Forensic science international quantitative analysis of proinflammatory cytokines (IL 1β, IL 6, TNF

Forensic Science International 113 (2000) 251–264 www.elsevier.com / locate / forsciint Quantitative analysis of proinflammatory cytokines (IL-1b, IL-6, TNF-a) in human skin wounds a, b a W Grellner *, T Georg , J Wilske a Institute of Forensic Medicine, Saarland University, Building 42, D-66421 Homburg /Saar, Germany b Institute of Medical Biometrics, Saarland University, Homburg /Saar, Germany Abstract Proinflammatory cytokines play an important role in the mediation of inflammation and trauma They could be useful for the determination of vitality and wound age In the present study, 144 human skin wounds due to sharp force were investigated The material was collected during operations (N596) and postmortem examinations (N548) The wound age varied from several seconds or minutes to days Control skin was available in each individual The tissue specimens were homogenized and extracted in a solution of PBS and protease inhibitors Interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-a) were measured by quantitative ELISA analysis Statistical evaluation was performed by the t-test using the quotients of levels (wound sample / control skin) In surgical specimens the cytokine levels revealed a clear tendency to increase with wound age IL-1b in early skin wounds (#30 min) and TNF-a after a wound age of 1–2 h demonstrated statistically significant changes in comparison with control skin (P,0.05) In autopsy samples with severe traumatization excessive elevation of cytokine levels was observed: IL-1b, IL-6 and TNF-a showed significant increases (P,0.001–0.05) in stab and incised wounds with very short survival times of less than min, but not in possibly supravital injuries Elevated IL-6 levels persisted in older wounds (.24 h, P,0.05) The quantitative analysis of proinflammatory cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular in the very early post-traumatic interval (classic stab wounds)  2000 Elsevier Science Ireland Ltd All rights reserved Keywords: Proinflammatory cytokines; Interleukin-1b; Interleukin-6; Tumour necrosis factor alpha; Wound age; Vitality Introduction Cytokines are mediators with multiple functions, including the initiation or influence of numerous biological processes such as inflammation, sepsis and wound healing [1–9] The proinflammatory cytokines interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) play key roles within the cytokine network as it is frequently called (see reviews in Refs [10–13]) This term refers to *Corresponding author Fax: 149-6841-166-314 the close relationship between various cytokines and their pathways Minute concentrations, rapid stimulation and short durations of activity characterize the autocrine / paracrine regulation of these peptides They are produced by a variety of cell types such as macrophages, thrombocytes and keratinocytes The features mentioned could make cytokines to an appropriate tool for the evaluation of vitality and early wound age In the present study, the proinflammatory mediators IL-1b, IL-6 and TNF-a were determined quantitatively in extracts of human skin wounds and 0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd All rights reserved PII: S0379-0738( 00 )00218-8 252 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 tested for their usefulness in the estimation of vitality and wound age Material and methods 2.1 Material In the present study, 144 human skin wounds due to sharp force were investigated The material was collected during operations (N596) and postmortem examinations (N548) Control skin from uninjured areas was available in each individual 2.1.1 Surgical material A total of 96 wound samples with control skin were collected; 54 from males and 42 from females, average age: 56.3615.4 years In dependence on weight and volume, the tissue could be used for the determination of one or more markers and was distributed as follows: IL-1b (N543), IL-6 (N549) and TNF-a (N546) The wound age varied from to 5.5 h with special emphasis on the early post-traumatic interval up to h (average: 73656 min) Four wound age classes were formed: #30 (N523), 31–60 (N531), 61–120 (N523) and 120 (N5 19) Most of the skin samples originated from the abdominal region (N584) 2.1.2 Autopsy material A total of 48 wound samples with control skin were collected; 33 from males and 15 from females, average age: 45.8616.4 years Because of the higher tissue weight, three markers could be determinded in most of the samples: IL-1b (N548), IL-6 (N547) and TNF-a (N544) The wound age varied from several seconds or minutes in homicides due to sharp force and injuries from railway suicides to a maximum of days in surgically treated wounds (average: 23.2651.2 h) Three wound age classes were formed: # ca (N532), 1–24 h (N56) and 24 h (N510) In the first group the