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Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine
Forensic Science International 113 (2000) 173–176
www.elsevier.com / locate / forsciint
Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine q ¨ a Jan Dressler a , Lutz Bachmann b , Premysel Strejc d , Rainer Koch c , Erich Muller a
Department of Legal Medicine, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany b Department of Surgery, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany c Department for Medical Informatics and Biometrics, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany d Department of Legal Medicine, Charles University Prague, Studnickova 4, CZ-12800 Prague 2, Czech Republic
Abstract The adhesion molecules identified in recent years can help improve the diagnosis of the wound age, especially of injuries with a short survival time. This is also indicative of the vitality of the wounds. The material investigated in the study originated from 465 skin wounds. The samples were taken from human autopsy material, during the surgical treatment of wounds (excision) of patients and from experimental incised wounds of mice. To judge the age of skin wounds the endothelial adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. Human skin wounds: strong positive staining was observed of ICAM-1 1.5 h at the earliest and 3.5 days at the latest, for the P-selectin 3 min at the earliest and 7 h at the latest, for the E-selectin 1 h at the earliest and 17 days at the latest and for VCAM-1 3 h at the earliest and 3.5 days at the latest after the time of injury. The L-selectin was expressed constitutively. Mice skin wounds: strong positive immunohistochemical reactions were found as a rule earlier than in human skin wounds. The detection of an increased expression of ICAM-1, VCAM-1 and P- and E-selectins can improve the wound age assessment in injuries with short survival times. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ICAM-1; VCAM-1; Selectins; Wound age; Immunohistochemistry
1. Introduction When carrying out an autopsy, the survival time of the deceased following injury is often unknown. Using conventional histological methods, the age of a wound and thus the vitality of the injury can only be assessed to a certain degree, primarily because of the lack of markers in the initial state of the wound healing process [5,8,16]. Adhesion molecules identified in recent years reveal a cascade of bonding q
IVth International Symposium of Advances in Legal Medicine (ISALM), September 22–25, 1999.
reactions, which lead to an emigration of granulocytes, lymphocytes and monocytes from the blood vessels to the tissue [9].
2. Materials and methods The material investigated in this study originated from 465 skin wounds. Of the samples, 194 were taken from autopsy material, 100 were extracted during the surgical treatment of wounds (excision) of patients and 140 were taken from experimental incised wounds of mice. In addition, 31 postmortem
0379-0738 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 00 )00258-9
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
174
skin injuries were investigated. The wound age varied between 3 min and 790 days. To judge the age of skin wounds the adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. The staining intensity was assessed semi-quantitatively. Differences in the distribution of the variables of the findings between types of wounds and wound age classes were statistically tested.
skin was injured. L-selectin was regularly detected on leukocytes in the control samples (n597) of uninjured skin. As a rule strong positive immunohistochemical reactions were found in mice skin wounds earlier than in human skin wounds. The immunohistochemical results for ICAM-1, VCAM-1, P- and E-selectins were significantly different between injured and uninjured skin (P,0.01).
3. Results 4. Discussion In injured skin (Table 1), the intercellular adhesion molecule 1 (ICAM-1) showed strong positive staining on the keratinocytes, especially on basal keratinocytes of the epidermis. With the passage of time since injury, the staining intensity for ICAM-1 on endothelial cells, in particular in the vicinity of inflammatory infiltrates, was enhanced. Strong positive staining was observed 1.5 h at the earliest and 3.5 days at the latest after the time of injury. ICAM1 could also be detected on the granulocytes and lymphocytes of these perivascular infiltrates. The vascular cell adhesion molecule 1 (VCAM-1) was found to be positive on endothelial cells of small and large blood vessels in skin wounds. VCAM-1 showed both an increase in the semi-quantitatively determined intensity of the immunohistological staining reaction and a higher number of blood vessels with positive reaction. Strong positive staining reactions were observed 3 h at the earliest and 3.5 days at the latest after the time of injury. Strong positive immunohistochemical reactions were observed for the P-selectin 3 min at the earliest and 7 h at the latest after the time of injury. For the E-selectin a positive staining was evident 1 h at the earliest and 17 days at the latest from the time the Table 1 Time dependence of adhesion molecules in human skin wounds Adhesion molecule
Earliest appearance
Longest appearance
ICAM-1 VCAM-1 P-selectin E-selectin L-selectin
1.5 h 3h 3 min 1h Ø
3.5 days 3.5 days 7h 17 days Ø
Ø: regular detected.
