Hhlish Joiirmil of ni-rimitohujy (1992) 126, 2 16-221.
Analysis of collagen gene expression by cultured fibroblasts in morphoea A.HATAMOCHI. M.ONO. M.ARAKAWA, K.TAKEDA AND H.IJEKI Di'iKirtnu'itt of liermiitokniii,
Kawasaki Medivu! School. Kunishiki.
japan
Accepted for publication 2 October 1991
Summary
Collagen gene expression was studied in dermal fibrobiasts derived from the intlammatory and sclerotic skin lesions of patients with localized or generalized morphoea. The levels of mRNA for type I collagen in early-passage fibroblasts derived from inflammatory lesions were higher than those obtained from the uninvolved skin, whereas those tibrobUists obtained from sclerotic lesions were unaltered. No alteration in type [ collagen mRNA levels was observed in late-passage fibroblasts derived from the inllammatory lesions. The relative rate of collagen to totiil protein synthesis in earlypassage tibroblasts derived from intlammatory lesions was higher than that of libroblasts from uninvolved skin, while no alteration or a slight decrease was observed In Hbroblasts from the sclerotic lesions. The data suggest that the inflammatory reactions induce increased collagen synthesis by fibroblasts in the skin in scleroderma.
In both systemic and localized scleroderma, excessive accumulation of collagen in dermal lesions is a characteristic feature.' Many studies hiive been carried out on the collagen metabolism in scleroderma, particularly of collagen synthesis. Most of these studies have used cultured skin fibroblasts and have shown that the fibroblasts from the involved skin of scleroderma patients show increased collagen synthesis compared with those from normal controls.-"'' One study, however, found no significant increase in collagen synthesis/ and it has been suggested that the state of synthesis depends on the stage of the disease.'"^ Inflammatory cell infiltration of dermal lesions is another feature of these diseases.^ A number of studies have tested the hypothesis that factors derived from infiainmatory cells, such as lymphocytes, have an infiuence on the proliferation and collagen synthesis of libroblasts in scleroderma.'""'' However, no study has directly investigated the relationship between infiamniiition in the dermal lesions of scleroderma and the collagen synthesis of fibroblasts. In localized scleroderma, affected and normal areas are present in the same individual and in both morphoea and generalized morphoea, inflammatory changes are usually present around the sclerotic lesions.'"' Fibroblasts derived from these conditions should be suitable for in-vHro studies of the skin lesions in scleroderma. In Correspondence: DrA.Hatamochi, Department of Dermatology. Kawasiiki Medical School. 577 Matsushim;i. Kurashiki 701-1)1. [apaii.
216
this study, we analysed the collagen synthesis by fibroblasts derived from sclerotic and inflammatory skin lesions in patients with morphoea or generalized morphoea to investigate the relationship between the inflammatory changes and collagen synthesis.
Patients and methods l^atients Case I. A 54-year-old man presented in December 1988 with localized sclerotic areas of skin on the abdomen and the posterior part of both lower It*gs. The abdominal lesion had developed within the previous (i months and those on the lower legs in the previous year. The patient had no other symptoms and routine investigations including antinuclear factor were either normal or negative. Skin biopsies were taken from a sclerotic patch on the abdomen and from the surrounding lilac-coioured edge as well as from the non-involved skin (Fig. 1). Case. 2. A 20-year-oId woman with a 1-year history of sclerotic skin was seen in June 1989. There was a hard smooth ivory-coloured, discrete plaque with no lilaccoloured edge on the left upper arm. Routine investigations were all within normal limits. Skin biopsies were taken from the centre of the sclercttic piaque on the left upper arm and from uninvoived skin on the right upper arm.
COLLAGEN EXPRESSION IN MORPHOEA
Unaffected skin site (U)
Inflammatory lesion (1)
Sclerotic lesion (S)
I . Diiigram of sclerotic patch surrounded by lilac-coloured edge on the abdomen of a palit-nt with generalized morphoea iCase 1), showiii!^ biopsy sites.