survival time could not exactly be defined, as there were ‘‘classic’’ homicides and suicides by stab and incised wounds where the involved persons were already found dead Due to the presence of severe injuries of greater blood vessels the above mentioned time interval of ‘‘up to several min’’ (#5 min) was concluded as the probable wound age In this group, there were also cases (railway suicides, emergency measurements in the agonal or early supravital period) with extremely short survival times or possibly supravital injury This small subgroup (N512) was additionally evaluated separately Most of the skin samples originated from the abdominal region (ca 25%), but other anatomical locations were also present (extremities, thorax) The postmortem interval was between h and 20 days (average: 74687 h) Individuals with known immunodeficiencies or immunotherapy were excluded from the study in both surgical and autopsy samples The wound specimens were taken parallel to the wound margin They measured approximately 0.5– 1.0 cm in length and 0.2–0.3 cm in width The subcutis was removed The skin samples were shock frozen in liquid nitrogen and could be stored at 2708C until analysis The average weight was 62632 mg in surgical samples and 84619 mg in postmortem specimens, respectively 2.2 Tissue extraction The complete extraction was performed under cooling conditions (#48C, in liquid nitrogen, on ice) because of the known susceptibility of cytokines We developed our own protocol according to procedures for the quantitation of stem cell factor (SCF) from placental tissue [14] and IL-8 from psoriatic skin [15] In brief, the frozen skin specimens were homogenized mechanically and by incubation in an oscillating mill under the permanent addition of liquid nitrogen (1–2 min) The resulting product, which resembled a powder, was transferred to cups, weighed (wet weight) and extracted in a ten-fold volume of phosphate buffered saline (PBS, pH 7.4) and protease inhibitors [2 mM phenylmethylsulfonyl fluoride (PMSF); mM ethylenediaminetetraacetate (EDTA); mg / ml of leupeptin, antipain, aprotinin and pepstatin A, respectively] The extraction took h at 48C under permanent agitation Then the samples were centrifuged for 20 at 15 000 g and W Grellner et al / Forensic Science International 113 (2000) 251 – 264 48C The resulting solution (soluble cytokine fraction) could be stored at 2708C until ELISA analysis In some samples, the sediments were re-extracted overnight in a solution of M guanidinium, pH 7.4, and the protease inhibitors were, as mentioned above, dialyzed for h to remove the guanidinium and finally centrifuged again The resulting solution contained the matrix associated cytokine fraction 253 P,0.05 were regarded as statistically significant The arithmetical means and the standard deviations were calculated The chi-square test and the correlations of Kendall and Spearman were used for testing the results on independence of age, gender, anatomical site and postmortem interval Results 2.3 Determination of protein content 3.1 General remarks In some samples the protein content (mg / ml) was determined by three-fold photometric measurements at 560 nm according to the microtiter plate method of the bicinchoninic acid (BCA) protein assay (Pierce, USA) [16–19] 2.4 ELISA Interleukin-1b (IL-1b), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-a) were measured by quantitative ELISA analysis (kits and protocols of BioSource, USA) using a microtiter plate reader (photometry at 450 nm) and an automatic calculation of results The respective cytokine levels were determined in wound samples and control specimens by means of double measurements requiring 100– 200 ml of extract The results had the dimension of pg / ml The detection limits were pg / ml for IL-1b, pg / ml for IL-6 and ,1 pg / ml for TNF-a, respectively 2.5 Evaluation and statistical analysis The following parameters were considered: age, gender, wound age, postmortem interval in autopsy cases, anatomical site of wounds, sample weight, protein content and ELISA results The following quotients were calculated and tested: pg of cytokine / mg of protein, pg of cytokine / g of tissue and pg of cytokine / mg of protein / g of tissue Testing all parameters, the ELISA results of the PBS extracts in pg / ml were finally related to the wet weight of skin samples (pg / g) These values were independent of artificial changes Statistical evaluation was performed by the t-test using the quotients of levels (wound sample / control skin) Values of The results of the quantitative analyses differed considerably between the wound samples from the surgical procedures and those from the postmortem examinations Both parts are therefore depicted separately (Sections 3.2 and 3.3) Common characteristics of this study were as follows: the decisive variable was the content of soluble cytokines as extracted by PBS It did