The expression patterns on keratinocytes and perivascular inflammatory cells (granulocytes, lymphocytes and monocytes) in injured and uninjured skin differ significantly. Of forensic significance is the distinction between earliest, regular and latest appearance [4] of this adhesion molecule in skin wounds. In our series, the immunohistochemical investigation revealed a strong ICAM-1 expression on endothelial cells and keratinocytes as early as 2 h after injury. The oldest wound with strong ICAM-1 expression was 3.5 days old. These result is comparable to the findings of Nwariaku et al. [10]. According to Pober and Cotran [13] and Abe et al. [1], the up-regulation of VCAM-1 begins after 2–4 h and can last several days. In vitro experiments conducted by Gemmell et al. [7] showed that VCAM-1 could be detected over a period of 6–48 h. Strong positive staining reactions were observed after 3 h at the earliest and in 3.5-day-old skin wounds at the latest in our study. Strong positive immunohistochemical reactions were observed for the P-selectin earliest 3 min after injury and latest after 7 h. These findings are in accordance with the results reported by Wyler [16], Silber et al. [14] and Ohnishi et al. [11], who observed the expression of P-selectin within a few minutes of intradermal injection of endotoxin or anti-ovalbumin in animals. Ortmann and Brinkmann [12] reported a strong immunohistochemical staining reaction for P-selectin on blood vessels in the lungs from autopsy cases with rapid occurrence of death (hanging, carbon monoxide and cyanide intoxication), but a decrease of stainability of the endothelial cells of the blood vessels in cases with protracted
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
occurrence of death as a result of pneumonia and septic shock. Pober and Cortan [13] found out that E-selectin is expressed on blood vessels 2–6 h after an inflammatory irritation and could be detected for a period of up to 2 days. This process is a new synthesis because E-selectin does not occur preformed in the endothelial cells. In the studies we undertook, E-selectin could not be detected in uninjured skin in 99% of the cases. The expression of E-selectin was evident in 1-h-old skin wounds at the earliest and 17 days after injury at the latest. The time dependence of Eselectin expression we found is more or less in line with the conclusions of Bevilacqua et al. [3], Nwariaku et al. [10] and Abe et al. [1]. L-selectin is unanimously mentioned as an adhesion molecule [15] and is inherently expressed by leukocytes. L-selectin was regularly detected on leukocytes when inflammatory infiltrates occurred in the investigated samples of injured and uninjured skin. The immunohistochemical detection of adhesion molecules does not make excessive demands on laboratories. The pretreatment (autoclaving) of the paraffin sections should pose no problems [2], and all antibodies and reagents are commercially available. It is important, however, that the experiments are made under standardized conditions, i.e. with monoclonal primary antibodies and a standardized staining kit.
5. Conclusions In 54% of the wounds were detected positive staining reactions on endothelial cells. In contrast were only 16% of the investigated blood vessels positive in uninjured skin. The immunohistochemical pattern showed a statistically difference. The moderate to strong expression of ICAM-1, VCAM-1 and selectins is a valuable indication of the vitality of the wound [6]. The samples taken from the wounds of autopsy cases were 9 days old at the most. Over this time we found no statistical differences in staining intensity of the adhesion molecules. The inclusion of the adhesion molecules in the immunohistochemical estimation of the time since injury is thought to be
175
useful [6], provided parallel investigations using established indicators.
Acknowledgements The authors are very much obliged to Helga Kunze for her excellent technical assistance.
