British Journal of Dermatology (1979) lOO, 359.
Characterization of dermal collagen in systemic sclerosis C.R.LOVELL,* A.C.NICHOLLS,t V.C.DUANCE4 AND AJ.BAILEYf
•
* Department of Medicine, University of Bristol, I Department of Animal Husbandry, University of Bristol, Langford, and t Meat Research Institute, Agricultural Research Council, Langford, Bristol Accepted for publication 7 July 1978
SUMMARY
The amount of dermal collagen is increased in systemic sclerosis. However, unlike certain inflammatory conditions, the relative proportions of Type I and Type III collagens are closely similar to those found in normal adult dermis. Similarly, no change in the distribution of the collagen types could be detected by immunofluorescent staining, although a considerable thickening of the epidermis was clearly evident in all the sclerotic lesions examined.
The pathological changes in systemic sclerosis include vascular narrowing with ischaemic atrophy of tissues and proliferation of collagen. Abnormal deposition of collagen leads to gross thickening of skin, often resulting in multiple contractures. Using electron microscopy, Bahr (1956) described the presence of an abnormal population of small collagen fibres in the dermis of patients with scleroderma and Hayes & Rodnan (1971) noted a 'beaded fllament' appearance of these fibres, similar to those of embryonic dermis. Despite marked macroscopic and microscopic changes in dermal collagen, little is known of any biochemical modifications. Harris & Sjoerdsma (1966) found a decreased amount of neutral salt soluble collagen in affected, as compared with normal, adult dermis. Uitto et al. (1971), however, demonstrated an increased proportion of neutral salt soluble collagen in the diseased dermal plaque, indicating either an increase in collagen synthesis, decreased cross-linking or decreased degradation. Studies on the cross-linking of the dermal collagen in scleroderma have confirmed that increased synthesis is taking place (Herbert et al, 1974). Recent studies have demonstrated that normal skin contains at least two genetically distinct isotypes of collagen. The major constituent, Type I, consists of two identical polypeptide chains (oti). and a third, non-identical oc2 chain. The other collagen. Type HI, contains three identical polypeptide chains, similar but not identical to the cf, chains of Type I collagen. Normal skin also contains a very small amount of basement membrane collagen defined histologically as the dermal-epidermal junction. Basement membrane collagen also occurs in the blood vessels but the Correspondence: Dr C.R. Lovell, Dermatology Department, Bristol Royal Infirmary, Bristol. 0007-0963/79/0400-0359 $02.00
«
.(;,. 1979 British Association of Dermatologists
359
36o
C.R.Lovell et al
biochemical constitution of these collagens has not been investigated. Type III collagen is a major constituent of embryonic dermis (about 50-60",,) but is present to a lesser extent (15-25'^,) in adult dermal collagen (Epstein & Munderloh, 1975). In certain inflammatory conditions the proportion of Type i n collagen increases (Bailey et al, 1975) and it was therefore considered of interest to see if a similar change occurs in systemic sclerosis. In this paper we describe the presence and location of the different genetic types of collagen in the affeaed dermis in systemic sclerosis in comparison with normal dermis. MATERIALS AND METHODS
Patients Samples of the skin were obtained from biopsy specimens, with the patient's informed consent, or at post mortem of patients with systemic sclerosis. Clinical details of the patients are tabulated in Table I. Age-matched samples of skin were obtained at post mortem or operation from subjects with no evidence of the disease. The skin was dissected free of adhering fat, diced into cubes of approximately "* and then chemically defatted using i :3 methanol: chloroform. Materials Pepsin was obtained from Worthington Biochemical Corp. (Freehold, N.J., U.S.A.) 3250 units/mg. Fluorescein conjugated anti-rabbit IgG was obtained from Burroughs Wellcome. Normal rabbit serum was obtained from Gibco. Determination of genetic types of collagen Pepsin digestion. Defatted samples of skin were extracted with physiological saline, pH 74 for 4 days at 4-C. The residue was incubated with pepsin in 0 5 M acetic acid at a substrate:enzyme ratio of 10:1 (w/w) for 18 h at 4' C (Chung & Miller, 1974). These conditions arc known to degrade most proteins but to leave the helical portion of collagens intact. The residue and supernatant were separated by centrifugation and further pepsin in 0-5 M acetic acid was added to the residue, the digestion being continued for a further i8 h at 4 C; this was repeated four times. Finally, the solubilized collagen was precipitated from the pooled supcrnatants by the addition of salt crystals to 0 9 M NaCl dialysed exhaustively against 0-5 M acetic acid and freeze-dried. The proportion of collagen extraaed was determined by hydroxyproline analysis on ponions of the solubilized material and residue. SDS polyacrylamide gel electrophoresis. To check the purity and identify the molecular types of collagen present the total digest was analysed by acrylamide gel electrophoresis. Samples of the pepsin-solubilized collagen were denatured in sodium dodecyl sulphate (SDS) at 38 C, with and without mercaptoethanol, to disrupt the intramolecular disulphide bonds of Type III collagen. The achain composition was determined by the polyacrylamide gel electrophoresis using the flat-bed techniques described previously (Bailey & Sims, 1976). In order to separate the 1 (III) and a (I) collagen chains the reduction of the 1 (III) to a III with //-mercaptoethanol was delayed until 10 min after the start of the electrophoresis. Cyanogen bromide digestion. Defatted samples of skin were homogenized and extracted with physiological saline, pH 74 for 4 days and the residue digested under nitrogen with cyanogen bromide (CNBr) in 70",, formic acid at 30 C for 4 h. The samples were diluted with deionized water and the CNBr and formic acid were removed by evaporation in vacuo at 30 C. The residue was redissolvcd in 0 5 M acetic acid and centrifuged.