not disappear in a possibly greater pool of matrix associated cytokines as extracted by guanidinium It had to be related to the weight of skin samples (pg / g) These results showed great interindividual variability of cytokine levels concerning also the normal values in the control skin (Table 1) Therefore, it was in addition necessary to calculate the quotients of levels (wound sample / control skin), other correlations or quotients did not give useful results Using this method, a good correlation between the quantitative results and the wound age was possible The results were statistically independent of age, gender, anatomical site or postmortem interval The partially performed determination of the protein content revealed the following values which were not used in the statistical evaluation: • surgical material: 1.4860.44 mg / ml in skin wounds (N578) and 1.3560.47 mg / ml in control skin (N574) Table Cytokine levels in normal human skin Cytokine Level in normal skin a (vital, pg / g) Level in normal skin a (postmortem, pg / g) IL-1b IL-6 TNF-a 127.56127.6 205.16159.0 1470.863186.6 329.66570.8 190.26384.3 627.161503.6 a Mean values and standard deviation 254 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 • autopsy material: 1.9560.60 mg / ml in skin wounds (N529) and 1.5360.47 mg / ml in control skin (N529) 3.2 Wound samples from surgical procedures In surgical specimens the cytokine levels revealed a clear tendency to increase with wound age In this group IL-1b and TNF-a proved to be the most valuable markers 3.2.1 Interleukin-1b The quotients of IL-1b varied from 0.41 to 3.44 with increasing tendency in wounds with an age of hours (Fig 1) When compared to normal skin, IL-1b showed a statistically significant decrease in the first wound age class (#30 min) with an average quotient of 0.7960.28 (P,0.05) The quotients exhibited then an almost linear increase with values of about 1.5 in wounds with an age of h (quotient: 1.5360.76, P50.056) (Fig 2, Table 2) 3.2.2 Interleukin-6 In comparison with IL-1b, the quotients of IL-6 demonstrated a considerably greater variability with values from 0.23 to 11.26 They also increased with higher wound age (Fig 3) The quotients amounted to approximately 1.0 (i.e., identical to control skin) in wounds aging up to h and doubled in the following wound age classes (61–120 min: 1.9562.49; 120 min: 2.0863.26) (Fig 4) However, as shown by the standard deviation, the variability was also considerable in posttraumatic intervals of more than h, so that the increase was not significant (Table 2) 3.2.3 Tumour necrosis factor-alpha The quotients of TNF-a were mostly greater than 1.0, the variation was between 0.37 and 8.43 (Fig 5) An increase with higher wound age was not observed, but TNF-a was the only cytokine with an average quotient of 1.8461.57 for all skin wounds (independent of wound age) which demonstrated high significance compared to control skin (P5 0.001) The various wound classes are shown in Fig 6: the TNF-a quotients elevated to 1.6161.20 already in the early post-traumatic interval (#30 min, P5 0.066) Apart from a statistically significant decrease Fig Distribution of IL-1b quotients in human skin wounds (surgical material) W Grellner et al / Forensic Science International 113 (2000) 251 – 264 255 Fig IL-1b in human skin wounds (surgical material) Mean quotients and standard deviations in the class from 61 to 120 (quotient: 1.3960.54, P,0.05) the quotients remained over (Fig 6, Table 2) average of 1.7260.92 in the shortly survived stab and incised wounds of homicides and suicides was highly significant (P,0.001) IL-1b fell back to values in normal skin in the medium wound age group from to 24 h (quotient: 1.2960.65) and disclosed the highest levels in the later wound age phase 24 h (quotient: 3.2364.87) Due to the high standard deviation statistical significance could not be reached 3.3 Wound samples from autopsies In autopsy samples with severe traumatization, an excessive elevation of cytokine levels was frequently observed exceeding the results of surgical samples by far IL-6 proved to be the ‘‘key marker’’ 3.3.2 Interleukin-6 The quotients of IL-6 demonstrated again – comparable to surgical wounds – the greatest range with values between 0.23 and 80.82 The maximum average IL-6 levels were detectable in the medium 3.3.1 Interleukin-1b The quotients of IL-1b ranged from 0.42 to 16.84 with an increasing tendency in higher wound age 24 h (Fig 7, Table 3) The rapid increase to an Table Proinflammatory cytokines: statistical significances for surgical samples a Wound age IL-1b IL-6 TNF-a a #30 31–60 120 61–120 P N P N P N P N ,0.05 n.s 0.066 11 15 n.s n.s 0.061 11 13 n.s n.s ,0.05 15 17 11 0.056 n.s n.s 10 10 P-values of quotients, n.s.5not significant 256 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 Fig Distribution of IL-6 quotients in human skin wounds (surgical