References [1] Y. Abe, K. Sugisaki, A.M. Dannenberg, Rabbit vascular endothelial adhesion molecules: ELAM-1 is most elevated in acute inflammation, whereas VCAM-1 and ICAM-1 predominate in chronic inflammation, J. Leukoc. Biol. 60 (1996) 692–703. ¨ [2] A. Bankfalvi, K. Riehemann, D. Ofner, R. Checci, J.M. ¨ Morgan, J. Piffko, W. Bocker, B. Jasani, K.W. Schmid, Feuchtes Autoklavieren, Pathologe 15 (1994) 345–349. [3] M.P. Bevilaqua, S. Stengelin, M.A. Gimbrone, B. Seed, Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins, Science 241 (1989) 1160–1165. [4] P. Betz, Histological and enzyme histochemical parameters for the age estimation of human skin wounds, Int. J. Legal Med. 107 (1994) 60–68. [5] P. Betz, Immunohistochemical parameters for the age estimation of human skin wounds, Am. J. Forensic Pathol. Med. 16 (1995) 203–209. ¨ ¨ [6] J. Dressler, Zur Bedeutung endothelialer Adhasionsmolekule, ¨ die immunhistochemische insbesondere der Selektine, fur Diagnostik des Wundalters, Postdoctoral Thesis, Dresden, 1998. [7] E. Gemmell, L.J. Walsh, N.W. Savage, G.J. Seymour, Adhesion molecule expression in chronic inflammatory periodontal disease tissue, J. Periodont. Res. 29 (1994) 46–53. [8] T. Kondo, T. Ohshima, The dynamics of inflammatory cytokines in the healing process of mouse skin wound: a preliminary study for possible wound age determination, Int. J. Legal Med. 108 (1996) 231–236. ¨ ¨ Neue Perspektiven [9] S. Martin, H. Kolb, Adhasionsmolekule: ¨ Therapie und Diagnostik, Serva News, 1995, 12–14. fur [10] F.E. Nwariaku, W.J. Mileski, E. Lightfoot, P.J. Sikes, P.E. Lipsky, Alterations in leukocyte adhesion molecule expression after burn injury, J. Trauma 39 (1995) 285–288. [11] M. Ohnishi, H. Koike, N. Kawamura, S.J. Tojo, M. Hayashi, S. Morooka, Role of P-selectin in the early stage of Arthus reaction, Immunopharmac. 34 (1996) 161–170. [12] C. Ortmann, B. Brinkmann, The expression of P-selectin in inflammatory and non-inflammatory lung tissue, Int. J. Legal Med. 110 (1997) 155–158. [13] J.S. Pober, R.S. Cotran, The role of endothelial cells in inflammation, Transplantation 50 (1990) 537–544. [14] A. Silber, W. Newman, K.A. Reimann, E. Hendricks, D.
176
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
Walsh, D.J. Ringler, Kinetic expression of endothelial adhesion molecules and relationship to leukocyte recruitment in two cutaneous models of inflammation, Lab. Invest. 70 (1994) 163–175. [15] L.M. Stoolmann, Adhesion molecules controlling lymphocyte migration, Cell 56 (1989) 907–910.
[16] D. Wyler, Determining the age and assessing the vitality of wounds by immunohistochemical detection of cell adhesion molecules, in: M. Oehmichen, H. Kirchner (Eds.), The Wound Healing Process — Forensic Pathology Aspects, ¨ ¨ Schmidt Romhild, Lubeck, 1996, pp. 133–138.
www.elsevier.com / locate / forsciint
Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine q ¨ a Jan Dressler a , Lutz Bachmann b , Premysel Strejc d , Rainer Koch c , Erich Muller a
Department of Legal Medicine, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany b Department of Surgery, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany c Department for Medical Informatics and Biometrics, Technical University Medical School, Fetscherstrasse 74, D-01307 Dresden, Germany d Department of Legal Medicine, Charles University Prague, Studnickova 4, CZ-12800 Prague 2, Czech Republic
Abstract The adhesion molecules identified in recent years can help improve the diagnosis of the wound age, especially of injuries with a short survival time. This is also indicative of the vitality of the wounds. The material investigated in the study originated from 465 skin wounds. The samples were taken from human autopsy material, during the surgical treatment of wounds (excision) of patients and from experimental incised wounds of mice. To judge the age of skin wounds the endothelial adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. Human skin wounds: strong positive staining was observed of ICAM-1 1.5 h at the earliest and 3.5 days at the latest, for the P-selectin 3 min at the earliest and 7 h at the latest, for the E-selectin 1 h at the earliest and 17 days at the latest and for VCAM-1 3 h at the earliest and 3.5 days at the latest after the time of injury. The L-selectin was expressed constitutively. Mice skin wounds: strong positive immunohistochemical reactions were found as a rule earlier than in human skin wounds. The detection of an increased expression of ICAM-1, VCAM-1 and P- and E-selectins can improve the wound age assessment in injuries with short survival times. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ICAM-1; VCAM-1; Selectins; Wound age; Immunohistochemistry
1. Introduction When carrying out an autopsy, the survival time of the deceased following injury is often unknown. Using conventional histological methods, the age of a wound and thus the vitality of the injury can only be assessed to a certain degree, primarily because of the lack of markers in the initial state of the wound healing process [5,8,16]. Adhesion molecules identified in recent years reveal a cascade of bonding q
IVth International Symposium of Advances in Legal Medicine (ISALM), September 22–25, 1999.
reactions, which lead to an emigration of granulocytes, lymphocytes and monocytes from the blood vessels to the tissue [9].
