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Altered biosynthesis of tumour necrosis factor (TNF) alpha is involved in postburn hypertrophic scars
118
Bums (1994) 20, (2), 118-121
Printed in Great Britain
Altered biosynthesis of tumour necrosis factor (TNF) alpha is involved in postburn hypertrophic scars D. Peruccio 1'2, C. CastagnolP 'z, M. Stella% S. D'Alfonso ~'2, P. Richiardi Momigliano l'z, G. Magliacani 3 and S. Teich Alasia 4 ~Dip. di Genetica, Biologia e Chimica Medica, Universith di Torino, ZCentro CNR Immunogenetica e Istocompatibilita, 3Divisione di Chirurgia Plastica, Centro Grandi Ustionati, C T O and 4Fondazione Piemontese per gli Studi e le Ricerche sulle Ustioni, Torino, Italy
The present study shows that the decrease of TNF alpha in postburn hypertrophic scars is due to a decrease in the steady-state level of TNF alpha mRNA and thus to an altered biosynthesis of the cytokine. Thirteen scars, including seven hypertrophic and six normotrophic scars, were tested for TNF alpha mRNA production by a semiquantitative reverse polymerase chain reaction (PCR) method. TNF beta and beta actin were tested as a control. Six out of six normotrophic scar samples amplified with primers for TNF alpha showed a positive PCR signal up to the 1 : 32 dilution. On the contrary all the hypertrophic tested samples (7/7) had a positive PCR signal only at the t : t or t : 2 dilution. All samples, both normotrophic and hypertrophic, were homogeneous as to TNF beta production.
Introduction Hypertrophic scars are one of the most serious problems for severely burned patients who survive their injuries. The physiopathology of these scars represents a major field of interest. In fact, a more complete knowledge of the pathogenetic factors involved in hypertrophic scarring may provide a biological basis for therapy improvement. Several studies underline the immunological aspects involved in this pathological process~-L Recently, morphological alterations were demonstrated in Langerhans cells4 that are the 'professional' antigen-presenting cells in the skin. The presence of abundant activated T lymphocytes among cells infiltrating the scars and the aberrant expression of HLA-DR on keratinocytes and ffbroblasts s'o, suggest that an altered cooperation between the immune system and wound healing might be important for pathological scarring aetiology. Additional support for this hypothesis comes from the observation that in hypertrophic scars the percentage of infiltrating cells, positively stained by an anti-TNF alpha antibody, was significantly reduced compared with normotrophic scars ~ Thus it is conceivable that this cytokine constitutes an important factor involved in hypertrophic scarring. However, the decrease of TNF alpha positive cells in hypertrophic samples could be due to a lowered functional activity of the cells normally involved in TNF production (macrophages, activated T-cells) or to the presence of TNF inhibitors, rather than to a decreased TNF alpha synthesis. In order to clarify this point it was decided to quantify TNF alpha mRNA in hypertrophic and in 9 1994 Butterworth-Heinemann Ltd 0305-4179/94/020118-04
normotrophic scars. TNF beta mRNA production was also tested as a control of T-cell functional state.
Materials and methods Patients Hypertrophic scar biopsies were obtained after informed consent from seven patients who had developed hypertrophic scars consequent to thermal injury. Selected patients had a burned surface area covering 10--40 per cent of the body and the pathological scars were still present at least 1 year after trauma. Scars were raised, erythematous and with telangectasies and bullae at their surface, so they were considered as active lesions. Normotrophic scars were taken from six patients whose lesions had an optimal healing. All the analysed biopsies were obtained from patients undergoing plastic surgery under general anaesthesia. No patient had any evidence of malignancy or infection. None of them was treated with immunomodulating agents before surgery.
RNA preparation RNA was extracted from 100 mg of tissue frozen in liquid nitrogen with the RNAzol method (Biotex, Houston, TX, USA). The same method was used to purify RNA from resting and PHA-activated peripheral blood mononuclear cells. The quality of RNA preparations was checked on a denaturing agarose gel and their concentration was quantiffed by measuring the optical density at 200 nm.
cDNA synthesis Total RNA was reverse transcribed into cDNA (first strand) according to the following protocol: 1.5 ~tg total RNA was preincubated for 5 min at 65~ chilled on ice for 5 min and then incubated at 37~ for 60 min in a total vol. of 25 p.1 of a reverse transcription mixture containing 50 mM Tris-HC1, pH 8.3; 75 mM KC1; 3 mM MgC12; 0.5 mM dNTP; 0.5 ~tg oligo (dT)~e; 20 U ribonuclease inhibitor (RNasin, Promega Corp., Madison, WI, USA); IOOU of Moloney routine leukemia virus reverse transcriptase (BRL, Bethesda Research Lab., MD, USA). The samples were then heated at 65~ for 5 min.