material) Fig IL-6 in human skin wounds (surgical material) Mean quotients and standard deviations W Grellner et al / Forensic Science International 113 (2000) 251 – 264 Fig Distribution of TNF-a quotients in human skin wounds (surgical material) Fig TNF-a in human skin wounds (surgical material) Mean quotients and standard deviations 257 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 258 Fig IL-1b in human skin wounds (autopsy material) Mean quotients and standard deviations wound age class from to 24 h (Fig 8, Table 3) Despite considerable variability, the increase of IL-6 in stab wounds from homicides and suicides with extremely short survival times was so excessive that it proved to be statistically significant (quotient: 7.08615.73, P,0.05) In one case (stab wound) an over 80-fold increase of IL-6 was measured compared to normal skin Despite even greater average quotients (9.21611.42) in the wound age class from to 24 h, the high standard deviation and the relatively small case number prevented statistical significance However, statistically significant results were again found in ‘‘older’’ skin wounds 24 h (quotient: 5.1064.34, P,0.05) Table Proinflammatory cytokines: statistical significances for autopsy samples a Wound age IL-1b IL-6 TNF-a a #5 24 h 1–24 h P N P N P N ,0.001 ,0.05 ,0.01 32 32 29 n.s n.s n.s 6 n.s ,0.05 n.s 10 10 P-values of quotients, n.s.5not significant 3.3.3 Tumour necrosis factor-alpha TNF-a revealed the maximum quotients similar to IL-6 in the medium wound age interval from to 24 h (Fig 9, Table 3) The values for all skin wounds varied from 0.67 to 10.05 Among the proinflammatory cytokines, TNF-a showed the absolute lowest but most constant increase in stab wounds with very short survival times so that this marker was markedly elevated in the earliest post-traumatic interval (quotient: 1.5560.99, P,0.01) The medium wound age class from to 24 h was characterized by greater absolute increases, but also by higher variations, so that statistical significance could not be demonstrated (quotient: 3.5963.41) TNF-a fell back to normal levels similar to the control skin in the late wound age group (quotient: 1.0460.33) 3.3.4 Final summary of postmortem investigations The quantitative analysis of proinflammatory cytokines demonstrated its advantages especially in the very early post-traumatic interval of less than (vitality) (Fig 10, Table 3) All three parameters IL-1b, IL-6 and TNF-a showed significant increases (P,0.001–0.05) in stab and incised wounds despite W Grellner et al / Forensic Science International 113 (2000) 251 – 264 Fig IL-6 in human skin wounds (autopsy material) Mean quotients and standard deviations Fig TNF-a in human skin wounds (autopsy material) Mean quotients and standard deviations 259 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 260 Fig 10 Summary of proinflammatory cytokines in human skin wounds (autopsy material) Mean quotients and significances the shortest survival times (homicides and suicides by sharp force with rapid exsanguination) Separate evaluation of the subgroup (N512) with extremely short survival times (railway suicides) or possibly supravital injuries (emergency measurements) exhibited only slightly elevated cytokine quotients (IL-1b: 1.5060.89; IL-6: 1.2960.57; TNFa: 1.5761.04) Statistically significant differences to the control skin were not found The medium post-traumatic interval from to 24 h was characterized by high levels of IL-6 and TNF-a Significant results were not demonstrable High and statistically significant IL-6 levels persisted in older wounds (.24 h) and also IL-1b remained elevated, whereas TNF-a fell back to basal values (Fig 10) Discussion After a traumatic event cytokines are primarily synthesized and / or released at the site of tissue destruction (autocrine / paracrine effect) Systemic levels follow in the later course of an inflammatory or traumatic process Therefore, it made sense to quantitate the cytokine response directly in the skin wound Despite vast literature data on cytokines in general, there are only a few comparable studies using this method The main problem is the establishment of an efficient extraction Cytokines act in minimal concentrations and are relatively unstable, so that it may be difficult to get quantities above the detection limit of ELISAs In addition, skin is an especially resistant material In our study, we used an intensive mechanical homogenization and an extraction in a solution of PBS and protease inhibitors By this way it was possible to obtain the pool of soluble cytokines within the dermal cells which is subject to rapid alterations after stimulation In order to estimate the relation between the soluble and the matrix associated cytokine pool, we firstly also used a re-extraction by guanidinium The results, however, showed that the soluble pool was much greater in most of the cases (relation: 