2. Materials and methods The material investigated in this study originated from 465 skin wounds. Of the samples, 194 were taken from autopsy material, 100 were extracted during the surgical treatment of wounds (excision) of patients and 140 were taken from experimental incised wounds of mice. In addition, 31 postmortem
0379-0738 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 00 )00258-9
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
174
skin injuries were investigated. The wound age varied between 3 min and 790 days. To judge the age of skin wounds the adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. The staining intensity was assessed semi-quantitatively. Differences in the distribution of the variables of the findings between types of wounds and wound age classes were statistically tested.
skin was injured. L-selectin was regularly detected on leukocytes in the control samples (n597) of uninjured skin. As a rule strong positive immunohistochemical reactions were found in mice skin wounds earlier than in human skin wounds. The immunohistochemical results for ICAM-1, VCAM-1, P- and E-selectins were significantly different between injured and uninjured skin (P,0.01).
3. Results 4. Discussion In injured skin (Table 1), the intercellular adhesion molecule 1 (ICAM-1) showed strong positive staining on the keratinocytes, especially on basal keratinocytes of the epidermis. With the passage of time since injury, the staining intensity for ICAM-1 on endothelial cells, in particular in the vicinity of inflammatory infiltrates, was enhanced. Strong positive staining was observed 1.5 h at the earliest and 3.5 days at the latest after the time of injury. ICAM1 could also be detected on the granulocytes and lymphocytes of these perivascular infiltrates. The vascular cell adhesion molecule 1 (VCAM-1) was found to be positive on endothelial cells of small and large blood vessels in skin wounds. VCAM-1 showed both an increase in the semi-quantitatively determined intensity of the immunohistological staining reaction and a higher number of blood vessels with positive reaction. Strong positive staining reactions were observed 3 h at the earliest and 3.5 days at the latest after the time of injury. Strong positive immunohistochemical reactions were observed for the P-selectin 3 min at the earliest and 7 h at the latest after the time of injury. For the E-selectin a positive staining was evident 1 h at the earliest and 17 days at the latest from the time the Table 1 Time dependence of adhesion molecules in human skin wounds Adhesion molecule
Earliest appearance
Longest appearance
ICAM-1 VCAM-1 P-selectin E-selectin L-selectin
1.5 h 3h 3 min 1h Ø
3.5 days 3.5 days 7h 17 days Ø
Ø: regular detected.
The expression patterns on keratinocytes and perivascular inflammatory cells (granulocytes, lymphocytes and monocytes) in injured and uninjured skin differ significantly. Of forensic significance is the distinction between earliest, regular and latest appearance [4] of this adhesion molecule in skin wounds. In our series, the immunohistochemical investigation revealed a strong ICAM-1 expression on endothelial cells and keratinocytes as early as 2 h after injury. The oldest wound with strong ICAM-1 expression was 3.5 days old. These result is comparable to the findings of Nwariaku et al. [10]. According to Pober and Cotran [13] and Abe et al. [1], the up-regulation of VCAM-1 begins after 2–4 h and can last several days. In vitro experiments conducted by Gemmell et al. [7] showed that VCAM-1 could be detected over a period of 6–48 h. Strong positive staining reactions were observed after 3 h at the earliest and in 3.5-day-old skin wounds at the latest in our study. Strong positive immunohistochemical reactions were observed for the P-selectin earliest 3 min after injury and latest after 7 h. These findings are in accordance with the results reported by Wyler [16], Silber et al. [14] and Ohnishi et al. [11], who observed the expression of P-selectin within a few minutes of intradermal injection of endotoxin or anti-ovalbumin in animals. Ortmann and Brinkmann [12] reported a strong immunohistochemical staining reaction for P-selectin on blood vessels in the lungs from autopsy cases with rapid occurrence of death (hanging, carbon monoxide and cyanide intoxication), but a decrease of stainability of the endothelial cells of the blood vessels in cases with protracted
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
occurrence of death as a result of pneumonia and septic shock. Pober and Cortan [13] found out that E-selectin is expressed on blood vessels 2–6 h after an inflammatory irritation and could be detected for a period of up to 2 days. This process is a new synthesis because E-selectin does not occur preformed in the endothelial cells. In the studies we undertook, E-selectin could not be detected in uninjured skin in 99% of the cases. The expression of E-selectin was evident in 1-h-old skin wounds at the earliest and 17 days after injury at the latest. The time dependence of Eselectin expression we found is more or less in line with the conclusions of Bevilacqua et al. [3], Nwariaku et al. [10] and Abe et al. [1]. L-selectin is unanimously mentioned as an adhesion molecule [15] and is inherently expressed by leukocytes. L-selectin was regularly detected on leukocytes when inflammatory infiltrates occurred in the investigated samples of injured and uninjured skin. The immunohistochemical detection of adhesion molecules does not make excessive demands on laboratories. The pretreatment (autoclaving) of the paraffin sections should pose no problems [2], and all antibodies and reagents are commercially available. It is important, however, that the experiments are made under standardized conditions, i.e. with monoclonal primary antibodies and a standardized staining kit.