Dermal collagen in systemic sclerosis
361
TABLE I. Clinical details of patients with systemic sclerosis
Site of biopsy
Patient
Age
Sex
A
40
F
Forearm
B
42
F
Forearm
C
45
F
Forearm
D
42
F
Forearm
E
53
F
Forearm
F
40
F
Forearm
G
50
F
Forearm
H
72
F
Forearm
I
58
M
Forearm
J
45
F
Forearm
K
44
F
Forearm
L
56
F
Forearm
History
Treatment at time of biopsy
22 years progressive dysphagia. 5 years Raynaud's Colchicine phenomenon and progressive tightening of skin 0'5 mg b.d. of fingers, hands and forearms. 4 years ago calcinotic nodule right middle finger 3 years Raynaud's phenomenon and pruritus of Nil hands with increasing stiffness of the fingers. 2 months tendon sheath involvement. No visceral involvement clinically. 2 years Raynaud's phenomenon with tightening of skin on forearms and fingers. 2 weeks progressive renal failure and malignant hypertension leading to death
D-penicillamine 750 mg Prednisolone 10 mg Anti-hypertensive drugs and diuretics
18 months diffuse thickening of skin of hands, forearms, neck and thighs, i month progressive D-penicillamine 500 mg renal failure, leading to death Diuretics 9 years swollen fact and hands, generalized D-penicillamine pruritus. Raynaud's. Minimal skin tethering. 500 mg. Topical Dysphagia corticosteroid ointments 1 year swelling of hands and forearms. Raynaud's. D-penicillamine 750 mg No systemic involvement. Sister has S.L.E. 1 year heartburn, dysphagia and constipation. D-penicillamine 500 mg 6 months taut fingers; spontaneous amputation Prednisolone 5 mg of one finger. Facial telangiectases. No dermal Colchicine 0'5 mg b.d. involvement of forearm 4 years tightening of skin over fingers with facial D-penicillamine 250 mg puckering, 2 months constipation. Raynaud's and ulceration of skin 2 years tightening of skin over fingers and foreNil arms. Raynaud's. No systemic involvement 15 years Raynaud's. 5 years facial telangiectases Colchicine 0 5 mg b.d. 2 years dysphagia and dyspnoea, i year tethering of forearm skin 8 years Raynaud's, tethering of skin of hands and D-penicillamine 125 mg forearms. Facial telangiectases. Involvement of Spironolactone 100 mg skin of neck and thighs Burinex K ii b.d. Prednisolone E.C. 75 mg Nil 6 years tethering of skin of fingers, hands and forearms, neck, trunk and thighs. Dyspnoea due to pulmonary fibrosis
(i) The CNBr peptidcs were analysed by disc gel SDS polyacrylamide electrophoresis using the method described by Furthmayr & Timpl (1971). After staining with Coomassie Blue the gels were analysed on a Joyce-Loebl Chromoscan. (ii) The cyanogen bromide peptides were also examined by CM-cellulose chromatography to obtain a more accurate quantification of the proportion of collagen types present. The soluble cyanogen
362
C.R.Lovell et al.
bromide peptides were applied to the column (i-8x 12 cm) of CM-cellulose (Whatman CM 52) equilibrated with 002 M sodium formate, pH 36, containing 001 M sodium chloride, at 44 C. The peptides were eluted by means of a linear gradient 001-017 ^ ^^Cl over i 1. The column effluent was monitored at 228 nm using a Cecil CE 212 spectrophotometer. Selected areas of the chromatographs (see results) were pooled, desalted on Biogel P-2 and further purified by chromatography on Agarose (Biogel A 05 M, 2 6 x 120 cm) equilibrated with i M calcium chloride. Preparation of antibodies. Collagens were extracted from human placenta by digestion with pepsin at io°C for 24 h. The solubilized collagens were fractionated by salt precipitation to give Type III collagen at i -5 M NaCl, Type I at 25 M NaCl and AB Type IV at 4 0 M. The procedure was repeated and the purity of the collagens determined by SDS-polyacrylamide gel electrophoresis. Antibodies to these collagens were raised in New Zealand White rabbits, and purified on immunoabsorbent columns of Types I, III and AB IV to remove non-specific antibodies, as previously described (Duance et al, 1977)Tissue staining. Transverse sections were stained with specific rabbit anti-human Type I, III and AB Type IV, washed extensively with phosphate buffered saline, and then stained with fluorescein conjugated anti-rabbit IgG as previously described (Duance et al., 1977). For controls non-immune rabbit serum was used in place of the specific anti-collagen antibody. The sections were viewed with a Leitz fluorescence microscope. RESULTS Collagen isotypes
Over 80/,, of dermal collagen was extracted from both normal and diseased dermis using repeated pepsin digests at 4 C. SDS-polyacrylamide gel electrophoresis revealed a preponderance of Type I
aid)-
FIGURE I. SDS-polyacrylamide slab gel electrophoresis of normal and diseased skin. Analysis of total pepsin digests: Tracks 1^3 systemic sclerosis skin—(i) without mercaptoethanol; (2) plus mercaptoethanol added 10 min after start of electrophoresis; (3) plus mercaptoethanol. Tracks 4-6 normal skin—(4) without mercaptoethanol; (5) plus mercaptoethanol added after 10 min eiectrophoresis; (6) plus mercaptoethanol.
Dermal collagen in systemic sclerosis
NORMAL
FIGURE 2. Densitometric traces of SDS-polyacrylamide disc gel electrophoresis of cyanogen bromide peptides of normal and diseased dermis. The peaks marked i(I), i(III) denote cyanogen bromide peptides of Types I and III collagen respectively (Traub & Piez, 1971).
FRACTION NUMBER
FIGURE 3. CM-celluIose chromatography of the cyanogen bromide peptides derived from (i) systemic sclerosis and (ii) normal skin. Conditions as described in text. Arrows mark application of sample and gradient.
363
C.R.Lovell et al.
364
40
50
70
80
90 FRACTION
100
110
120
130
NUMBER
FIGURE 4. Molecular sieve chromatography of the cyanogen bromide peptides from regions (i)a and (ii)a of Fig. 3. (i) systemic sclerosis skin (ii) normal skin. Conditions as described in the text.
05 03 02 01
FRACTION
NUMBER
FIGURE 5. Molecular sieve chromatography of the cyanogen bromide peptides from regions (i)b and (ii)b of Fig. 3. (i) systemic sclerosis (ii) normal skin. Conditions as described in the text.
Dermal collagen in systemic sclerosis
FIGURE 6. Immunofluorescent localization of collagen isotypes in skin from normal and systemic sclerosis subjects, (a) staining of normal skin with anti-human Type III; (b) immunofluorescent localization of the epidermal-dermal basement membrane with anti-human AB Type IV; (c) antiAB Type IV staining of systemic sclerosis skin. Note gross thickening of epidermis in (c) compared to the normal skin in (b).
365
366
C.R.Lovell et al.