0.6–5.8) and that the re-extraction had no additional advantage Slight variations in the extraction yield did not influence the final result, as the control skin of the same individual was measured in the same examination and the W Grellner et al / Forensic Science International 113 (2000) 251 – 264 quotients between wound levels and levels in control skin were used The absolute cytokine concentrations did not allow an evaluation of the wound age, as the interindividual variation was considerable It was useful to relate the concentrations to the skin weight, an additional consideration of the protein content like Kondo and Ohshima [20] had no advantage One revealing result of our study was the marked graduation of the cytokine reactivity between surgical and autopsy material These differences were obviously due to the considerably more severe traumatization in autoptic skin wounds (stab wounds of homicides) with additional manifestation of hemorrhagic shock By contrast, during operations, preservation of the tissue and avoidance of severe laceration is attempted In the surgical skin wounds, up to 11-fold increases (IL-6) could be observed (IL-1b: up to 3.5-fold; TNF-a: up to 8.5-fold) On the whole, the levels of proinflammatory cytokines increased with higher wound age In comparison with normal skin significant differences, however, were only found for IL-1b in the early post-traumatic interval (#30 min) and for TNF-a in the wound age class from to h On an average the levels doubled in the various wound age groups when compared to controls In skin wounds collected at autopsies, much higher levels were detected Frequently excessive increases were observed even in (stab) wounds with very short survival times The postmortem ‘‘key marker’’ IL-6 exhibited up to 81-fold increases (IL1b: up to 17-fold; TNF-a: up to 10-fold) All three parameters demonstrated statistically significant elevations of levels in the very early wound age group up to In the case of positive reactions the proinflammatory cytokines can be regarded as very useful vitality markers for stab and incised wounds This is supported by the fact that wounds with extremely short survival times did not disclose significant changes of cytokine content Thus, these markers are characterized by a good discriminating function between vital and supravital injuries (good specificity) On the other hand a negative result in the analysis of cytokines can not exclude vitality (poorer sensitivity) Former quantitative studies on cytokines were mainly conducted by means of blood / serum levels and cell cultures or animal experiments [21–34] To our knowledge extracts of human organs have only 261 been used for the determination of IL-8 (skin) [15] and stem cell factor (placenta) [14] Furthermore, Kondo and Ohshima [20] presented the single study under medicolegal aspects: in a murine animal model experimentally-incised wounds were examined quantitatively for IL-1a, IL-1b, IL-6 and TNF-a Their data from a controlled animal experiment with maximum average cytokine quotients of approximately resemble our results in surgical human wounds However, increases were observed much later than in our study and statistical significances were not mentioned Kondo and Ohshima observed maximum levels for IL-1b and TNFa not before h and for IL-6 even not before 12 h after wounding This is obviously not in concordance with the forensic reality, since massive increases can be detected in severely traumatized tissues of homicide victims even after very short survival times Thus, the local cytokine analysis at the wound site can be quite useful, at least as far as macroscopically ‘‘fresh’’ skin is available after postmortem intervals of up to approximately week There are various reasons for the rapid increase of proinflammatory factors In the first place local release from preformed stores comes into question It is known that in particular multipotent keratinocytes, mast cells, sweat glands and partly macrophages contain higher amounts of various cytokines (e.g [21,35–40]) Numerous mediators are stored there as inactive precursors or in the active form [37,41,42] The influence of the severe traumatization with finally fatal outcome must not be underestimated as well: it leads to a considerable activation of the cytokine cascade exceeding other stimuli in quantity and kinetic by far In massive trauma factors such as hemorrhagic shock with systemic elevation of proinflammatory cytokines, local ischemia and hypoxia become relevant, inducing the activation of cytokines [43,44] In addition, the transudation of the tissue with blood components (soluble cytokines in the serum, additional release from cellular elements such as monocytes) may contribute to the rapid quantitative increase of proinflammatory mediators as well However, the negative results of possibly supravital injuries point to the fact that