 Gil J, Martinez-Hernandez A. The oonnective tissue of the rat lung: electron immunohistochemical studies. J Histocbem Cytocbem 1984; 32:230-38

2 Toribta C , Villager B, Kuhn C , McDonald JA. Ultrasbuctural disbibution of 6bronectin in normal and fibrotic human lung. Lab Invest 1985; 52:399-408 3 Sannes PL. Cytochemistry and immunocytochemistry of basement membranes and other extraceUular matrices. In: A oom· prebensive treatise on pulmonary toxioology. \btl. Comparative pulmonary biology of the normal lung. Caldwell, NJ: Telford Press, Inc (in press) 4 Sannes PL. Immunobistochemical localization of chondroitin sulfate in extraceUular matrices of rat lung. J Histocbem Cytocbem 1990; 38:1063 5 IC.atsuyama T, Spicer SS. A cation-retaining layer in the alveolarcapillary membrane. Lab Invest 1977; 36:428-35 6 Vaccaro CA, Brody JS. Ultrasbuctural localization and characterization of proteogl~s in the pulmonary alveolus. Am Rev Respir Dis 1979; 120:901-10 7 Van ICuppeveltTHMSM, Cremers FPM,ICuyper CMA. Staining proteoglycans In mouse lung alveoli. I . Ultrastructural localIzation of anionic sites. Histochem J 1984; 16:657-69 8 Sannes PL. Dilferences In basement membrane microdomains type I and type II pneumocytes in the rat and rabbit lung. J Histochem Cytocbem 1984; 32:827-33 9 Dobbs LG, Gonzalez R, Williams MC. An improved method f'or isolating type n ceUs in high yield and purity. Am Rev Respir Dis 1986; 134:141-45 10 Frenette GP, Ruddon Rw, JCnesidd RF, Naser JA, Peter BP. Biosynthesis and deposition of a oonoovalent laminin-beparan sulfate proteoglycan complex and other basal lamina components by human malignant cell line. J Bioi Cbem 1989; 264:3078-88

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Collagen Synthesis and Degradation by Systemic Sclerosis Lung Fibroblasts* Responses to Transforming Growth Factor-p

N . K.Harr-Von, M.R.C.P.; A C. Argent, M . Sc.;

R. J. McAnulty, Ph.D .; C. M . BltJclc, M.D .; 8 . Corrin, M.D.; and G. J. Laurent, Ph.D.

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atients with systemic sclerosis (SSe) frequently develop flbrosing alveolitis., and investigation of such patients often allows the identification of 6brosing alveolitis early in its natural history.' In preliminary ultrastructural studies of patients with early lung disease we have demonstrated swollen and pyknotic endothelial cells., thickened endothelial basement membrane, interstitial edema, and bare epithelial basement membranes with proliferating type II pneumocytes.• These observations suggest that injury to the endothelium and epithelium initiates a sequence of inflammatory events that ultimately results in the deposition of excess collagen in lung interstitium. To investigate whether this environment selects lung fibroblasts that are phenotypically high producers ofcollagen, we compared the rates ofcollagen synthesis by lung fibroblasts from 6 patients with SSe and 6 control cell lines. •From the Biochemistry Unit (N .IC.H ., A.C .A., R.J.McA, G.J.L .) and the Department Pathology (B.C .), Natiooal Heart and Lung Institute, Brompton Hospital, and the Department ofRbeumatol= ogy (C.M.B.), Royal Free Hospital, London, Emdand. Supported by a grant from the Chest, Heart and Stroke Association of Great Britain.

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CHEST I 99 I 3 I MARCH, 1991 I SUpplement

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Cells were grown to confluence and incubated for 24 h in media containing ascorbate, L-proline, and p-aminoproprionitrile supplemented with varying concentrations of calf serum and transforming growth facto~P (I'GF-p). Collagen synthesis and degradation of newly synthesized collagen were measured by estimating protein-bound and free hydroxyproline, respectively, in the culture medium by highpressure liquid chromatography. 3 A wide range of values was obtained in both groups. The mean (range), expressed as pmol hydroxyproline/J.Lg DNA/h for SSe lines was 16.8 (3.338). This value was similar to that of controls, 17.1 (4-39.3). Enhanced collagen synthesis occurred in response to increasing concentrations of serum and TGF-P in both groups, but the mean response to TGF-~. expressed as percent baseline synthesis rates in 0.4% calf serum, was greater in SSe cell lines than in controls (125 vs 31%). This increase was due in part to a reduction in the degradation of newly synthesized collagen by SSe cell lines. More than half of the newly synthesized collagen was degraded rapidly, but again,

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groups did not differ (mean values 55 and 62%). These results suggest (1) if SSe lung fibroblasts are producing excess collagen in vivo, this phenotype is not consistently maintained for all cell lines in vitro, and (2) a potential role may exist for TGF-P in the pathogenesis of lung fibrosis in SSe. REFERENCES

1 Harrison NK, Glanville AR, Strickland 8 , Haslam PL, Corrin B, Addis BJ, et al. Pulmonary involvement in systemic sclerosis: the detection of early changes by thin section cr scan, broncboalveolar lavage and -rc-DTPA clearance. Respir Med 1989; 83:403-14 2 Harrison NK, Soosay G , Dewar A, Myers AR, Black CM, Corrin 8 . Morphology of the lungs in systemic sclerosis: comparison with cryptogenic fibrosing alveolitis and identification of early ultrastructural abnormalities. Am Rev Respir Dis 1990; 141:A318 3 Campa JS, McAnulty RJ, Laurent GJ. Application ofhigb-pressure liquid cbromatogJBpby to studies of collagen production by isolated cells in culture. Anal Biocbem 1990; 186:257-63