collagen, but a significant proportion of Type III. The patterns were similar in both normal and diseased skin. There was a suggestion using this gel technique that an increased amount of Type III was present in systemic sclerosis. Two faint bands were also seen, the mobility corresponding to the Type IV A & B collagen bands found in placental basement membranes (Duance et al., 1977) and rheumatoid synovium (Lovell et al.., 1977). These bands were present in both normal and diseased tissue (Fig. i) in approximately the same amounts. CNBr peptide analysis (i) Cyanogen bromide peptides of dermal collagen were separated by SDS disc gel electrophoresis to provide a semi-quantitative analysis of the proponions of Types I and III but gave identical patterns for both normal and diseased dermis (Fig. 2), indicating the absence of any change in the amounts of Type I and III collagen in the diseased condition. Biopsies from twelve different patients were compared with sex and age matched controls. (ii) Accurate quantification of the collagen types was carried out by analysis of the cyanogen bromide peptides on CM-cellulosc column. However, in confirmation of the SDS gels, the chromatograms from normal and diseased skin were also very similar (Fig. 3i and ii). In order to determine the amount of each isotype of collagen present in each case the regions marked a—representing 5:i(I)CB3 and 3!i(III)CB4 (Miller review, 1976) and b—representing ai(I)CB8 and ai(III)CB8 (Epstein & Munderloh, 1975) were pooled, desalted and separated on agarose. The regions marked a gave one major peak corresponding to a molecular weight of approximately 13,000 (Fig. 4a, b); the region marked b gave three peaks (Fig. 5a, b) one of which had a molecular weight of approximately 24,000 and a second with a molecular weight of about 10,000. The peak of higher molecular weight was assumed to be products of partial cleavage by cyanogen bromide. The 24,000 dalton peak was identified as .ai(I)CB8 by molecular weight and amino acid composition and the 10,000 dalton peak was assigned as ai(III)CB8 by molecular weight; unfortunately, insufficient material was obtained for an accurate amino acid analysis. The proportion of Types I and III collagens were determined in two ways: (i) by integration of peak areas representing 0(i(I)CB8 and xi(ni)CB8 in normal and diseased skin (Epstein & Munderloh, 1975) and (2) by measuring the threonine:valine ratio in the 13,000 dalton peak from region a of the CM-cellulose chromatogram as suggested by Butler et al. (1975). By the first method the diseased skin was found to contain about 10",, Type III and normal skin about 12/,:, Type III. However, by the second method, the ratio of threonine to valine showed a much higher proportion of Type HI; about 23",, Type HI in diseased skin and 25",, Type HI in normal (Table 2). This discrepancy between the two methods of determining the ratio of the collagen types may be accounted for by several factors. Firstly, only u.v. absorbance was used to determine the ratio in the first method and although some account was made for the differing size of ai(I)CB8 and :fi(III)CB8 the extinction coefficients for each peptide at the monitoring wave length could be different. Also the CB peptides eluting either side of ai(I)CB8 in the CM-cellulose, i.e. ai(I)CB7 and a2CB4, each have a molecular weight approximately the same as aiCB8 (24,000) so that any overlap by them in the chromatogram would result in an increase of the size of 31CB8 on the agarose and hence a decrease in the proportion of Type HI. In order to reduce this overlap only the main peak of ai(I)CB8 was pooled and as xr(HI)CB8 appears to elute slightly earlier than 5;i(I)CB8, this might have resulted in slight losses of the total ai(HI)CB8 present in the sample. However, despite the discrepancy in absolute values of Type I and Type HI, both methods clearly showed that the proportion of Type IH present in normal and systemic sclerosis skin was the same. This technique was time consuming and required considerable amounts of material, hence could not be carried out on each patient. For rapid comparison of biopsy material from a significant number of patients the CNBr peptide pattern on acrylamide gel was utilized.
Dermal collagen in systemic sclerosis
367
TABLE 2. Amino acid analyses of normal and systemic sclerosis skin cyanogen bromide (CB) peptides Normal >B3 + 3ri(in)CB4
Systemic sclerosis 3ti(I)CB3 + =(i(Iir)CB4
ctiCB^ ca(III)CB4 (Butler et al., 1975)
—
—
I5'8
15
19
6-8 1-8
158 6-6 17
6
7 4
Ser
33
yz
HSer Glu Pro Gly Ala Val
3
0-4
0-6
I
15-6 15-7 40-8
15 15
49
56
21
14
35 —
15-5 15-3 41 4 18-4 37 —
0 I II 20
4
0
0-5 4-0
0-7 4-0
0 0
0 0
3
5
0-2
0-16
0
2'5
3
0-2
26 0-4
0 I
0-3
0-5
53
5*2
46
7-5
O-I
0-2
0
0
56
5S
6
3
3Hyp 4Hyp Asp Thr
Met
He Leu Tyr Phe Hyl Lys His Arg •Ratio of Thr: Val = «i(III):.i(I)
t87
Normal Systemic sclerosis
0-34:1 0'3i:i
0
25" „ Type III 23" „ Type III
* Corrected for differences in molecular chain composition in Type I and Type III.
Immunofluorescent localization of collagen types
Normal dermis. The antibodies against Type I collagen reacted with all parts of the dermis as observed by other workers (Meigel et al, 1977). In contrast to this overall staining. Type III staining is restricted to thread-like structures throughout the dermis reminiscent of the reticulin network observed following silver staining. The staining of Type III collagen also appears to be more concentrated around the epidermal-dermal junction. Pronounced staining of the epidermal-dermal junction and blood vessels also occurs (Fig. 6a). Since the large blood vessels are known to contain a high proportion of Type III collagen, it is not surprising that the small blood vessels also contain Type III collagen. Similar results have been obtained by Meigel et al. (1977) using anti-bovine Types I and III. In our hands bovine antibodies are much less reactive against human tissues (Duance et al, 1978). When stained with anti-AB Type IV the epidermal-dermal junctionisclearly delineated and apart from the small blood vessels no other parts of the dermis react with this basement membrane collagen antibody (Fig. 6b). Systemic sclerosis dermis. No difference could be demonstrated in the distribution of the anti-Types I, III and AB-IV in biopsies from normal subjects and those with systemic sclerosis. A distinct difference observed in every case examined was the thickening of the epidermis, as reported previously by a number of workers. This thickening is clearly shown in Fig. 6c.