5. Conclusions In 54% of the wounds were detected positive staining reactions on endothelial cells. In contrast were only 16% of the investigated blood vessels positive in uninjured skin. The immunohistochemical pattern showed a statistically difference. The moderate to strong expression of ICAM-1, VCAM-1 and selectins is a valuable indication of the vitality of the wound [6]. The samples taken from the wounds of autopsy cases were 9 days old at the most. Over this time we found no statistical differences in staining intensity of the adhesion molecules. The inclusion of the adhesion molecules in the immunohistochemical estimation of the time since injury is thought to be
175
useful [6], provided parallel investigations using established indicators.
Acknowledgements The authors are very much obliged to Helga Kunze for her excellent technical assistance.
References [1] Y. Abe, K. Sugisaki, A.M. Dannenberg, Rabbit vascular endothelial adhesion molecules: ELAM-1 is most elevated in acute inflammation, whereas VCAM-1 and ICAM-1 predominate in chronic inflammation, J. Leukoc. Biol. 60 (1996) 692–703. ¨ [2] A. Bankfalvi, K. Riehemann, D. Ofner, R. Checci, J.M. ¨ Morgan, J. Piffko, W. Bocker, B. Jasani, K.W. Schmid, Feuchtes Autoklavieren, Pathologe 15 (1994) 345–349. [3] M.P. Bevilaqua, S. Stengelin, M.A. Gimbrone, B. Seed, Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins, Science 241 (1989) 1160–1165. [4] P. Betz, Histological and enzyme histochemical parameters for the age estimation of human skin wounds, Int. J. Legal Med. 107 (1994) 60–68. [5] P. Betz, Immunohistochemical parameters for the age estimation of human skin wounds, Am. J. Forensic Pathol. Med. 16 (1995) 203–209. ¨ ¨ [6] J. Dressler, Zur Bedeutung endothelialer Adhasionsmolekule, ¨ die immunhistochemische insbesondere der Selektine, fur Diagnostik des Wundalters, Postdoctoral Thesis, Dresden, 1998. [7] E. Gemmell, L.J. Walsh, N.W. Savage, G.J. Seymour, Adhesion molecule expression in chronic inflammatory periodontal disease tissue, J. Periodont. Res. 29 (1994) 46–53. [8] T. Kondo, T. Ohshima, The dynamics of inflammatory cytokines in the healing process of mouse skin wound: a preliminary study for possible wound age determination, Int. J. Legal Med. 108 (1996) 231–236. ¨ ¨ Neue Perspektiven [9] S. Martin, H. Kolb, Adhasionsmolekule: ¨ Therapie und Diagnostik, Serva News, 1995, 12–14. fur [10] F.E. Nwariaku, W.J. Mileski, E. Lightfoot, P.J. Sikes, P.E. Lipsky, Alterations in leukocyte adhesion molecule expression after burn injury, J. Trauma 39 (1995) 285–288. [11] M. Ohnishi, H. Koike, N. Kawamura, S.J. Tojo, M. Hayashi, S. Morooka, Role of P-selectin in the early stage of Arthus reaction, Immunopharmac. 34 (1996) 161–170. [12] C. Ortmann, B. Brinkmann, The expression of P-selectin in inflammatory and non-inflammatory lung tissue, Int. J. Legal Med. 110 (1997) 155–158. [13] J.S. Pober, R.S. Cotran, The role of endothelial cells in inflammation, Transplantation 50 (1990) 537–544. [14] A. Silber, W. Newman, K.A. Reimann, E. Hendricks, D.
176
J. Dressler et al. / Forensic Science International 113 (2000) 173 – 176
Walsh, D.J. Ringler, Kinetic expression of endothelial adhesion molecules and relationship to leukocyte recruitment in two cutaneous models of inflammation, Lab. Invest. 70 (1994) 163–175. [15] L.M. Stoolmann, Adhesion molecules controlling lymphocyte migration, Cell 56 (1989) 907–910.
[16] D. Wyler, Determining the age and assessing the vitality of wounds by immunohistochemical detection of cell adhesion molecules, in: M. Oehmichen, H. Kirchner (Eds.), The Wound Healing Process — Forensic Pathology Aspects, ¨ ¨ Schmidt Romhild, Lubeck, 1996, pp. 133–138.