Case 3. A 3()-year-old man with a 3-month history of sclerotic areas of skin on the right arm and surrounded by slight erythema was seen in July 1 989, Skin hiopsies were tuken Irtmi the centre of the sclerotic pliique on the right upper arm and from uninvolved skin on the left upper arm. Case 4. A 71-year-old woman with a 1-year history of an area of sclerotic skin on the left breast was seen in January 1991. The borders of the sclerotic plaque were lilac-coloured. A skin biopsy was taken from the lilaccoloured lesions on the left breast. Cell culture Dermal libroblasts were cultured using standard methods. Primary cultures of dermal libroblasts were established by growing the cells on plastic dishes in Dulbecco's modilied Eagle's medium (DMEM) with glutamine (O-f) mg/ml). supplemented with penicillin (KK) IJ/ml). streptomycin (50 /ig/ml), and 20% foetal bovine serum (FBS). I-ollowing treatment of the primary cultures with 0-2% trypsin and 0 7 mM EDTA the cells were passaged and the medium in the 25 cm-^ plastic flasks with 10% FBS was changed every third day. Measurcim'ttt oj tnRNA expression Early passages (2 population doubling levels. PDL) of libroblasts derived from three regions of the patient with generalized morphoea (U. unaffected: I, inllanimatory: S. sclerotic) (Case 1). as well as late passages (1 5 PDL) from two regions (U. 1), were inoculated on to 1 50 x 25 mm plastic dishes, and cultures that reached confluence were used. Fibroblasts (II. I. S) from patients with
217
morphoea (Cases 2. 3 and 4) and from a healthy 59year-old woman were also inoculated and used. The culture medium was removed and the cells washed twice with cold phosphate-buffered saline (PBS). Next a 5 M guanidine thiocyanate (GTC) solution containing 0 75% mercaptoethanol was used for the RNA extraction. The total RNA obtained was layered over 5 7 M caesium chloride and puriHed by ultracentrifugation. Total RNA was diluted stepwise (4. 2. 1.0-5 /ig) and transferred to nitrocellulose filters by the dot-blot method and, after baking, was hybridized with cDNA probes. Northern blotting was also performed as previously reported.' ^ As DNA probes, the 1-5 kb fragment of plasmid Hf-f)77 was usedforai (1) collagen,'^ the 1 Okb fragment of piasmid FN 771 for fibronectin.'~ and the 0-5 kb fragment of plasmid pHFA-1 for/?-actin."* Measurement oj collagen synthesis Fibroblasts were hioculated on to 3 5 x 1 0 mm plastic dishes. After the cells reached confluence, the culture medium was replaced by DMEM without serum containing ascorbic acid (50 /(g/ml) and /i-aminopropionitrile (50/ig/ml). L-(2,3-'H)-proline, 5/jCi/ml(specificactivity 27 7 Ci/mmol, NHN Chemicals. Boston. MA. U.S.A.) was added, and the cells were cultured in a COj incubator for 24 h. Part of each dish was used for the cell count, and the rest for the measurement of collagen synthesis. After culturing was completed, pheuylmethylsulphonyl fluoride was added to each dish to a final concentration of 0-5 mM, and the culture medium was removed. After washing with cold PBS (2 ml), the cells were scraped off with a rubber policeman and subjected to ultrasonic treatment. Bovine serum albumin (BSA) was added to the cell lysate as a carrier protein, and then trichloroacetic acid {TCA) at a final concentration of 10% was added to precipitate protein. This procedure was repeated three times. The precipitate obtained was dissolved in 0-05 M NaOH. After neutralization using collagenase form 111 (Advanced Biofactures Corporation, Lynbrook, NY, U.S.A.), collagenase-susceptible protein (CSP) and noncollagen ase-susceptible protein (NCSP) were separated according to the method of Peterkofsky and Diegelniann.' "^ and radioactivity was determined with a scintillation counter. The value obtained was converted to the value per cell count and was taken as the capacity of collagen synthesis and that of non-coliagenous protein synthesis. The ratio of collagen synthesis to total protein synthesis was then calculated according to the formula of Diegelmann and Peterkofsky:^" CSP/(CSP+5-4 x NCSP) X 100. The results were expressed as percentages.