Peruccio et al.: TNF alpha in postburn scars Polymerase chain reaction (PCR) Four microlitres of several dilutions of cDNA were amplified with TNF alpha, TNF beta and beta actin primers in a Perkin-Elmer Cetus 480 thermal cycler in a total volume of 50 pJ containing 1 x PCR buffer (10 mM Tris HC1, pH 8.3, 50 mM KC1) 2raM (for TNF alpha primers) or 1.5 mM (for TNF beta and beta Actin primers) MgC1 v 0.2 mM dNTP, 0.25 U Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA, USA). TNF alpha primers (Perkin-Elmer Cetus, Emeryville, CA, USA) were used at a final concentration of 0.15 I,tM. The amplification consisted of 28 cycles (95~ for 30 s, 55~ for 30 s, 72~ for 60 s). TNF beta and beta actin primers (Clontech, Palo Alto, CA, USA) were used at a final concentration of 0.3 btM. Beta-actin amplification involved 20 cycles (94~ for 1 min, 62~ for I min, 72~ for I rain). TNF beta PCR conditions were I min at 94~ 2 min at 60~ 3 min at 72~ for 30 cycles. Amplified samples were electrophoresed in 1.2 per cent agarose gel and stained with ethidium bromide. Instant photographs of the gels were obtained with Polaroid 57 sheet film. TNF alpha detection in PBL- and PHA-activated cells TNF alpha was detected in supematants of cell cultures of PBL and PHA activated cells, by a sandwich enzyme immunoassay (Biokine TNF Test Kit, T Cell Sciences, Inc., Cambridge, MA, USA) as previously described 7. The detection limit, as stated by the manufacturer, was 10 pg TNF alpha/ml. Samples below the detection level were assumed to have a value of 0.
119 The amount of RNA expression was analysed with a semiquantitative reverse PCR method consisting of amplification of serially diluted cDNA. Pilot experiments were performed to set conditions which would detect differences in the cytokine mRNA signal. For this purpose RNA from resting and PHA activated mononuclear cells of the same individual was reverse transcribed. Sequential dilutions of the obtained cDNA were then amplified with specific primers. In the example reported in Figure I, TNF alpha signal in the sample obtained from resting cells was faint and disappeared at a dilution of I : 16, whereas a strongly positive signal was still observed at a dilution of I : 32 in the PHA activated sample. The beta actin signal was equal to both samples (Figure I). An ELISA test revealed a parallel strong increase of TNF alpha protein in the supematant of PHA stimulated cells in comparison to resting cells supematant. Thus under the chosen conditions a difference in mRNA amount was clearly detectable. The same procedure was applied to the analysis of RNA extracted from the scar biopsies. RNA extracts were reverse-transcribed into cDNA and then amplified with primers for TNF alpha. TNF beta and beta-actin were used as controls. The PCR signal for beta actin cDNA was comparable in hypertrophic and normotrophic scars (Figure2), indicating
Results Thirteen scar biopsies, including seven hypertrophic and six normotrophic samples, were tested for mRNA production of TNF alpha, beta, and beta actin. All the samples had been previously analysed by immunohistochemical techniques for the presence of positive infiltrating cells stained by anti-TNF alpha and beta monoclonal antibodiesL All tested hypertrophic scars showed a significantly lower amount of TNF alpha positive infiltrating cells compared to normotrophic biopsies. On the contrary, the amount of TNF beta positive cells was higher in the hypertrophic scars, equal to the percentage of activated infiltrating T cells.
Figure 1. TNF alpha and beta actin PCR amplification of sequential dilutions of cDNA. Dilutions were 1 : 1, 1 : 4, 1 : 16, 1 : 32 for TNF alpha and 1 : 1, 1 : 4, 1 : 16, 1 : 32, 1 : 128 for beta actin. 1, PBL sample; 2, PHA sample.
N 1
t
Figure 2. Beta actin PCR amplification of cDNA sequential dilutions (1 : 1, 1 : 4, 1 : 16, 1 : 32, 1 : 128). H, hypertrophic scar samples; N, normotrophic scar samples.
I20
Figure 3. TNF beta PCR amplification of cDNA sequential dilutions (1 : 1, 1 : 4, 1 : 16, 1 : 32). H, hypertrophic scar samples; N, normotrophic scar samples.