368
C.R.Lovell et al DISCUSSION
Traditionally it has been held that in systemic sclerosis, as well as in morphoea, there is a progressive increase in dermal collagen associated with more or less destruction of dermal appendages (e.g. Lever, 1961; Gardner, 1976). Others (Milne, 1972; Black, Bottoms & Shuster, 1970) suggest there is dermal shrinkage but that the normal micro-architecture of the skin is maintained, i.e. it is normal skin in a contracted state. An increase in dermal collagen may reflect either increased rate of synthesis or a decreased rate of degradation. Although the latter possibility remains to be explored, there is considerable indirect evidence of increased collagen synthesis. Uitto et al. (1969) have found increased levels of protocollagen proline hydroxylase, an enzyme involved in collagen synthesis, in diseased skin compared with skin obtained from normal subjects. Increased incorporation of '*C-proline as ' *C-hydroxyproline into explanted skin samples in vitro has been demonstrated in clinically involved skin (Le Roy, 1974) and in clinically normal skin (Perlish, Bashey & Stephens, 1976) from patients with the disease. Indeed, the presence of excess labile aldimine intermolecular cross-links, which would account for the increased solubility, has been demonstrated in 'active' dermal lesions in our previous studies on systemic sclerosis (Herbert et al, 1974). In newly formed granulation tissue of dermal wounds there is a marked increase in Type III collagen (Bailey et al., 1975) and a reappearance of the stable keto intermolecular cross-link found in embryonic skin collagen (Bailey, Bazin & Delaunay, 1973). In systemic sclerosis, however, we found no increase in the proportion of Type III collagen in the dermis, the proportion of the two collagen types remaining similar to that of normal dermis. Furthermore, in this disease an aldimine cross-link is present, similar to that found in dermal collagen in childhood, rather than the 'keto' type crosslink found in embryonic skin and granulation tissue. The analyses on the proportions of Type I and III collagens were carried out on a large number of patients by CNBr peptide electrophoresis, and confirmed by the more exact, but time and material consuming, CM-ccllulose technique on two subjects. More importantly, the distribution of the collagen is also unchanged. Although active lesions were analysed it is possible that a transient increase in Type III could occur during an initial infiammatory stage. On the basis of these results the clinical impression of toughness of the skin is not readily accountable. Clearly there is increased collagen synthesis, which could lead to toughness due to a tighter packing of the fibres, or the binding to the deeper structures could be increased. Alternatively the grossly thickened epidermis or subcutaneous oedema could lead to the impression of rigidity in the dermis. Although there is no change in the proportion of collagen types synthesised by fibroblasts in systemic sclerosis, abnormal interaction between fibroblasts and other cells may lead to increased collagen synthesis. Alternatively the proliferation of dermal collagen may be due to an external stimulating factor, rather than an inherent abnormality of the fibroblasts themselves. This is supported to some extent by the finding that cultured fibroblasts from subjects with systemic sclerosis do not synthesize excessive amounts of collagen (Gibbon, unpublished data). Scleroderma-like changes are seen in metabolic disorders, such as scurvy, prophyria cutanea tarda and carcinoid syndrome, and also following exposure to vinyl chloride (acro-osteolysis) (Jayson et al, 1976). Unlike these disorders, a metabolic abnormality which might stimulate collagen synthesis has not yet been described in systemic sclerosis. ACKNOWLEDGMENTS
We thank the Consultant Dermatologists of the Bristol Royal Infirmary, and Mrs A.Swan and Mrs M.Gibson for technical assistance. This work was supported in part by grants from the Nuifield Foundation and the Hadwen Trust.
Dermal collagen in systemic sclerosis
369
REFERENCES BAHR, G.F. (1956) Eleklronenoptische Untersuchungen bei Sklerodermie Zugleich ein Beitrag zur Kenntis des Bindegewebes der Haut. Aerztliche Forschung, 10, 255. BAILEY, A.J., BAZIN, S. & DELAUNAY, A. (1973) Changes in the nature of the collagen during development and resorption of granulation tissue. Biochimica et Biophysica Acta, 328, 383. BAILEY, A.J., SIMS, T.J., LE LOUS, M . & BAZIN, S. (1975) Collagen polymorphism in experimental granulation
tissue. Biochemical and Biophysical Research Communications, 66, ir6o. BAILEY, A.J. & SIMS, T.J. (1976) Chemistry of the collagen cross-links. Biochemical Journal, 153, 211. BLACK, M.M., BOTTOMS, E . & SHUSTER, S. (1970) Skin collagen content and thickness in systemic sclerosis. British Journal of Dermatology, 88, 552. BUTLER, W.T., BIBKEDAL-HANSEN, H . , BEEGLE, W.T., TAYLOR, R.E. & CHUNG, E . (1975) Proteins of the perio-
dentium. Identificationof collagens with the [ai (i)lj and [«i (in)]., structures in bovine periodental ligament. Journal of Biological Chemistry, 250, 8907. CHUNG, E. & MILLER, E.J, (1974) Collagen polymorphism: characterisation of molecules with the chain composition [ai(iii)]3 in human tissues. Science, 183, 1200. DUANCE, V . C , RESTALL, D.J., BEARD, H,, BOURNE, F,J. & BAILEY, A.J. (1977) The location of three collagen types
in skeletal muscle, FEBS Letters, 79, 245. EPSTEIN, E.H. & MUNDERLOH, H . N , (1975) Isolation and characterisation of CNBr peptides of human [aiii]^ collagen and tissue distribution of [ar]: and [xllll., collagen. Journal of Biological Chemistry, 250, 9304. FURTHMAYR, H . & TIMPL, R. (1971) Characterisation of collagen peptides by sodium dodecyl sulphate-polyacrylamide electrophoresis. Analytical Biochemistry, 41, 510. GARDNER, D.L. (1965) Pathology of the Connective Tissue Diseases, p. 171. Edward Arnold, London. HARRIS, E.D., JR & SJOERDSMA, A, (1966) Collagen profile in various chemical conditions. Lancet, ii, 707, HAYES, R.L. & RODNAN, G . P . (1971) The ultrastructure of skin in progressive systemic sclerosis (scleroderma). American Journal of Pathology, 63, 433. HERBERT, CM,, JAYSON, M , I , V . , LINDBERG, K . & BAILEY, A.J. (1974) Biosynthesis and maturation of skin
collagen in scleroderma and effect of D-penicillamine. Lancet, i, 187. LE ROY, E.C. (1974) Increased collagen synthesis by scleroderma skin fibroblasts in vitro. A possible defect in the regulation or activation of the scleroderma fibroblast. Journal of Clinical Investigation, 54, 880, LEVER, W , F , (1961) Histopathology of the Skin, 3rd edn, p, 392, Pitman Medical, London. LOVELL, C.R., NICHOLLS, A.C, JAYSON, M . L V . J . & BAILEY, A.J. (1978) Changes in the collagen of synovial
membrane in rheumatoid arthritis and effect of D-penicillamine. Clinical Science and Molecular Medicine, 55,31MEIGEL, W.N., GAY, S, & WEBER, L . (1977) Dermal architecture and collagen type distribution. Archives of Dermatological Research, 259, i. MILLER, E.J. (1976) Biochemical characteristics and biological significance of genetically distinct collagens. Molecular and Cellular Biochemistry, 13, 165. MILNE, J.A. (1972) Introduction to the Diagnostic Histopathology of the Skin, p. 178. Edward Arnold, London, PERLISH, J.S., BASHEY, R.I, & STEPHENS, R.E. (1976) Connective tissue synthesis by cultured scleroderma fibroblasts. i. In vitro collagen synthesis by normal and scleroderma dermal fibroblasts. Arthritis and Rheumatism, 19, 891. TRAUB, W . & PIEZ, K.A, (1971) Chemistry and structure of collagen. Advances in Protein Chemistry, 25, 243. UITTO, J., HALNE, J,, HANNUKSELA, M . , PELTOKALIO, P. & KIVIRIKKO, K , I , (1969) Protocollagen proline hydroxy-
lase activity in the skin of normal human subjects and of patients with scleroderma. Scandinavian Journal of Clinical and Laboratory Investigation, 23, 241. UITTO, J,, OHLENSCHEXGER, K, & LORENZEN, I. (1971) Solubility of skin collagen in normal human subjects and in patients with generalised scleroderma. Clinica et Chimica Acta, 31, 13,
Characterization of dermal collagen in systemic sclerosis C.R.LOVELL,* A.C.NICHOLLS,t V.C.DUANCE4 AND AJ.BAILEYf
•
* Department of Medicine, University of Bristol, I Department of Animal Husbandry, University of Bristol, Langford, and t Meat Research Institute, Agricultural Research Council, Langford, Bristol Accepted for publication 7 July 1978
SUMMARY
The amount of dermal collagen is increased in systemic sclerosis. However, unlike certain inflammatory conditions, the relative proportions of Type I and Type III collagens are closely similar to those found in normal adult dermis. Similarly, no change in the distribution of the collagen types could be detected by immunofluorescent staining, although a considerable thickening of the epidermis was clearly evident in all the sclerotic lesions examined.