218
A.HATAMOCHI et ai
The significance of differences was tested using the ranksum test.-'
Table 2. Collagen synthesis and collagen m-RNA levels in flbmblasts from ii patient with niorphoea (2D-year-old woman, Cnsv 2), Fibroblasts derived from the sclerotic (S) lesion and from the unaffected (lU skin site were examined
Results ,,,-RNAt
In Case 1 there were no abnormal findings in the uninvoived skin apart from a slightly thickened dermis with dense hundles of collagen. In the inflammatory lesion there was homogenization of the connective tissue in the reticular and lower papillary dermis with a perivuscular intiltrate with mononuclear cells in the deeper dermis. In the sclerotic area the homogenization of the connective tissue was more marked. In Cases 2 and 5 there were no abnormal findings in the uninvoived skin, but in the sclerotic lesions there was hdmoyenization of the connective tissue in the reticular and lower levels of the papillary dermis but with no perivascular infiltrate. The hiopsy taken from the inflammatory lesion in Case 4 showed an atrophic epidermis with thickened collagen hundles in the reticular and middle dermis and a perivascular infiltrate with mononuclear cells.
The values obtained u.sing u dunsitometer on the dot blots are shown in Tables 1-3. In Case 1 among the Table 1. m-RNA levels of a| (I) collagen, fibmnectin and /i-actin in librobliists frum a patient with gentTiilized morphoea {Case 1). I'ibrobliists derived from the unaffected (HI skin site unii i'roni intlainmalory (1) and si'Ierntif IS) Ifsions were exiimineil, Viilues were expressed as a percentage of the level of Hbrobliists derived frnin tbe unaffected skin site. The values given are the average of two indcpeniJenl experiments. All values were normalized for the same RNA
collagen
Fibronectin
^-actin
Passage 2 Passage 15
100 100
100 100
100 100
Passage 2 Passage 15
160 102
162 108
98 102
Passage 2 Passage 15
90 ISTD
105 ND
97 ND
u I
S
ND. not determined.
S(3Pni:|
9K±() 2t
o(i(t) collagen (%)
j8-actin (%)
96 100
99 100
•% Collagen synthesis was determined wiili li)iir dishes, and values given are the rncan±SF.M. t Not signilicantly different from (he value oltJ |/'>()-l)S). t Values of m-RNA were expressed as Ihe percentage of the value of U.
Table J. Collagen synthesis and collagen ni-KNA levels In libroblasls from a patient with morphoea (3()-year-oid man. Case 3). Kibniblasts derived from the sclerotic IS) lesion find from Ihe iinaffec-te(l (I') skin site were examined
% Collagen synthesis* (meaniSEM) S(3PDL) U(3PDM
Steadii-stati' level i>l iolkiffen niRNA
iinitninl of
% Collagen synthesis* (mean±SFA1)
(I) collagen (%) 94 100
^actfn (%) lOi
'% Collagen symbesis was determined witb four disbes. and values given arc the me;tn±SI\M. t Not signiiicantly ditTerent from the value of I) (J'>()-OS). :|: Values ol' m-RNA were expressed as the percentage of the value of 11,
early-passage (2 PDL) cells (Fig. 2a). values for otj (1) collagen and fibronectin mRN As were about f>()% higher for fibrohlasts derived from the inflammatory lesions than for tho.se derived from the uninvoived skin, in fibroblasts derived from sclerotic skin, levels of at] (I) collagen and fibronectin mRNA were similar to those derived from uninvoived skin. As an internal control, jiactin was used and its expression was similar in fibroblasts derived from both inflammatory and sclerotic lesions and from uninvoived skin. In the later passage (IS PDL) cells (Fig. 2b). levels of a, (I) collagen and fibronectin mRNA were the same in fibroblasts derived from the inflammatory lesion as in those from uninvoived skin. No qualitative differences in ot\ (I) collagen, fibronectin and /i-actin mRNA transcripts were detected by Northern blot between fibroblasts derived from inflammatory, sclerotic and uninvoived skin (data not shown). In Cases 2 and 3. there were no significant differences in levels of S] (I) collagen and ^-actin mKNA
COLLAGEN EXPRESSION IN MORPHOEA
ta)
ai(l)Collagen
RNA .