Figure 4, TNF alpha PCR amplification of cDNA sequential
dilutions. Dilutions were I : 1, 1 : 4, 1 : 16, 1 : 32 for normotrophic scars (N) and I : 1, 1 : 2, 1 : 4 for hypertrophic scars (H). The H1 sample was diluted up to I : 8.
that the efficiency of retrotranscription and consequently the amount of cDNA used was homogeneous among the samples. Analysis of TNF beta mRNA showed an equally high PCR signal (1:16) in all hypertrophic and normotrophic tested samples (Figure 3). A strikingly different result was obtained for TNF alpha (Figure4). In fact 6/6 normotrophic scars amplified with primers for TNF alpha showed a positive PCR signal up to a 1:32 cDNA dilution, whereas all the hypertrophic tested samples had a positive PCR signal at a 1 : I or a I : 2 dilution. Discussion
In a previous report Castagnoli et al.o showed that in hypertrophic scar samples the percentage of TNF alpha positive cells among infiltrates was significantly lower than in normotrophic scars (8 per cent versus 35.4 per cent). Several mechanisms might be responsible for the decrease of TNF alpha positive cells in hypertrophic samples. For instance, the detection of TNF alpha positive cells could be hampered by the presence of TNF inhibitors such as TNF-binding proteinL Another possible mechanism could be a lowered functional activity of cells infiltrating hypertrophic scars.
Burns (I994) Vol. 20/No. 2
In the present study, we analysed the amount of TNF alpha mRNA in hypertrophic scars by means of a reverse PCR method, demonstrating that the decrease in TNF alpha is due to an altered biosynthesis leading to a decrease in the steady-state level of TNF alpha mRNA. The PCR, by amplifying specific sequences with remarkable efficiency, permits analysis of sequences of interest even when the initial amount of material is extremely small. This is a major advantage of this technique compared to other reported methods. However the PCR reaction is hard[y amenable to an exact quantitation. A semiquantitative estimate of the starting template can be obtained either by serial dilutions of it or by determining the number of cycles necessary to reach plateau amplificationL In the present study cDNA was tested in PCR at serial dilutions. Moreover, two controls were always introduced: beta actin which was assumed to be a non-inducible marker for verifying the efficiency and extent of reverse PCR transcription and TNF beta to test the presence of RNA from infiltrating cells. The finding of a significant decrease of TNF alpha mRNA transcription was made by comparing results of cytokine and beta actin mRNA. The fact that all hypertrophic scar samples showed a comparable signal pattern, strikingly different from that of normotrophic scars, gives strength to the results. Our findings are in contrast with those obtained by McCauty et al. ~~ who demonstrated an increase in TNF alpha in the serum of black-skinned patients with keloids, associated with a decrease in TNF beta. However, it must be noted that our reported findings directly concern the involved tissue. Since TNF levels in the serum are contributed to by many different tissues, it is possible that the observations by the above authors are the expression of entirely different phenomena. TNF alpha and beta are multipotent cytokines 11, released by irnmunocompetent cells, that are capable of up- or down-regulating fibroblast and keratinocyte activity. Several reports indicate that TNF alpha may play a predominantly catabolic role in situ during dermal fibrotic responses, both directly by inhibiting fibronectin production and indirectly by significantly increasing dermal fibroblast elaboration of collagenase and proteoglycanase activitieslL TNF alpha has been reported to act on fibroblasts both as a growth inducer and as a growth inhibitor 13. Hypertophic scars are characterized by the presence of fibroblasts actively synthesizing extracellular matrix components 14. It has been demonstrated that cultured fibroblasts, derived from the early phases of traumatic wound tissue or from sites of pathological fibrosis, such as hypertrophic scars, rheumatoid synovium or sclerodermatous skin, display activated phenotypes characterized by increased production of the connective tissue matrix components (collagen, glycosaminoglycans) and altered collagenase and prostaglandin E2 production ~s. Moreover, these fibroblasts retain their activated phenotypes for many generations in vitro in the absence of in situ stimulators, suggesting that fibroblast-inhibitory factors or cytokines present only in postgranulation fibrotic tissue are required for fibroblasts deactivation. Our results point to TNF alpha as a possible factor, the presence of which is important for a normal regulation of cellular activity during wound healing. It will be interesting to compare TNF alpha production in all the phases of wound healing, to analyse possible variations during scarring processes. Moreover, it will be interesting to analyse other cytokine production to verify the presence of factors
Peruccio et al.: TNF alpha in postburn scars
121
modulating TNF alpha biosynthesis in hypertrophic scars. The study of the regulatory pathways and molecules involved in hypertrophic scarring will contribute to a better understanding of normal wound healing and facilitate development of new strategies for the management of hypertrophic scars and other fibrotic conditions.