The pathological changes in systemic sclerosis include vascular narrowing with ischaemic atrophy of tissues and proliferation of collagen. Abnormal deposition of collagen leads to gross thickening of skin, often resulting in multiple contractures. Using electron microscopy, Bahr (1956) described the presence of an abnormal population of small collagen fibres in the dermis of patients with scleroderma and Hayes & Rodnan (1971) noted a 'beaded fllament' appearance of these fibres, similar to those of embryonic dermis. Despite marked macroscopic and microscopic changes in dermal collagen, little is known of any biochemical modifications. Harris & Sjoerdsma (1966) found a decreased amount of neutral salt soluble collagen in affected, as compared with normal, adult dermis. Uitto et al. (1971), however, demonstrated an increased proportion of neutral salt soluble collagen in the diseased dermal plaque, indicating either an increase in collagen synthesis, decreased cross-linking or decreased degradation. Studies on the cross-linking of the dermal collagen in scleroderma have confirmed that increased synthesis is taking place (Herbert et al, 1974). Recent studies have demonstrated that normal skin contains at least two genetically distinct isotypes of collagen. The major constituent, Type I, consists of two identical polypeptide chains (oti). and a third, non-identical oc2 chain. The other collagen. Type HI, contains three identical polypeptide chains, similar but not identical to the cf, chains of Type I collagen. Normal skin also contains a very small amount of basement membrane collagen defined histologically as the dermal-epidermal junction. Basement membrane collagen also occurs in the blood vessels but the Correspondence: Dr C.R. Lovell, Dermatology Department, Bristol Royal Infirmary, Bristol. 0007-0963/79/0400-0359 $02.00
«
.(;,. 1979 British Association of Dermatologists
359
36o
C.R.Lovell et al
biochemical constitution of these collagens has not been investigated. Type III collagen is a major constituent of embryonic dermis (about 50-60",,) but is present to a lesser extent (15-25'^,) in adult dermal collagen (Epstein & Munderloh, 1975). In certain inflammatory conditions the proportion of Type i n collagen increases (Bailey et al, 1975) and it was therefore considered of interest to see if a similar change occurs in systemic sclerosis. In this paper we describe the presence and location of the different genetic types of collagen in the affeaed dermis in systemic sclerosis in comparison with normal dermis. MATERIALS AND METHODS
Patients Samples of the skin were obtained from biopsy specimens, with the patient's informed consent, or at post mortem of patients with systemic sclerosis. Clinical details of the patients are tabulated in Table I. Age-matched samples of skin were obtained at post mortem or operation from subjects with no evidence of the disease. The skin was dissected free of adhering fat, diced into cubes of approximately "* and then chemically defatted using i :3 methanol: chloroform. Materials Pepsin was obtained from Worthington Biochemical Corp. (Freehold, N.J., U.S.A.) 3250 units/mg. Fluorescein conjugated anti-rabbit IgG was obtained from Burroughs Wellcome. Normal rabbit serum was obtained from Gibco. Determination of genetic types of collagen Pepsin digestion. Defatted samples of skin were extracted with physiological saline, pH 74 for 4 days at 4-C. The residue was incubated with pepsin in 0 5 M acetic acid at a substrate:enzyme ratio of 10:1 (w/w) for 18 h at 4' C (Chung & Miller, 1974). These conditions arc known to degrade most proteins but to leave the helical portion of collagens intact. The residue and supernatant were separated by centrifugation and further pepsin in 0-5 M acetic acid was added to the residue, the digestion being continued for a further i8 h at 4 C; this was repeated four times. Finally, the solubilized collagen was precipitated from the pooled supcrnatants by the addition of salt crystals to 0 9 M NaCl dialysed exhaustively against 0-5 M acetic acid and freeze-dried. The proportion of collagen extraaed was determined by hydroxyproline analysis on ponions of the solubilized material and residue. SDS polyacrylamide gel electrophoresis. To check the purity and identify the molecular types of collagen present the total digest was analysed by acrylamide gel electrophoresis. Samples of the pepsin-solubilized collagen were denatured in sodium dodecyl sulphate (SDS) at 38 C, with and without mercaptoethanol, to disrupt the intramolecular disulphide bonds of Type III collagen. The achain composition was determined by the polyacrylamide gel electrophoresis using the flat-bed techniques described previously (Bailey & Sims, 1976). In order to separate the 1 (III) and a (I) collagen chains the reduction of the 1 (III) to a III with //-mercaptoethanol was delayed until 10 min after the start of the electrophoresis. Cyanogen bromide digestion. Defatted samples of skin were homogenized and extracted with physiological saline, pH 74 for 4 days and the residue digested under nitrogen with cyanogen bromide (CNBr) in 70",, formic acid at 30 C for 4 h. The samples were diluted with deionized water and the CNBr and formic acid were removed by evaporation in vacuo at 30 C. The residue was redissolvcd in 0 5 M acetic acid and centrifuged.