0.5
Fibronectin 4
2
1 0.5
219
A-Actin 4
2
•
i
1 0.5
U I I'igurf 2 . Blot quiintiiication o l a i (I) i-ollagen. tibnmectin a n d /J-actin m-RNAs in lihroblasts frorn a patient with [imrphofii (Case 11. Total RNAs wvvv obtiiiiiL'd trom (a) early- a n d (b) latepassayf dermal libroblasts derived from tbe iiniilVei-k'd iV\ skin, and from intlammatnry II) and .si-kimiii- iS) Icsimis. Seriiil dilutions of lotal RNA !4, 1. I, l)-5 /ly) were dotted o n lo a nitrocellulose tilter. baked, a n d hybridized with cDNA probes.
s (b)
a i (I) Collagen RNA (pg)
1 0.5
/i-Actin
Fibronectin 4
2
1 0.5
4
2
1 0.5
between tibroblasts from sclerotic lesions and uninvolved skin. CoUafjcn synthesis
15 -
The ratio of collagen synthesis to total protein synthesis was significantly higher in dermal iibroblasts derived from uninvolved skin, with a difference of about 20%. The ratio in tibroblasts derived frotn the sclerotic lesion was significantly lower than in those from uninvolved skin, with a difference of about 20% (Fig. 5). In Cases 2 and 3. the ratio of collagen synthesis to total protein synthesis was similar in fibroblasts derived from sclerotic lesions and uninvolved skin (Tables 2 and J). However, the ratio was significantly higher in fibroblasts from the inflammatory lesion of Case 4 than in those derived from the skin of a normal individual, with a difierence of about 70% {Table 4).
10 -
Table 4. Collagen synthesis in Bbroblasts from inflammatory skin of a patient with morphoea i 71-year-old woman. Case 4). Fibroblasts derived from intlamtnatory skin (l)and those derived from the skin of a tuirmal individual (S9-year-o!d woman) (N) were examUied
m-RNA
5 -
U
I
S
Figure i, Katio of eollagcn synthesis to total protein synthesis in libroblasts from a patient with generalized morphoea (Case 1). h'ibroblasts derived from unaffected (U) skin, and from intlammatory (I) and stlerotic (S) lesions were examined. Rach determination was done with four dishes, and the values given are the mean ±SEM. ' Difference Irnm the ratio in libroblasts derived from tht- unaffected skin: /'
Analysis of collagen gene expression by cultured fibroblasts in morphoea A.HATAMOCHI. M.ONO. M.ARAKAWA, K.TAKEDA AND H.IJEKI Di'iKirtnu'itt of liermiitokniii,
Kawasaki Medivu! School. Kunishiki.
japan
Accepted for publication 2 October 1991
Summary
Collagen gene expression was studied in dermal fibrobiasts derived from the intlammatory and sclerotic skin lesions of patients with localized or generalized morphoea. The levels of mRNA for type I collagen in early-passage fibroblasts derived from inflammatory lesions were higher than those obtained from the uninvolved skin, whereas those tibrobUists obtained from sclerotic lesions were unaltered. No alteration in type [ collagen mRNA levels was observed in late-passage fibroblasts derived from the inllammatory lesions. The relative rate of collagen to totiil protein synthesis in earlypassage tibroblasts derived from intlammatory lesions was higher than that of libroblasts from uninvolved skin, while no alteration or a slight decrease was observed In Hbroblasts from the sclerotic lesions. The data suggest that the inflammatory reactions induce increased collagen synthesis by fibroblasts in the skin in scleroderma.
In both systemic and localized scleroderma, excessive accumulation of collagen in dermal lesions is a characteristic feature.' Many studies hiive been carried out on the collagen metabolism in scleroderma, particularly of collagen synthesis. Most of these studies have used cultured skin fibroblasts and have shown that the fibroblasts from the involved skin of scleroderma patients show increased collagen synthesis compared with those from normal controls.-"'' One study, however, found no significant increase in collagen synthesis/ and it has been suggested that the state of synthesis depends on the stage of the disease.'"^ Inflammatory cell infiltration of dermal lesions is another feature of these diseases.^ A number of studies have tested the hypothesis that factors derived from infiainmatory cells, such as lymphocytes, have an infiuence on the proliferation and collagen synthesis of libroblasts in scleroderma.'""'' However, no study has directly investigated the relationship between infiamniiition in the dermal lesions of scleroderma and the collagen synthesis of fibroblasts. In localized scleroderma, affected and normal areas are present in the same individual and in both morphoea and generalized morphoea, inflammatory changes are usually present around the sclerotic lesions.'"' Fibroblasts derived from these conditions should be suitable for in-vHro studies of the skin lesions in scleroderma. In Correspondence: DrA.Hatamochi, Department of Dermatology. Kawasiiki Medical School. 577 Matsushim;i. Kurashiki 701-1)1. [apaii.