Acknowledgements This work was supported by Fondazione Piemontese per gli Studi e le Ricerche sulle Ustioni, by MURST 60 per cent, and by CNR, Progetto Finalizzato BTBS. C. Castagnoli was supported by a fellowship from Fondazione Piemontese per gli Studi e le Richerche sulle Ustioni. S. D'Alfonso is a postgraduate student of Dottorato di Ricerca in Genetica Umana. Helpful suggestions of Dr Roberto Tosi during the preparation of the manuscript are gratefully acknowledged.
References 1 Janssen de Limpens AMP and Cormane RH. Studies on the immunological aspects of keloids and hypertrophic scars. Arch Dermatol Res 1982; 274: 259. 2 Ketchum LD, Cohen IK and Masters FW. Hypertrophic scars and keloids - a collective review. Plast Reconstr Surg 1974; 53: 140. 3 Kisher CW, Shetlar MR, Shettar C and Chavpil M. Immunoglobuins in hypertrophic scars and keloids. Plast Reconstr Surg 1983; 71: 82I. 4 Cracco C, SteUa M, Teich Alasia S and Filogamo G. Comparative study of Langerhans cells in normal and pathogical scars. Hypertrophic scars. Eur ] Histochem 1991; 36: 53. 5 Castagnoli C, Stella M, Magliacani G, Teich Alasia S and Richiardi P. Anomalous expression of HLA class II molecules on keratinocytes and fibroblasts in hypertrophic scars
consequent to thermal injury. Clin Exp Immunol 1990; 82: 350. 6 Castagnoli C, Stella M, Berthod C, Magliacani G and Momigliano Richiardi P. TNF Production and hypertrophic scarring. Celllmmunol 1993; 147: 51. 7 Roccatello D, D'Alfonso S and Peruccio D et al. Induction of mRNA for Tumor Necrosis Factor atpha in hemodiatysis. l~'dney Int 1993; 43: (supp139), 144. 8 Lantz M, Gullberg U, Nilsson E and Olsson I. Characterization in vitro of a human TNF-binding protein. ] Clin Invest 1990; 86: I396. 9 Wang AM, Doyle V and Mark DF. Quantitation of mRNA by the polymerase chain reaction. ProcNatI Acad Sci USA 1989; 86: 9717. 10 McCauly RL, Chopra V, Li Y, Hemdon DN and Robson MC Altered cytokine production in black patients with keloids. JClin Immunol 1992; 12: 300. I1 Tracey KJ, Vlassara H and Cerami A. Cachectin/Tumor Necrosis Factor. Lancet 1989; i: 1122. 12 Duncan MR and Berman B. Differential regulation of collagene, glycosaminoglycan, fibronectin and collagenase activity production in cultured human adult dermal fibroblasts by interleukin 1 alpha and beta and TNF alpha and beta. ] Invest Dermatol 1989; 92: 699. 13 Kovacs E. Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol Today 1991; 12: 17. 14 Datubo-Brown DD. Keloids: a review of the literature. Br J Plast Surg I990; 43: 70. 15 Abergel RP, Pizzurro D, Meeker CA et al. Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures. ] Invest Dermatol 1985; 84: 384. Paper accepted 26 August 1993. Correspondence should be addressed to: Dr Carlotta Castagnoli, Dip. Genetica, Biologia e Chimica Medica, Via Santena I9, 10126 Torino, Italy.
JAMES LAING MEMORIAL ESSAY The British Bum Association has instituted a memorial essay to be awarded annually in memory of James Ellsworth Laing, Burn Surgeon, founder member of the British Burn Association and a former Editor of this Journal. There is a prize of up to s for the winning essay. The subject for the tenth essay is:
Quality A s s u r a n c e
in B u r n P a t i e n t C a r e
The essay should be confined to not more than 100(~ words and correspondingly less if up to 6 Figures and/or Tables are included. The substance of the essay should not already have been published since the winning essay will be published in this Journal. The essays will be assessed anonymously. All persons interested in the problems associated with burning injury are eligible to submit an essay. The deadline for submission of an essay (4 copies) is 31 December 1994. Completed essays and any queries should be sent to: The Secretary of the British Burn Association, Dr J.N. Kearney, Ph.D., Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Wakefield WF1 4DG, West Yorkshire, UK.