Dermal collagen in systemic sclerosis
361
TABLE I. Clinical details of patients with systemic sclerosis
Site of biopsy
Patient
Age
Sex
A
40
F
Forearm
B
42
F
Forearm
C
45
F
Forearm
D
42
F
Forearm
E
53
F
Forearm
F
40
F
Forearm
G
50
F
Forearm
H
72
F
Forearm
I
58
M
Forearm
J
45
F
Forearm
K
44
F
Forearm
L
56
F
Forearm
History
Treatment at time of biopsy
22 years progressive dysphagia. 5 years Raynaud's Colchicine phenomenon and progressive tightening of skin 0'5 mg b.d. of fingers, hands and forearms. 4 years ago calcinotic nodule right middle finger 3 years Raynaud's phenomenon and pruritus of Nil hands with increasing stiffness of the fingers. 2 months tendon sheath involvement. No visceral involvement clinically. 2 years Raynaud's phenomenon with tightening of skin on forearms and fingers. 2 weeks progressive renal failure and malignant hypertension leading to death
D-penicillamine 750 mg Prednisolone 10 mg Anti-hypertensive drugs and diuretics
18 months diffuse thickening of skin of hands, forearms, neck and thighs, i month progressive D-penicillamine 500 mg renal failure, leading to death Diuretics 9 years swollen fact and hands, generalized D-penicillamine pruritus. Raynaud's. Minimal skin tethering. 500 mg. Topical Dysphagia corticosteroid ointments 1 year swelling of hands and forearms. Raynaud's. D-penicillamine 750 mg No systemic involvement. Sister has S.L.E. 1 year heartburn, dysphagia and constipation. D-penicillamine 500 mg 6 months taut fingers; spontaneous amputation Prednisolone 5 mg of one finger. Facial telangiectases. No dermal Colchicine 0'5 mg b.d. involvement of forearm 4 years tightening of skin over fingers with facial D-penicillamine 250 mg puckering, 2 months constipation. Raynaud's and ulceration of skin 2 years tightening of skin over fingers and foreNil arms. Raynaud's. No systemic involvement 15 years Raynaud's. 5 years facial telangiectases Colchicine 0 5 mg b.d. 2 years dysphagia and dyspnoea, i year tethering of forearm skin 8 years Raynaud's, tethering of skin of hands and D-penicillamine 125 mg forearms. Facial telangiectases. Involvement of Spironolactone 100 mg skin of neck and thighs Burinex K ii b.d. Prednisolone E.C. 75 mg Nil 6 years tethering of skin of fingers, hands and forearms, neck, trunk and thighs. Dyspnoea due to pulmonary fibrosis
(i) The CNBr peptidcs were analysed by disc gel SDS polyacrylamide electrophoresis using the method described by Furthmayr & Timpl (1971). After staining with Coomassie Blue the gels were analysed on a Joyce-Loebl Chromoscan. (ii) The cyanogen bromide peptides were also examined by CM-cellulose chromatography to obtain a more accurate quantification of the proportion of collagen types present. The soluble cyanogen
362
C.R.Lovell et al.
bromide peptides were applied to the column (i-8x 12 cm) of CM-cellulose (Whatman CM 52) equilibrated with 002 M sodium formate, pH 36, containing 001 M sodium chloride, at 44 C. The peptides were eluted by means of a linear gradient 001-017 ^ ^^Cl over i 1. The column effluent was monitored at 228 nm using a Cecil CE 212 spectrophotometer. Selected areas of the chromatographs (see results) were pooled, desalted on Biogel P-2 and further purified by chromatography on Agarose (Biogel A 05 M, 2 6 x 120 cm) equilibrated with i M calcium chloride. Preparation of antibodies. Collagens were extracted from human placenta by digestion with pepsin at io°C for 24 h. The solubilized collagens were fractionated by salt precipitation to give Type III collagen at i -5 M NaCl, Type I at 25 M NaCl and AB Type IV at 4 0 M. The procedure was repeated and the purity of the collagens determined by SDS-polyacrylamide gel electrophoresis. Antibodies to these collagens were raised in New Zealand White rabbits, and purified on immunoabsorbent columns of Types I, III and AB IV to remove non-specific antibodies, as previously described (Duance et al, 1977)Tissue staining. Transverse sections were stained with specific rabbit anti-human Type I, III and AB Type IV, washed extensively with phosphate buffered saline, and then stained with fluorescein conjugated anti-rabbit IgG as previously described (Duance et al., 1977). For controls non-immune rabbit serum was used in place of the specific anti-collagen antibody. The sections were viewed with a Leitz fluorescence microscope. RESULTS Collagen isotypes
Over 80/,, of dermal collagen was extracted from both normal and diseased dermis using repeated pepsin digests at 4 C. SDS-polyacrylamide gel electrophoresis revealed a preponderance of Type I
aid)-
FIGURE I. SDS-polyacrylamide slab gel electrophoresis of normal and diseased skin. Analysis of total pepsin digests: Tracks 1^3 systemic sclerosis skin—(i) without mercaptoethanol; (2) plus mercaptoethanol added 10 min after start of electrophoresis; (3) plus mercaptoethanol. Tracks 4-6 normal skin—(4) without mercaptoethanol; (5) plus mercaptoethanol added after 10 min eiectrophoresis; (6) plus mercaptoethanol.
Dermal collagen in systemic sclerosis
NORMAL
FIGURE 2. Densitometric traces of SDS-polyacrylamide disc gel electrophoresis of cyanogen bromide peptides of normal and diseased dermis. The peaks marked i(I), i(III) denote cyanogen bromide peptides of Types I and III collagen respectively (Traub & Piez, 1971).
FRACTION NUMBER
FIGURE 3. CM-celluIose chromatography of the cyanogen bromide peptides derived from (i) systemic sclerosis and (ii) normal skin. Conditions as described in text. Arrows mark application of sample and gradient.
363
C.R.Lovell et al.
364
40
50
70
80
90 FRACTION
100
110
120
130
NUMBER
FIGURE 4. Molecular sieve chromatography of the cyanogen bromide peptides from regions (i)a and (ii)a of Fig. 3. (i) systemic sclerosis skin (ii) normal skin. Conditions as described in the text.
05 03 02 01
FRACTION
NUMBER
FIGURE 5. Molecular sieve chromatography of the cyanogen bromide peptides from regions (i)b and (ii)b of Fig. 3. (i) systemic sclerosis (ii) normal skin. Conditions as described in the text.
Dermal collagen in systemic sclerosis
FIGURE 6. Immunofluorescent localization of collagen isotypes in skin from normal and systemic sclerosis subjects, (a) staining of normal skin with anti-human Type III; (b) immunofluorescent localization of the epidermal-dermal basement membrane with anti-human AB Type IV; (c) antiAB Type IV staining of systemic sclerosis skin. Note gross thickening of epidermis in (c) compared to the normal skin in (b).
365
366
C.R.Lovell et al.