216
this study, we analysed the collagen synthesis by fibroblasts derived from sclerotic and inflammatory skin lesions in patients with morphoea or generalized morphoea to investigate the relationship between the inflammatory changes and collagen synthesis.
Patients and methods l^atients Case I. A 54-year-old man presented in December 1988 with localized sclerotic areas of skin on the abdomen and the posterior part of both lower It*gs. The abdominal lesion had developed within the previous (i months and those on the lower legs in the previous year. The patient had no other symptoms and routine investigations including antinuclear factor were either normal or negative. Skin biopsies were taken from a sclerotic patch on the abdomen and from the surrounding lilac-coioured edge as well as from the non-involved skin (Fig. 1). Case. 2. A 20-year-oId woman with a 1-year history of sclerotic skin was seen in June 1989. There was a hard smooth ivory-coloured, discrete plaque with no lilaccoloured edge on the left upper arm. Routine investigations were all within normal limits. Skin biopsies were taken from the centre of the sclercttic piaque on the left upper arm and from uninvoived skin on the right upper arm.
COLLAGEN EXPRESSION IN MORPHOEA
Unaffected skin site (U)
Inflammatory lesion (1)
Sclerotic lesion (S)
I . Diiigram of sclerotic patch surrounded by lilac-coloured edge on the abdomen of a palit-nt with generalized morphoea iCase 1), showiii!^ biopsy sites.
Case 3. A 3()-year-old man with a 3-month history of sclerotic areas of skin on the right arm and surrounded by slight erythema was seen in July 1 989, Skin hiopsies were tuken Irtmi the centre of the sclerotic pliique on the right upper arm and from uninvolved skin on the left upper arm. Case 4. A 71-year-old woman with a 1-year history of an area of sclerotic skin on the left breast was seen in January 1991. The borders of the sclerotic plaque were lilac-coloured. A skin biopsy was taken from the lilaccoloured lesions on the left breast. Cell culture Dermal libroblasts were cultured using standard methods. Primary cultures of dermal libroblasts were established by growing the cells on plastic dishes in Dulbecco's modilied Eagle's medium (DMEM) with glutamine (O-f) mg/ml). supplemented with penicillin (KK) IJ/ml). streptomycin (50 /ig/ml), and 20% foetal bovine serum (FBS). I-ollowing treatment of the primary cultures with 0-2% trypsin and 0 7 mM EDTA the cells were passaged and the medium in the 25 cm-^ plastic flasks with 10% FBS was changed every third day. Measurcim'ttt oj tnRNA expression Early passages (2 population doubling levels. PDL) of libroblasts derived from three regions of the patient with generalized morphoea (U. unaffected: I, inllanimatory: S. sclerotic) (Case 1). as well as late passages (1 5 PDL) from two regions (U. 1), were inoculated on to 1 50 x 25 mm plastic dishes, and cultures that reached confluence were used. Fibroblasts (II. I. S) from patients with
217
morphoea (Cases 2. 3 and 4) and from a healthy 59year-old woman were also inoculated and used. The culture medium was removed and the cells washed twice with cold phosphate-buffered saline (PBS). Next a 5 M guanidine thiocyanate (GTC) solution containing 0 75% mercaptoethanol was used for the RNA extraction. The total RNA obtained was layered over 5 7 M caesium chloride and puriHed by ultracentrifugation. Total RNA was diluted stepwise (4. 2. 1.0-5 /ig) and transferred to nitrocellulose filters by the dot-blot method and, after baking, was hybridized with cDNA probes. Northern blotting was also performed as previously reported.' ^ As DNA probes, the 1-5 kb fragment of plasmid Hf-f)77 was usedforai (1) collagen,'^ the 1 Okb fragment of piasmid FN 771 for fibronectin.'~ and the 0-5 kb fragment of plasmid pHFA-1 for/?-actin."