The title and author(s) of the winning essay will be announced in April 1995.
Bums (1994) 20, (2), 118-121
Printed in Great Britain
Altered biosynthesis of tumour necrosis factor (TNF) alpha is involved in postburn hypertrophic scars D. Peruccio 1'2, C. CastagnolP 'z, M. Stella% S. D'Alfonso ~'2, P. Richiardi Momigliano l'z, G. Magliacani 3 and S. Teich Alasia 4 ~Dip. di Genetica, Biologia e Chimica Medica, Universith di Torino, ZCentro CNR Immunogenetica e Istocompatibilita, 3Divisione di Chirurgia Plastica, Centro Grandi Ustionati, C T O and 4Fondazione Piemontese per gli Studi e le Ricerche sulle Ustioni, Torino, Italy
The present study shows that the decrease of TNF alpha in postburn hypertrophic scars is due to a decrease in the steady-state level of TNF alpha mRNA and thus to an altered biosynthesis of the cytokine. Thirteen scars, including seven hypertrophic and six normotrophic scars, were tested for TNF alpha mRNA production by a semiquantitative reverse polymerase chain reaction (PCR) method. TNF beta and beta actin were tested as a control. Six out of six normotrophic scar samples amplified with primers for TNF alpha showed a positive PCR signal up to the 1 : 32 dilution. On the contrary all the hypertrophic tested samples (7/7) had a positive PCR signal only at the t : t or t : 2 dilution. All samples, both normotrophic and hypertrophic, were homogeneous as to TNF beta production.
Introduction Hypertrophic scars are one of the most serious problems for severely burned patients who survive their injuries. The physiopathology of these scars represents a major field of interest. In fact, a more complete knowledge of the pathogenetic factors involved in hypertrophic scarring may provide a biological basis for therapy improvement. Several studies underline the immunological aspects involved in this pathological process~-L Recently, morphological alterations were demonstrated in Langerhans cells4 that are the 'professional' antigen-presenting cells in the skin. The presence of abundant activated T lymphocytes among cells infiltrating the scars and the aberrant expression of HLA-DR on keratinocytes and ffbroblasts s'o, suggest that an altered cooperation between the immune system and wound healing might be important for pathological scarring aetiology. Additional support for this hypothesis comes from the observation that in hypertrophic scars the percentage of infiltrating cells, positively stained by an anti-TNF alpha antibody, was significantly reduced compared with normotrophic scars ~ Thus it is conceivable that this cytokine constitutes an important factor involved in hypertrophic scarring. However, the decrease of TNF alpha positive cells in hypertrophic samples could be due to a lowered functional activity of the cells normally involved in TNF production (macrophages, activated T-cells) or to the presence of TNF inhibitors, rather than to a decreased TNF alpha synthesis. In order to clarify this point it was decided to quantify TNF alpha mRNA in hypertrophic and in 9 1994 Butterworth-Heinemann Ltd 0305-4179/94/020118-04
normotrophic scars. TNF beta mRNA production was also tested as a control of T-cell functional state.
Materials and methods Patients Hypertrophic scar biopsies were obtained after informed consent from seven patients who had developed hypertrophic scars consequent to thermal injury. Selected patients had a burned surface area covering 10--40 per cent of the body and the pathological scars were still present at least 1 year after trauma. Scars were raised, erythematous and with telangectasies and bullae at their surface, so they were considered as active lesions. Normotrophic scars were taken from six patients whose lesions had an optimal healing. All the analysed biopsies were obtained from patients undergoing plastic surgery under general anaesthesia. No patient had any evidence of malignancy or infection. None of them was treated with immunomodulating agents before surgery.
RNA preparation RNA was extracted from 100 mg of tissue frozen in liquid nitrogen with the RNAzol method (Biotex, Houston, TX, USA). The same method was used to purify RNA from resting and PHA-activated peripheral blood mononuclear cells. The quality of RNA preparations was checked on a denaturing agarose gel and their concentration was quantiffed by measuring the optical density at 200 nm.
cDNA synthesis Total RNA was reverse transcribed into cDNA (first strand) according to the following protocol: 1.5 ~tg total RNA was preincubated for 5 min at 65~ chilled on ice for 5 min and then incubated at 37~ for 60 min in a total vol. of 25 p.1 of a reverse transcription mixture containing 50 mM Tris-HC1, pH 8.3; 75 mM KC1; 3 mM MgC12; 0.5 mM dNTP; 0.5 ~tg oligo (dT)~e; 20 U ribonuclease inhibitor (RNasin, Promega Corp., Madison, WI, USA); IOOU of Moloney routine leukemia virus reverse transcriptase (BRL, Bethesda Research Lab., MD, USA). The samples were then heated at 65~ for 5 min.
