The glycosaminoglycan composition of skins from scleroderma patients has been analyzed by several groups with apparently conflicting results, while each ...
Biomedical Research 3 (1) 70—82, 1982
GLYCOSAMINOGLYCAN METABOLISM BY SCLERODERMA EIBROBLASTS IN CULTURE
YOSHIFUMI NINOMIYA1, RYU-ICI-IIRO HATA1, YUTAKA NAGAI1, SHINGO TAJIMA2, TAKEJI NISHIKAWA2 and HITOSHI HATANO2 ‘Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai, Chiyodaku, Tokyo 101, and ZDepartment of Dermatology, Keio University School of Medicine, Shinjuku, Tokyo I60, Japan
ABSTRACT
Increased amounts of derinatan sulfate accumulated in scleroderma skin of fibrotic stage, compared with control skin. Cultures of dividing skin fibroblasts from normal and scleroderma patients have permitted estimations of synthetic and degradative activity of each glycosaininoglycan using [3H]glucosamine incorporation and pulsechase experiments. Hyaluronic acid production in scleroderma fibroblasts was almost the same level as the control fibroblasts, although the degradation rate was slower than in control cells. On the other hand, sulfated glycosamnogiycan (derinatan sulfate, chondroitin sulfate and heparan sulfate) synthesis in scleroderma fibroblasts was lower than in control cells. Each prelabeled sulfated glycosaminoglycan was secreted into the medium without significant degradation in scleroderma fibroblasts, in contrast to control cells where it was secreted with partial degradation. These results suggest that scleroderma fibroblasts manifest a diminished level of degradative activity of each glycosaininoglycan. r
Scleroderma is a generalized connective tissue disorder characterized by inflammatory, fibrotic and degenerative tissue changes (5, 28). The nature of the pathologic process of this disorder remains unknown. Recently, LeRoy reported that scleroderma fibroblasts were characterized by an elevated synthesis and accumulation of collagen (19, 20). On the other hand, Perlish er til. (27) demonstrated that. scleroderma fibroblasts failed to show a significant increase in collagen synthesis when they were compared to control fibroblasts. The data that the scleroderma fibroblasts are more sensitive to stimulation of synthetic activity by serum than normal fibroblasts was also reported (1). It is likely that the contradictory results reported by these authors might be due to the fact that the biopsies were obtained at different stages of the progressive disease. The glycosaminoglycan composition of skins from scleroderma patients has been analyzed by
several groups with apparently conflicting results, while each group reported increases in total glycosaminoglycanconcentration. Fleischmajer and Perlish (7), Tajima er (ii. (33) and Ishikawa a.nd Horiuchi (14) found that the increase was primarily in the dermatan sulfate fraction, whereas Uitto er 0/. (34) found the increase predominantly in the chondroitin 4- and/or 6sulfate fractions. This paper describes an increased accumulation of derrnatan sulfate in scleroderma skins of fibrotic stage compared to control skins. This increase might come from the decreased degradative activity rather than increased synthetic activity. MATERIALS AND METHODS
Paii'eizr.s' Four female patients with systemic sclerosis were included in this study. All patients whose
GLYCOSAMINOGLYCANS IN SCLERODERMA FIBROBLASTS Table 1
71
Sub/'ect.s' and Biopsied J1/[aieifals E.i'ami'ned
Case No.
Age (years)
SS
Normal
G‘\L)1-R1.»-)I‘\-)i—¢-|I§.xL\Jl-.)l—*
47 57 56 41 72 20 36 33 27 23
Sex
*'1lT"l'1T1"fi1' "l'*1' "l1 'l
Durauon (years)
Stage
Biopsy Siifl
23 5 11 1 _. M M _-_ M _...