collagen, but a significant proportion of Type III. The patterns were similar in both normal and diseased skin. There was a suggestion using this gel technique that an increased amount of Type III was present in systemic sclerosis. Two faint bands were also seen, the mobility corresponding to the Type IV A & B collagen bands found in placental basement membranes (Duance et al., 1977) and rheumatoid synovium (Lovell et al.., 1977). These bands were present in both normal and diseased tissue (Fig. i) in approximately the same amounts. CNBr peptide analysis (i) Cyanogen bromide peptides of dermal collagen were separated by SDS disc gel electrophoresis to provide a semi-quantitative analysis of the proponions of Types I and III but gave identical patterns for both normal and diseased dermis (Fig. 2), indicating the absence of any change in the amounts of Type I and III collagen in the diseased condition. Biopsies from twelve different patients were compared with sex and age matched controls. (ii) Accurate quantification of the collagen types was carried out by analysis of the cyanogen bromide peptides on CM-cellulosc column. However, in confirmation of the SDS gels, the chromatograms from normal and diseased skin were also very similar (Fig. 3i and ii). In order to determine the amount of each isotype of collagen present in each case the regions marked a—representing 5:i(I)CB3 and 3!i(III)CB4 (Miller review, 1976) and b—representing ai(I)CB8 and ai(III)CB8 (Epstein & Munderloh, 1975) were pooled, desalted and separated on agarose. The regions marked a gave one major peak corresponding to a molecular weight of approximately 13,000 (Fig. 4a, b); the region marked b gave three peaks (Fig. 5a, b) one of which had a molecular weight of approximately 24,000 and a second with a molecular weight of about 10,000. The peak of higher molecular weight was assumed to be products of partial cleavage by cyanogen bromide. The 24,000 dalton peak was identified as .ai(I)CB8 by molecular weight and amino acid composition and the 10,000 dalton peak was assigned as ai(III)CB8 by molecular weight; unfortunately, insufficient material was obtained for an accurate amino acid analysis. The proportion of Types I and III collagens were determined in two ways: (i) by integration of peak areas representing 0(i(I)CB8 and xi(ni)CB8 in normal and diseased skin (Epstein & Munderloh, 1975) and (2) by measuring the threonine:valine ratio in the 13,000 dalton peak from region a of the CM-cellulose chromatogram as suggested by Butler et al. (1975). By the first method the diseased skin was found to contain about 10",, Type III and normal skin about 12/,:, Type III. However, by the second method, the ratio of threonine to valine showed a much higher proportion of Type HI; about 23",, Type HI in diseased skin and 25",, Type HI in normal (Table 2). This discrepancy between the two methods of determining the ratio of the collagen types may be accounted for by several factors. Firstly, only u.v. absorbance was used to determine the ratio in the first method and although some account was made for the differing size of ai(I)CB8 and :fi(III)CB8 the extinction coefficients for each peptide at the monitoring wave length could be different. Also the CB peptides eluting either side of ai(I)CB8 in the CM-cellulose, i.e. ai(I)CB7 and a2CB4, each have a molecular weight approximately the same as aiCB8 (24,000) so that any overlap by them in the chromatogram would result in an increase of the size of 31CB8 on the agarose and hence a decrease in the proportion of Type HI. In order to reduce this overlap only the main peak of ai(I)CB8 was pooled and as xr(HI)CB8 appears to elute slightly earlier than 5;i(I)CB8, this might have resulted in slight losses of the total ai(HI)CB8 present in the sample. However, despite the discrepancy in absolute values of Type I and Type HI, both methods clearly showed that the proportion of Type IH present in normal and systemic sclerosis skin was the same. This technique was time consuming and required considerable amounts of material, hence could not be carried out on each patient. For rapid comparison of biopsy material from a significant number of patients the CNBr peptide pattern on acrylamide gel was utilized.
Dermal collagen in systemic sclerosis
367
TABLE 2. Amino acid analyses of normal and systemic sclerosis skin cyanogen bromide (CB) peptides Normal >B3 + 3ri(in)CB4
Systemic sclerosis 3ti(I)CB3 + =(i(Iir)CB4
ctiCB^ ca(III)CB4 (Butler et al., 1975)
—
—
I5'8
15
19
6-8 1-8
158 6-6 17
6
7 4
Ser
33
yz
HSer Glu Pro Gly Ala Val
3
0-4
0-6
I
15-6 15-7 40-8
15 15
49
56
21
14
35 —
15-5 15-3 41 4 18-4 37 —
0 I II 20
4
0
0-5 4-0
0-7 4-0
0 0
0 0
3
5
0-2
0-16
0
2'5
3
0-2
26 0-4
0 I
0-3
0-5
53
5*2
46
7-5
O-I
0-2
0
0
56
5S
6
3
3Hyp 4Hyp Asp Thr
Met
He Leu Tyr Phe Hyl Lys His Arg •Ratio of Thr: Val = «i(III):.i(I)
t87
Normal Systemic sclerosis
0-34:1 0'3i:i
0
25" „ Type III 23" „ Type III
* Corrected for differences in molecular chain composition in Type I and Type III.
Immunofluorescent localization of collagen types
Normal dermis. The antibodies against Type I collagen reacted with all parts of the dermis as observed by other workers (Meigel et al, 1977). In contrast to this overall staining. Type III staining is restricted to thread-like structures throughout the dermis reminiscent of the reticulin network observed following silver staining. The staining of Type III collagen also appears to be more concentrated around the epidermal-dermal junction. Pronounced staining of the epidermal-dermal junction and blood vessels also occurs (Fig. 6a). Since the large blood vessels are known to contain a high proportion of Type III collagen, it is not surprising that the small blood vessels also contain Type III collagen. Similar results have been obtained by Meigel et al. (1977) using anti-bovine Types I and III. In our hands bovine antibodies are much less reactive against human tissues (Duance et al, 1978). When stained with anti-AB Type IV the epidermal-dermal junctionisclearly delineated and apart from the small blood vessels no other parts of the dermis react with this basement membrane collagen antibody (Fig. 6b). Systemic sclerosis dermis. No difference could be demonstrated in the distribution of the anti-Types I, III and AB-IV in biopsies from normal subjects and those with systemic sclerosis. A distinct difference observed in every case examined was the thickening of the epidermis, as reported previously by a number of workers. This thickening is clearly shown in Fig. 6c.