Peruccio et al.: TNF alpha in postburn scars Polymerase chain reaction (PCR) Four microlitres of several dilutions of cDNA were amplified with TNF alpha, TNF beta and beta actin primers in a Perkin-Elmer Cetus 480 thermal cycler in a total volume of 50 pJ containing 1 x PCR buffer (10 mM Tris HC1, pH 8.3, 50 mM KC1) 2raM (for TNF alpha primers) or 1.5 mM (for TNF beta and beta Actin primers) MgC1 v 0.2 mM dNTP, 0.25 U Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA, USA). TNF alpha primers (Perkin-Elmer Cetus, Emeryville, CA, USA) were used at a final concentration of 0.15 I,tM. The amplification consisted of 28 cycles (95~ for 30 s, 55~ for 30 s, 72~ for 60 s). TNF beta and beta actin primers (Clontech, Palo Alto, CA, USA) were used at a final concentration of 0.3 btM. Beta-actin amplification involved 20 cycles (94~ for 1 min, 62~ for I min, 72~ for I rain). TNF beta PCR conditions were I min at 94~ 2 min at 60~ 3 min at 72~ for 30 cycles. Amplified samples were electrophoresed in 1.2 per cent agarose gel and stained with ethidium bromide. Instant photographs of the gels were obtained with Polaroid 57 sheet film. TNF alpha detection in PBL- and PHA-activated cells TNF alpha was detected in supematants of cell cultures of PBL and PHA activated cells, by a sandwich enzyme immunoassay (Biokine TNF Test Kit, T Cell Sciences, Inc., Cambridge, MA, USA) as previously described 7. The detection limit, as stated by the manufacturer, was 10 pg TNF alpha/ml. Samples below the detection level were assumed to have a value of 0.
119 The amount of RNA expression was analysed with a semiquantitative reverse PCR method consisting of amplification of serially diluted cDNA. Pilot experiments were performed to set conditions which would detect differences in the cytokine mRNA signal. For this purpose RNA from resting and PHA activated mononuclear cells of the same individual was reverse transcribed. Sequential dilutions of the obtained cDNA were then amplified with specific primers. In the example reported in Figure I, TNF alpha signal in the sample obtained from resting cells was faint and disappeared at a dilution of I : 16, whereas a strongly positive signal was still observed at a dilution of I : 32 in the PHA activated sample. The beta actin signal was equal to both samples (Figure I). An ELISA test revealed a parallel strong increase of TNF alpha protein in the supematant of PHA stimulated cells in comparison to resting cells supematant. Thus under the chosen conditions a difference in mRNA amount was clearly detectable. The same procedure was applied to the analysis of RNA extracted from the scar biopsies. RNA extracts were reverse-transcribed into cDNA and then amplified with primers for TNF alpha. TNF beta and beta-actin were used as controls. The PCR signal for beta actin cDNA was comparable in hypertrophic and normotrophic scars (Figure2), indicating
Results Thirteen scar biopsies, including seven hypertrophic and six normotrophic samples, were tested for mRNA production of TNF alpha, beta, and beta actin. All the samples had been previously analysed by immunohistochemical techniques for the presence of positive infiltrating cells stained by anti-TNF alpha and beta monoclonal antibodiesL All tested hypertrophic scars showed a significantly lower amount of TNF alpha positive infiltrating cells compared to normotrophic biopsies. On the contrary, the amount of TNF beta positive cells was higher in the hypertrophic scars, equal to the percentage of activated infiltrating T cells.
Figure 1. TNF alpha and beta actin PCR amplification of sequential dilutions of cDNA. Dilutions were 1 : 1, 1 : 4, 1 : 16, 1 : 32 for TNF alpha and 1 : 1, 1 : 4, 1 : 16, 1 : 32, 1 : 128 for beta actin. 1, PBL sample; 2, PHA sample.
N 1
t
Figure 2. Beta actin PCR amplification of cDNA sequential dilutions (1 : 1, 1 : 4, 1 : 16, 1 : 32, 1 : 128). H, hypertrophic scar samples; N, normotrophic scar samples.
I20
Figure 3. TNF beta PCR amplification of cDNA sequential dilutions (1 : 1, 1 : 4, 1 : 16, 1 : 32). H, hypertrophic scar samples; N, normotrophic scar samples.