Fibrotic Fibrotic Fibrotic Fibrotic -_-H H -_ _#-
r-forearm r-forearm l-forearm 1-forearm 1-forearm r-forearm r-forearm r-forearm r-forearm r-forearm
SS, systemic sclerosis
age ranged from 41 to 57 years old, met the preliminary criteria for the classification of systemic sclerosis (32). As shown in Table 1, the duration of disease was from 1 year to 23 years. All had abnormal thickening and tightness of the skin (scleroderma), Raynaud’s phenomenon and involvement of at least one other organ system such as lung fibrosis and esophageal dysfunction in addition to the skin involvement. Subjects with healthy skin from 6 normal individuals were selected for controls and the care was taken to match the age, race, sex and site of biopsy. Tissue Ciiiture Spindle shaped skin biopsies were performed from the extensor sites of forearm from normal and scleroderma patients and transported to the laboratory in sterile culture bottles. A portion of the biopsied skin was fixed for histological examination. Under sterile conditions, fat and hair were carefully removed and washed with Hanks’ solution (Nissui Seiyaku Co., Tokyo) containing penicillin (50 U/ml) and streptomycin (50 pg/ml) to remove serum. The remaining materials were weighed and divided into 3 pieces. The first and second pieces were cut to less than 1 mm-cubes and subjected to evaluate the synthetic rate of collagen and glycosaminoglycan in tissue culture level. The third one was cut into small pieces and attached to the bottom of a 100-mm plastic culture dish (Falcon Plastic Inc., Los Angeles, CA) to get fibroblasts. The first piece minced (5.4-50.7 mg) was placed in 15 mm-Falcon organ culture dish containing lml of Dulbecco’s minimum essential medium (MEM) (Grand Island Biological Co., Grand Island, NY) supplemented with or
without 10% fetal calf serum (GIBCO, New York). After preincubation for 30 min, 100 /.tCi of D-[1-3H]glucosamine hydrochloride (3.2 Ci/mmol, Radiochemical Centre, Amersham, U.K.) was added and cultured in a humidified incubator under 5% CO2-95% air at 37°C for 24 h as described previously (24). The incubat.ion was stopped by cooling on ice and adding an unlabeled glucosamine and the resulting mixture was stored at ~—20°C until analysis. Cell Cu/i‘i1i‘e The third explants of each biopsy specimen were grown in Dulbecco’s MEM supplemented with 10% fetal calf serum in the incubator. The medium was replaced twice a week. The explant was surrounded first by epithelial-like cells, followed by growth of fibroblasts. Cells were harvested from the explant cultures by 0.1 % trypsin (1: 250, DIFCO Lab., Detroit, M1) solution for 15 min and filtered through several sheets of cheese cloth. Cells were propagated in monolayer subcultures on plastic dishes in a split ratio of 1: 4. Cell numbers were counted using a heinocytometer. Cell viability was monitored by staining with trypan blue solution. Cells in the second or third subcultures were frozen in liquid nitrogen. GIycosam iiioglycrm. Me i‘ab0li'sm. Three controls and 3 scleroderma fibroblast. cultures matched for passage level (subculture 4 or 5), site of biopsy, sex and age of the subjects were used in parallel to investigate glycosaminoglycan metabolic rate. At the desired cell growth phases, cultured
72
cells were washed twice with serum-free Dulbecco’s MEM, preincubated for 30 min, and then incubated in the same medium containing 10 ,uCi/ml of [3H]glucosamine for 2—48 h. The incubation was stopped by adding unlabeled glucosamine and cooling on ice. The medium and cell layer were stored at —20°C until analysis. For pulse-chase experiments, labeled medium was removed and remaining cells were rinsed twice and cultured with the fresh medium containing no radio-active materials as described previously (24). Isolation and Analysis of G/ycosanii'n0g/ycans Labeled glycosaminoglycans were isolated from the stored fractions as described previously (13, 22-24). The stored medium fraction was first lyophilized, then dissolved in 3ml of chilled water. The cell layer pellet was suspended in the same volume of water. Both fractions were brought to 0.5 M NaOH and kept at 4°C overnight. After adjusting pH to 6-8, an equal volume of 0.1 M Tris-HCl buffer, pH 7.8, containing 5 mM CaCl2 was added to each fraction. The resulting solution was boiled for 30 min, then subjected to Pronase (Kakenkagaku Co., Tokyo) digestion, followed by deproteinization with trichloroacetic acid (final 10%), dialysis, and lyophilization to isolate crude glycosaminoglycans. The isolated glycosaminoglycans were subjected to two-dimensional electrophoresis on cellulose acetate membranes and stained with Alcian blue to identify each components (11). For quantitative determination of glycosaminoglycan components, each spot to be determined was cut off, placed in a 1 ml dioxane and dissolved. As controls, unstained portions of the membrane was cut off and treated similarly. The amount of glycosaminoglycan was determined by measuring the optical density of the solution at 615 nm (12). When only radioactivity was determined, an aliquot of the isolated glycosaminoglycans supplemented with 1-2 /.cg each of hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, heparan sulfate and heparin as carriers was subjected to two-dimensional electrophoresis on a cellulose acetate membrane and stained. Glycosaminoglycan spots were cut and directly dissolved in Bray’s solution.
