Immunohistochemical analysis of dendritic cells in skin lesions: Correlations with survival time
Introduction
Deciding whether a skin wound occurred before or after death may be hard for a forensic pathologist when the survival time is short and an inflammatory reaction has not yet started. During the past 50 years, the development of histochemistry, enzymology and biochemistry and their application to investigations on wounds has offered partial solution to the problem [1]. The behavior of mast cells is among the data which can be of help in that respect [2], [3].
Dendritic cells (DCs) are another cell type involved in the response to injury even independent of antigen challenge, e.g. in the arterial wall [4], [5], [6]. Therefore DC modifications might be useful to recognize vital from post mortem wounds, but no information is available on their behavior shortly upon skin wounding.
Dendritic cells have been primarily considered for their role in specific immune responses. They uptake not-self proteins through endocytosis, subject them to controlled hydrolysis and load antigenic fragments on class I and class II molecules of the major histocompatibility complex (MHC-I and MHC-II, respectively), which are transferred to the cell surface to be recognized by T cells and stimulate the latter cells. MHC-II, in particular, are involved in the stimulation of helper and perhaps regulatory cells, since they interact with CD4 cell surface molecules which are expressed by those lymphocyte subtypes [7]. These molecules are characteristically expressed by cells of the immune system and – with high intensity – by DCs, as part of their equipment of professional antigen presenting cells [7]. DCs also express other invariant pattern recognition receptors for non-peptide antigens [8]. Besides immune responses, DCs can participate to natural immune responses by stimulating the recruitment of neutrophils [9] and natural killer cells [10] and by secreting type I interferon [11]. The molecules actually secreted depend on the cell differentiation and maturation step and on the type of stimulation [12] and can regulate the differentiation, migration and function of autochthonous and blood-born cells in inflamed tissues [13]. The influence of DCs on other cells can be mediated also by the stimulation of lymphocytes to secrete further cytokines, tissue coagulation factor and matrix metalloproteases [14].
Skin DCs are found both in the epidermis and dermis. In the human epidermis Langerhans cells (LCs) are characterized by the expression of CD1a and CD207/langerin, two invariant pattern recognition receptors [7], [15]. Dermal DCs are connective tissue DCs and express the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) [16] and CD11c [7]. In an ex vivo model of human skin burn injury the tissue became devoid of LCs and dermal DCs and the cells from neighbor tissue were defective stimulators of mixed lymphocyte reaction, 24 h after injury [17]. However, burning may not be a valuable general model for trauma and the behavior of LC and dermal DC in the early phases upon mechanical injury remains to be defined.
Therefore, we have addressed the behavior of epidermal and dermal DCs upon wounding as a step to understand the control mechanisms of injury response in the human skin and to elucidate the possible advantages of analyzing these cells to estimate wound age. In the epidermis we have evaluated on separate sections MHC-II+ and CD1a+ cells, since not all epidermal MHC-II+ express CD1a [18] and only those which express this marker deserve to be counted as LCs.
Section snippets
Tissue specimens
Samples of 90 skin wounds occurred before death were taken from the corpses of 80 subjects, died of traumatic events (car accident, fall, homicide, hanging) within one day from the injury and undergoing autopsy at the Section of Forensic Medicine of the University of Florence (Italy) on appointment of the court of justice, in the course of five years. The corpses belonged to 56 men and 24 women, aged between 3 and 86 years (mean age: 50), of caucasian origin. They were routinely kept at +4 °C
Controls
No significant difference was found for any parameter between intact skin 5 cm from wounds and that 20 cm from wounds (Fig. 1 and Table 1). The controls for histochemical reactions were always unstained.
Epidermis
Upon wounding there was an increase in MHC-II+ cells in the epidermis followed by a slow decrease until the number became less than in controls (Fig. 1A). The cells expressing CD1a increased less (yet significantly), earlier and for a shorter period of time than those expressing MHC-II (Fig. 1B).
Discussion
This study showed, upon skin wounding: (1) an increase in the number of epidermal CD1a+ cells and in that of MHC-II+ cells in the epidermis, followed by protracted decrease; (2) these variations in number were different for timing and extent between the two labels; (3) infiltration of MHC-II+, DC-SIGN+ DCs in the dermis; (4) an increase followed by protracted decrease in mast cell degranulation, which accompanied the expected variations in mast cell number; (5) a marked decrease in the number
Conclusion
The present findings support a primary role in the injury response of human skin not only for mast cells but also for epidermal and dermal DCs, which may concur to the activation of local and systemic response since early upon wounding given their very early and intense reaction. Therefore the evaluation of DCs in the epidermis and dermis can be proposed as complementary to other investigations to discriminate between wounds occurred in life and those occurred after death and to estimate the
Acknowledgements
The technical help of Mr. P. Venturi and Mr. W. Calugi is gratefully acknowledged. Financial support was granted by the University of Florence, the Ente Cassa di Risparmio di Firenze (grant no. 3681 to S.B.), and Foemina Foundation.
S. Bacci designed the project, generated descriptive and quantitative data, took primary responsibility for writing the manuscript and was in part responsible for financial support. B. De Fraia and A. Bonelli gave advice for the selection of cadavers and the
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