Figure 4, TNF alpha PCR amplification of cDNA sequential
dilutions. Dilutions were I : 1, 1 : 4, 1 : 16, 1 : 32 for normotrophic scars (N) and I : 1, 1 : 2, 1 : 4 for hypertrophic scars (H). The H1 sample was diluted up to I : 8.
that the efficiency of retrotranscription and consequently the amount of cDNA used was homogeneous among the samples. Analysis of TNF beta mRNA showed an equally high PCR signal (1:16) in all hypertrophic and normotrophic tested samples (Figure 3). A strikingly different result was obtained for TNF alpha (Figure4). In fact 6/6 normotrophic scars amplified with primers for TNF alpha showed a positive PCR signal up to a 1:32 cDNA dilution, whereas all the hypertrophic tested samples had a positive PCR signal at a 1 : I or a I : 2 dilution. Discussion
In a previous report Castagnoli et al.o showed that in hypertrophic scar samples the percentage of TNF alpha positive cells among infiltrates was significantly lower than in normotrophic scars (8 per cent versus 35.4 per cent). Several mechanisms might be responsible for the decrease of TNF alpha positive cells in hypertrophic samples. For instance, the detection of TNF alpha positive cells could be hampered by the presence of TNF inhibitors such as TNF-binding proteinL Another possible mechanism could be a lowered functional activity of cells infiltrating hypertrophic scars.
Burns (I994) Vol. 20/No. 2
In the present study, we analysed the amount of TNF alpha mRNA in hypertrophic scars by means of a reverse PCR method, demonstrating that the decrease in TNF alpha is due to an altered biosynthesis leading to a decrease in the steady-state level of TNF alpha mRNA. The PCR, by amplifying specific sequences with remarkable efficiency, permits analysis of sequences of interest even when the initial amount of material is extremely small. This is a major advantage of this technique compared to other reported methods. However the PCR reaction is hard[y amenable to an exact quantitation. A semiquantitative estimate of the starting template can be obtained either by serial dilutions of it or by determining the number of cycles necessary to reach plateau amplificationL In the present study cDNA was tested in PCR at serial dilutions. Moreover, two controls were always introduced: beta actin which was assumed to be a non-inducible marker for verifying the efficiency and extent of reverse PCR transcription and TNF beta to test the presence of RNA from infiltrating cells. The finding of a significant decrease of TNF alpha mRNA transcription was made by comparing results of cytokine and beta actin mRNA. The fact that all hypertrophic scar samples showed a comparable signal pattern, strikingly different from that of normotrophic scars, gives strength to the results. Our findings are in contrast with those obtained by McCauty et al. ~~ who demonstrated an increase in TNF alpha in the serum of black-skinned patients with keloids, associated with a decrease in TNF beta. However, it must be noted that our reported findings directly concern the involved tissue. Since TNF levels in the serum are contributed to by many different tissues, it is possible that the observations by the above authors are the expression of entirely different phenomena. TNF alpha and beta are multipotent cytokines 11, released by irnmunocompetent cells, that are capable of up- or down-regulating fibroblast and keratinocyte activity. Several reports indicate that TNF alpha may play a predominantly catabolic role in situ during dermal fibrotic responses, both directly by inhibiting fibronectin production and indirectly by significantly increasing dermal fibroblast elaboration of collagenase and proteoglycanase activitieslL TNF alpha has been reported to act on fibroblasts both as a growth inducer and as a growth inhibitor 13. Hypertophic scars are characterized by the presence of fibroblasts actively synthesizing extracellular matrix components 14. It has been demonstrated that cultured fibroblasts, derived from the early phases of traumatic wound tissue or from sites of pathological fibrosis, such as hypertrophic scars, rheumatoid synovium or sclerodermatous skin, display activated phenotypes characterized by increased production of the connective tissue matrix components (collagen, glycosaminoglycans) and altered collagenase and prostaglandin E2 production ~s. Moreover, these fibroblasts retain their activated phenotypes for many generations in vitro in the absence of in situ stimulators, suggesting that fibroblast-inhibitory factors or cytokines present only in postgranulation fibrotic tissue are required for fibroblasts deactivation. Our results point to TNF alpha as a possible factor, the presence of which is important for a normal regulation of cellular activity during wound healing. It will be interesting to compare TNF alpha production in all the phases of wound healing, to analyse possible variations during scarring processes. Moreover, it will be interesting to analyse other cytokine production to verify the presence of factors
Peruccio et al.: TNF alpha in postburn scars
121
modulating TNF alpha biosynthesis in hypertrophic scars. The study of the regulatory pathways and molecules involved in hypertrophic scarring will contribute to a better understanding of normal wound healing and facilitate development of new strategies for the management of hypertrophic scars and other fibrotic conditions.