368
C.R.Lovell et al DISCUSSION
Traditionally it has been held that in systemic sclerosis, as well as in morphoea, there is a progressive increase in dermal collagen associated with more or less destruction of dermal appendages (e.g. Lever, 1961; Gardner, 1976). Others (Milne, 1972; Black, Bottoms & Shuster, 1970) suggest there is dermal shrinkage but that the normal micro-architecture of the skin is maintained, i.e. it is normal skin in a contracted state. An increase in dermal collagen may reflect either increased rate of synthesis or a decreased rate of degradation. Although the latter possibility remains to be explored, there is considerable indirect evidence of increased collagen synthesis. Uitto et al. (1969) have found increased levels of protocollagen proline hydroxylase, an enzyme involved in collagen synthesis, in diseased skin compared with skin obtained from normal subjects. Increased incorporation of '*C-proline as ' *C-hydroxyproline into explanted skin samples in vitro has been demonstrated in clinically involved skin (Le Roy, 1974) and in clinically normal skin (Perlish, Bashey & Stephens, 1976) from patients with the disease. Indeed, the presence of excess labile aldimine intermolecular cross-links, which would account for the increased solubility, has been demonstrated in 'active' dermal lesions in our previous studies on systemic sclerosis (Herbert et al, 1974). In newly formed granulation tissue of dermal wounds there is a marked increase in Type III collagen (Bailey et al., 1975) and a reappearance of the stable keto intermolecular cross-link found in embryonic skin collagen (Bailey, Bazin & Delaunay, 1973). In systemic sclerosis, however, we found no increase in the proportion of Type III collagen in the dermis, the proportion of the two collagen types remaining similar to that of normal dermis. Furthermore, in this disease an aldimine cross-link is present, similar to that found in dermal collagen in childhood, rather than the 'keto' type crosslink found in embryonic skin and granulation tissue. The analyses on the proportions of Type I and III collagens were carried out on a large number of patients by CNBr peptide electrophoresis, and confirmed by the more exact, but time and material consuming, CM-ccllulose technique on two subjects. More importantly, the distribution of the collagen is also unchanged. Although active lesions were analysed it is possible that a transient increase in Type III could occur during an initial infiammatory stage. On the basis of these results the clinical impression of toughness of the skin is not readily accountable. Clearly there is increased collagen synthesis, which could lead to toughness due to a tighter packing of the fibres, or the binding to the deeper structures could be increased. Alternatively the grossly thickened epidermis or subcutaneous oedema could lead to the impression of rigidity in the dermis. Although there is no change in the proportion of collagen types synthesised by fibroblasts in systemic sclerosis, abnormal interaction between fibroblasts and other cells may lead to increased collagen synthesis. Alternatively the proliferation of dermal collagen may be due to an external stimulating factor, rather than an inherent abnormality of the fibroblasts themselves. This is supported to some extent by the finding that cultured fibroblasts from subjects with systemic sclerosis do not synthesize excessive amounts of collagen (Gibbon, unpublished data). Scleroderma-like changes are seen in metabolic disorders, such as scurvy, prophyria cutanea tarda and carcinoid syndrome, and also following exposure to vinyl chloride (acro-osteolysis) (Jayson et al, 1976). Unlike these disorders, a metabolic abnormality which might stimulate collagen synthesis has not yet been described in systemic sclerosis. ACKNOWLEDGMENTS
We thank the Consultant Dermatologists of the Bristol Royal Infirmary, and Mrs A.Swan and Mrs M.Gibson for technical assistance. This work was supported in part by grants from the Nuifield Foundation and the Hadwen Trust.
Dermal collagen in systemic sclerosis
369
REFERENCES BAHR, G.F. (1956) Eleklronenoptische Untersuchungen bei Sklerodermie Zugleich ein Beitrag zur Kenntis des Bindegewebes der Haut. Aerztliche Forschung, 10, 255. BAILEY, A.J., BAZIN, S. & DELAUNAY, A. (1973) Changes in the nature of the collagen during development and resorption of granulation tissue. Biochimica et Biophysica Acta, 328, 383. BAILEY, A.J., SIMS, T.J., LE LOUS, M . & BAZIN, S. (1975) Collagen polymorphism in experimental granulation
tissue. Biochemical and Biophysical Research Communications, 66, ir6o. BAILEY, A.J. & SIMS, T.J. (1976) Chemistry of the collagen cross-links. Biochemical Journal, 153, 211. BLACK, M.M., BOTTOMS, E . & SHUSTER, S. (1970) Skin collagen content and thickness in systemic sclerosis. British Journal of Dermatology, 88, 552. BUTLER, W.T., BIBKEDAL-HANSEN, H . , BEEGLE, W.T., TAYLOR, R.E. & CHUNG, E . (1975) Proteins of the perio-
dentium. Identificationof collagens with the [ai (i)lj and [«i (in)]., structures in bovine periodental ligament. Journal of Biological Chemistry, 250, 8907. CHUNG, E. & MILLER, E.J, (1974) Collagen polymorphism: characterisation of molecules with the chain composition [ai(iii)]3 in human tissues. Science, 183, 1200. DUANCE, V . C , RESTALL, D.J., BEARD, H,, BOURNE, F,J. & BAILEY, A.J. (1977) The location of three collagen types
in skeletal muscle, FEBS Letters, 79, 245. EPSTEIN, E.H. & MUNDERLOH, H . N , (1975) Isolation and characterisation of CNBr peptides of human [aiii]^ collagen and tissue distribution of [ar]: and [xllll., collagen. Journal of Biological Chemistry, 250, 9304. FURTHMAYR, H . & TIMPL, R. (1971) Characterisation of collagen peptides by sodium dodecyl sulphate-polyacrylamide electrophoresis. Analytical Biochemistry, 41, 510. GARDNER, D.L. (1965) Pathology of the Connective Tissue Diseases, p. 171. Edward Arnold, London. HARRIS, E.D., JR & SJOERDSMA, A, (1966) Collagen profile in various chemical conditions. Lancet, ii, 707, HAYES, R.L. & RODNAN, G . P . (1971) The ultrastructure of skin in progressive systemic sclerosis (scleroderma). American Journal of Pathology, 63, 433. HERBERT, CM,, JAYSON, M , I , V . , LINDBERG, K . & BAILEY, A.J. (1974) Biosynthesis and maturation of skin
collagen in scleroderma and effect of D-penicillamine. Lancet, i, 187. LE ROY, E.C. (1974) Increased collagen synthesis by scleroderma skin fibroblasts in vitro. A possible defect in the regulation or activation of the scleroderma fibroblast. Journal of Clinical Investigation, 54, 880, LEVER, W , F , (1961) Histopathology of the Skin, 3rd edn, p, 392, Pitman Medical, London. LOVELL, C.R., NICHOLLS, A.C, JAYSON, M . L V . J . & BAILEY, A.J. (1978) Changes in the collagen of synovial
membrane in rheumatoid arthritis and effect of D-penicillamine. Clinical Science and Molecular Medicine, 55,31MEIGEL, W.N., GAY, S, & WEBER, L . (1977) Dermal architecture and collagen type distribution. Archives of Dermatological Research, 259, i. MILLER, E.J. (1976) Biochemical characteristics and biological significance of genetically distinct collagens. Molecular and Cellular Biochemistry, 13, 165. MILNE, J.A. (1972) Introduction to the Diagnostic Histopathology of the Skin, p. 178. Edward Arnold, London, PERLISH, J.S., BASHEY, R.I, & STEPHENS, R.E. (1976) Connective tissue synthesis by cultured scleroderma fibroblasts. i. In vitro collagen synthesis by normal and scleroderma dermal fibroblasts. Arthritis and Rheumatism, 19, 891. TRAUB, W . & PIEZ, K.A, (1971) Chemistry and structure of collagen. Advances in Protein Chemistry, 25, 243. UITTO, J., HALNE, J,, HANNUKSELA, M . , PELTOKALIO, P. & KIVIRIKKO, K , I , (1969) Protocollagen proline hydroxy-
lase activity in the skin of normal human subjects and of patients with scleroderma. Scandinavian Journal of Clinical and Laboratory Investigation, 23, 241. UITTO, J,, OHLENSCHEXGER, K, & LORENZEN, I. (1971) Solubility of skin collagen in normal human subjects and in patients with generalised scleroderma. Clinica et Chimica Acta, 31, 13,