* Measurement oj collagen synthesis Fibroblasts were hioculated on to 3 5 x 1 0 mm plastic dishes. After the cells reached confluence, the culture medium was replaced by DMEM without serum containing ascorbic acid (50 /(g/ml) and /i-aminopropionitrile (50/ig/ml). L-(2,3-'H)-proline, 5/jCi/ml(specificactivity 27 7 Ci/mmol, NHN Chemicals. Boston. MA. U.S.A.) was added, and the cells were cultured in a COj incubator for 24 h. Part of each dish was used for the cell count, and the rest for the measurement of collagen synthesis. After culturing was completed, pheuylmethylsulphonyl fluoride was added to each dish to a final concentration of 0-5 mM, and the culture medium was removed. After washing with cold PBS (2 ml), the cells were scraped off with a rubber policeman and subjected to ultrasonic treatment. Bovine serum albumin (BSA) was added to the cell lysate as a carrier protein, and then trichloroacetic acid {TCA) at a final concentration of 10% was added to precipitate protein. This procedure was repeated three times. The precipitate obtained was dissolved in 0-05 M NaOH. After neutralization using collagenase form 111 (Advanced Biofactures Corporation, Lynbrook, NY, U.S.A.), collagenase-susceptible protein (CSP) and noncollagen ase-susceptible protein (NCSP) were separated according to the method of Peterkofsky and Diegelniann.' "^ and radioactivity was determined with a scintillation counter. The value obtained was converted to the value per cell count and was taken as the capacity of collagen synthesis and that of non-coliagenous protein synthesis. The ratio of collagen synthesis to total protein synthesis was then calculated according to the formula of Diegelmann and Peterkofsky:^" CSP/(CSP+5-4 x NCSP) X 100. The results were expressed as percentages.
218
A.HATAMOCHI et ai
The significance of differences was tested using the ranksum test.-'
Table 2. Collagen synthesis and collagen m-RNA levels in flbmblasts from ii patient with niorphoea (2D-year-old woman, Cnsv 2), Fibroblasts derived from the sclerotic (S) lesion and from the unaffected (lU skin site were examined
Results ,,,-RNAt
In Case 1 there were no abnormal findings in the uninvoived skin apart from a slightly thickened dermis with dense hundles of collagen. In the inflammatory lesion there was homogenization of the connective tissue in the reticular and lower papillary dermis with a perivuscular intiltrate with mononuclear cells in the deeper dermis. In the sclerotic area the homogenization of the connective tissue was more marked. In Cases 2 and 5 there were no abnormal findings in the uninvoived skin, but in the sclerotic lesions there was hdmoyenization of the connective tissue in the reticular and lower levels of the papillary dermis but with no perivascular infiltrate. The hiopsy taken from the inflammatory lesion in Case 4 showed an atrophic epidermis with thickened collagen hundles in the reticular and middle dermis and a perivascular infiltrate with mononuclear cells.
The values obtained u.sing u dunsitometer on the dot blots are shown in Tables 1-3. In Case 1 among the Table 1. m-RNA levels of a| (I) collagen, fibmnectin and /i-actin in librobliists frum a patient with gentTiilized morphoea {Case 1). I'ibrobliists derived from the unaffected (HI skin site unii i'roni intlainmalory (1) and si'Ierntif IS) Ifsions were exiimineil, Viilues were expressed as a percentage of the level of Hbrobliists derived frnin tbe unaffected skin site. The values given are the average of two indcpeniJenl experiments. All values were normalized for the same RNA
collagen
Fibronectin
^-actin
Passage 2 Passage 15
100 100
100 100
100 100
Passage 2 Passage 15
160 102
162 108
98 102
Passage 2 Passage 15
90 ISTD
105 ND
97 ND
u I
S
ND. not determined.
S(3Pni:|
9K±() 2t
o(i(t) collagen (%)
j8-actin (%)
96 100
99 100
•% Collagen synthesis was determined wiili li)iir dishes, and values given are the rncan±SF.M. t Not signilicantly different from (he value oltJ |/'>()-l)S). t Values of m-RNA were expressed as Ihe percentage of the value of U.