Y. NINOMIYA er al.
C/iemical and Enzymatic Treai‘ineni‘s of Glyc0sami'n.0g/ycans In order to identify the glycosaminoglycans, isolated materials were subjected to various chemical and enzymatic treatments. Glycosaminoglycan samples containing 5-8>} (666599 I° ° ° ° f f fif
HA
DS
CS
32,687
9,404 (100) 8,793 ( 94) 8,728 ( 93) 252 ( 3) 8,439 ( 90)
3,633 (100) 3,726 (103) 450
(100) 777 ( 2) 252 ( 1) 570 ( 2) 35,132 (107)
15
( 12)
338
( 9)
3,408
( 94)
16,612 (100) 15,563 ( 94) 17,468 (105) 15,894 ( 96) 932 ( 6)
Aliquots of labeled glycosaminoglycans obtained from a confluent culture of scleroderma fibroblasts were subjected to chemical and enzymatic treatments (see Materials and Methods), then analyzed by two-dimensional electrophoresis on cellulose acetate membranes after adding carrier glycosaminoglycans. The figures in parentheses are percentages of total radioactivity in individual components. HA, hyaluronic acid; DS, dermatan sulfate; CS, chondroitin sulfate; HS, heparan sulfate _
GLYCOSAMINOGLYCANS IN SCLERODERMA FIBROBLASTS
tion, the fibroblast. population had overgrown any remnant of epithelial-like cells. This resulted in confluent monolayers lacking overlapping foci. We could not find any significant differences between control and scleroderma fibroblasts from morphological points of view. A two day lag phase following inoculation of both normal and scleroderma cultures preceded the initiation of exponential growth (Fig. 1). During the subsequent 5 day intervals, the cell
tem, because we have an idea that dermal fibroblasts play a major role in glycosaminoglycan metabolism in normal and pathological conditions such as scleroderma. Fibroblast migration from control and scleroderma tissue explants occurred after 5 days of incubation in Dulbecco’s MEM supplemented with 10% fetal calf serum. Prior to the appearance of fibroblasts, the explant was surrounded by epithelial-like cells. By 2 weeks of incuba-
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Fig. 2 Electrophoresis patterns of glycosaminoglycans synthesized by scleroderma fibroblasts in culture. Fibroblasts derived from scleroderma (case 3) were cultured in Dulbecco’s MEM supplemented with 10% fetal calf serum until confluent phase. After preincubation with serum-free medium for 30 min, 10 ,u-Ci/ml of [3H]g|ucosamine was added to the medium and incubated for 24 h. Glycosaminoglycans were purified from the cell layer fraction and subjected to two-dimensional electrophoresis on a cellulose-acetate membrane using 0.1 M pyridine-0.45 M formic acid, pH 3, at 1 mA/ cm for 1.5 h in the first dimension and 0.1 M barium acetate, pH 8, at 1 mA/cm for 5 h in the second (12). The membrane was stained with Alcian blue (A). The membrane (A) was dried up and surveyed for radioactivity using /3-chromatogram camera (B). Typical electrophoresis pattern of six kinds of standard glycosaminoglycans is also shown (C). C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; HA, hyaluronic acid; HS, heparan sulfate; HP, heparin. The arrow indicates the origin.
76
Y. NINOMIYA er a/.
number in control cultures increased sharply, demonstrating a rapid proliferation phase that ended with stationary phase 7 days after inoculation. The population doubling time calculated for control cultures was 22f;2 h. In contrast, the proliferative phase of scleroderma fibroblast cultures was extended, resulting in a population doubling time of 36i2 h. Furthermore, the stationary scleroderma cell population was approximately 80% that of the stationary control culture. Using microscopic observation, morphological differences could not be found between control and scleroderma fibroblasts.
G/ycosaniinoglycan. Coniponenrs Sym‘/iesized by Cit/ti1i'ed Fibroblasts Fig. 2A shows electrophoresis pattern of glycosaminoglycans synthesized by fibroblasts derived from scleroderma skin. Radioactivity incorporated into glycosaminoglycans (Fig. 2B) was found to correspond well to those in Fig. 2A, although the relative ratios of each component were different. In order to identify the glycosaminoglycans synthesized, further characterization was done using substrate-specific chemical and enzymatic
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