Acknowledgements This work was supported by Fondazione Piemontese per gli Studi e le Ricerche sulle Ustioni, by MURST 60 per cent, and by CNR, Progetto Finalizzato BTBS. C. Castagnoli was supported by a fellowship from Fondazione Piemontese per gli Studi e le Richerche sulle Ustioni. S. D'Alfonso is a postgraduate student of Dottorato di Ricerca in Genetica Umana. Helpful suggestions of Dr Roberto Tosi during the preparation of the manuscript are gratefully acknowledged.
References 1 Janssen de Limpens AMP and Cormane RH. Studies on the immunological aspects of keloids and hypertrophic scars. Arch Dermatol Res 1982; 274: 259. 2 Ketchum LD, Cohen IK and Masters FW. Hypertrophic scars and keloids - a collective review. Plast Reconstr Surg 1974; 53: 140. 3 Kisher CW, Shetlar MR, Shettar C and Chavpil M. Immunoglobuins in hypertrophic scars and keloids. Plast Reconstr Surg 1983; 71: 82I. 4 Cracco C, SteUa M, Teich Alasia S and Filogamo G. Comparative study of Langerhans cells in normal and pathogical scars. Hypertrophic scars. Eur ] Histochem 1991; 36: 53. 5 Castagnoli C, Stella M, Magliacani G, Teich Alasia S and Richiardi P. Anomalous expression of HLA class II molecules on keratinocytes and fibroblasts in hypertrophic scars
consequent to thermal injury. Clin Exp Immunol 1990; 82: 350. 6 Castagnoli C, Stella M, Berthod C, Magliacani G and Momigliano Richiardi P. TNF Production and hypertrophic scarring. Celllmmunol 1993; 147: 51. 7 Roccatello D, D'Alfonso S and Peruccio D et al. Induction of mRNA for Tumor Necrosis Factor atpha in hemodiatysis. l~'dney Int 1993; 43: (supp139), 144. 8 Lantz M, Gullberg U, Nilsson E and Olsson I. Characterization in vitro of a human TNF-binding protein. ] Clin Invest 1990; 86: I396. 9 Wang AM, Doyle V and Mark DF. Quantitation of mRNA by the polymerase chain reaction. ProcNatI Acad Sci USA 1989; 86: 9717. 10 McCauly RL, Chopra V, Li Y, Hemdon DN and Robson MC Altered cytokine production in black patients with keloids. JClin Immunol 1992; 12: 300. I1 Tracey KJ, Vlassara H and Cerami A. Cachectin/Tumor Necrosis Factor. Lancet 1989; i: 1122. 12 Duncan MR and Berman B. Differential regulation of collagene, glycosaminoglycan, fibronectin and collagenase activity production in cultured human adult dermal fibroblasts by interleukin 1 alpha and beta and TNF alpha and beta. ] Invest Dermatol 1989; 92: 699. 13 Kovacs E. Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol Today 1991; 12: 17. 14 Datubo-Brown DD. Keloids: a review of the literature. Br J Plast Surg I990; 43: 70. 15 Abergel RP, Pizzurro D, Meeker CA et al. Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures. ] Invest Dermatol 1985; 84: 384. Paper accepted 26 August 1993. Correspondence should be addressed to: Dr Carlotta Castagnoli, Dip. Genetica, Biologia e Chimica Medica, Via Santena I9, 10126 Torino, Italy.
JAMES LAING MEMORIAL ESSAY The British Bum Association has instituted a memorial essay to be awarded annually in memory of James Ellsworth Laing, Burn Surgeon, founder member of the British Burn Association and a former Editor of this Journal. There is a prize of up to s for the winning essay. The subject for the tenth essay is:
Quality A s s u r a n c e
in B u r n P a t i e n t C a r e
The essay should be confined to not more than 100(~ words and correspondingly less if up to 6 Figures and/or Tables are included. The substance of the essay should not already have been published since the winning essay will be published in this Journal. The essays will be assessed anonymously. All persons interested in the problems associated with burning injury are eligible to submit an essay. The deadline for submission of an essay (4 copies) is 31 December 1994. Completed essays and any queries should be sent to: The Secretary of the British Burn Association, Dr J.N. Kearney, Ph.D., Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Wakefield WF1 4DG, West Yorkshire, UK.
The title and author(s) of the winning essay will be announced in April 1995.