Table J. Collagen synthesis and collagen ni-KNA levels In libroblasls from a patient with morphoea (3()-year-oid man. Case 3). Kibniblasts derived from the sclerotic IS) lesion find from Ihe iinaffec-te(l (I') skin site were examined
% Collagen synthesis* (meaniSEM) S(3PDL) U(3PDM
Steadii-stati' level i>l iolkiffen niRNA
iinitninl of
% Collagen synthesis* (mean±SFA1)
(I) collagen (%) 94 100
^actfn (%) lOi
'% Collagen symbesis was determined witb four disbes. and values given arc the me;tn±SI\M. t Not signiiicantly ditTerent from the value of I) (J'>()-OS). :|: Values ol' m-RNA were expressed as the percentage of the value of 11,
early-passage (2 PDL) cells (Fig. 2a). values for otj (1) collagen and fibronectin mRN As were about f>()% higher for fibrohlasts derived from the inflammatory lesions than for tho.se derived from the uninvoived skin, in fibroblasts derived from sclerotic skin, levels of at] (I) collagen and fibronectin mRNA were similar to those derived from uninvoived skin. As an internal control, jiactin was used and its expression was similar in fibroblasts derived from both inflammatory and sclerotic lesions and from uninvoived skin. In the later passage (IS PDL) cells (Fig. 2b). levels of a, (I) collagen and fibronectin mRNA were the same in fibroblasts derived from the inflammatory lesion as in those from uninvoived skin. No qualitative differences in ot\ (I) collagen, fibronectin and /i-actin mRNA transcripts were detected by Northern blot between fibroblasts derived from inflammatory, sclerotic and uninvoived skin (data not shown). In Cases 2 and 3. there were no significant differences in levels of S] (I) collagen and ^-actin mKNA
COLLAGEN EXPRESSION IN MORPHOEA
ta)
ai(l)Collagen
RNA .
0.5
Fibronectin 4
2
1 0.5
219
A-Actin 4
2
•
i
1 0.5
U I I'igurf 2 . Blot quiintiiication o l a i (I) i-ollagen. tibnmectin a n d /J-actin m-RNAs in lihroblasts frorn a patient with [imrphofii (Case 11. Total RNAs wvvv obtiiiiiL'd trom (a) early- a n d (b) latepassayf dermal libroblasts derived from tbe iiniilVei-k'd iV\ skin, and from intlammatnry II) and .si-kimiii- iS) Icsimis. Seriiil dilutions of lotal RNA !4, 1. I, l)-5 /ly) were dotted o n lo a nitrocellulose tilter. baked, a n d hybridized with cDNA probes.
s (b)
a i (I) Collagen RNA (pg)
1 0.5
/i-Actin
Fibronectin 4
2
1 0.5
4
2
1 0.5
between tibroblasts from sclerotic lesions and uninvolved skin. CoUafjcn synthesis
15 -
The ratio of collagen synthesis to total protein synthesis was significantly higher in dermal iibroblasts derived from uninvolved skin, with a difference of about 20%. The ratio in tibroblasts derived frotn the sclerotic lesion was significantly lower than in those from uninvolved skin, with a difference of about 20% (Fig. 5). In Cases 2 and 3. the ratio of collagen synthesis to total protein synthesis was similar in fibroblasts derived from sclerotic lesions and uninvolved skin (Tables 2 and J). However, the ratio was significantly higher in fibroblasts from the inflammatory lesion of Case 4 than in those derived from the skin of a normal individual, with a difierence of about 70% {Table 4).
10 -
Table 4. Collagen synthesis in Bbroblasts from inflammatory skin of a patient with morphoea i 71-year-old woman. Case 4). Fibroblasts derived from intlamtnatory skin (l)and those derived from the skin of a tuirmal individual (S9-year-o!d woman) (N) were examUied
m-RNA
5 -
U
I
S
Figure i, Katio of eollagcn synthesis to total protein synthesis in libroblasts from a patient with generalized morphoea (Case 1). h'ibroblasts derived from unaffected (U) skin, and from intlammatory (I) and stlerotic (S) lesions were examined. Rach determination was done with four dishes, and the values given are the mean ±SEM. ' Difference Irnm the ratio in libroblasts derived from tht- unaffected skin: /'