intact circulation is required and that sole passive transudation (e.g., postmortem injury) is not sufficient It seems to be evident that all these factors lead to 262 W Grellner et al / Forensic Science International 113 (2000) 251 – 264 an earlier positive reaction in a quantitative biochemical cytokine analysis than in an immunohistochemical method where mainly cytokines bound on the cell surface are detected The time intervals for the earliest reactions as stated in this study are consequently markedly smaller than the comparable reaction times observed in immunohistochemistry (own data, publication in preparation) Our findings are in concordance with the results of the clinical and experimental literature on the role of proinflammatory factors in trauma and inflammation or sepsis The serum levels of TNF-a, IL-1a and IL-6 increase after operation and trauma [3] IL-1a, IL-1b, IL-6, TNF-a, TGF-a and TGF-b could be detected in wound fluid; monocytes and macrophages produce 10–20 times more IL-1b than IL-1a [45–49] The literature data mainly refer to measurements in body fluids (serum, plasma, wound fluid, cell culture supernatants) They clearly show up-regulation of the blood levels of various factors, especially of IL-6, following inflammatory or traumatic stimulation [21–34] The temporal occurrence of the various mediators is almost invariably protracted compared to our results (dimension of hours) It is revealing that also in the clinical literature a close relation between the severity of trauma or disease course and the cytokine concentrations is stated Similar to our study the most significant increases were described for the stimulator of the acute phase reaction, IL-6 [30–32] IL-6 can be regarded as a key mediator for trauma, injuries and operations [33,50–53] Systemic levels follow locally elevated levels in wound fluids [32] In sepsis, excessively elevated IL-6 levels were observed persisting partially for days [6–9,28,54– 57] On the other hand, a rapid down-regulation after stimulation is also well-known: in experimental animals in endotoxic shock TNF-a reached a maximum after 90 and fell back to basal levels after 210 [26,27] Generally, it must be considered that plenty of natural diseases may influence the (mainly systemic) levels of proinflammatory cytokines (see reviews in Refs [11,13,58]) The local determination of the mediators in the skin wound and, in particular, the relation of the results to normal skin of the same individual prevents theoretically possible falsifications Further influencing factors for systemic cytokine levels are as follows: genetic disposition [59], biological age [60,61], psychological stress [62] and pregnancy [63] Higher age and psychological stress are able to reduce the cytokine activation and production In our study we could not find a statistical dependence on age, but some observations in single cases point into this direction In any case, it is evident that numerous factors influence the complex cytokine network and explain the high variation However, the chosen methodological procedure and the type of evaluation with the formation of quotients between wound samples and control skin avoids some of the possible pitfalls and provides a good basis for a usable cytokine quantitation Considering the prerequisites mentioned, the quantitative analysis of proinflammatory cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular in the very early post-traumatic interval (classic stab wounds, vitality), but also in the later phases of the wound healing process Acknowledgements The experimental work presented in this paper was performed at the Institute of Forensic Medicine of the University of Cologne, Cologne, Germany It was supported by grants from the ‘‘Maria-Pesch¨ Stiftung’’ and the ‘‘Verein der Freunde und Forderer ¨ ¨ der Universitat zu Koln’’ The authors are very grateful to Prof Dr M Paulsson, director of the Institute of Biochemistry II, University of Cologne, for inestimable support in the development of biochemical techniques and advice in the selection of parameters for a useful evaluation References [1] N.T Bennett, G.S Schultz, Growth factors and wound healing: 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and IL- 6 increase after operation and trauma [3] IL- 1a, IL- 1b, IL- 6, TNF- a, TGF-a and TGF-b could be detected in wound fluid; monocytes and macrophages produce 10–20 times more IL- 1b than IL- 1a [45–49] The literature data... elevation of proinflammatory cytokines, local ischemia and hypoxia become relevant, inducing the activation of cytokines [43,44] In addition, the transudation of the tissue with blood components (soluble cytokines in the serum, additional release from cellular elements such as monocytes) may contribute to the rapid quantitative increase of proinflammatory mediators as well However, the negative results of. .. the surgical skin wounds, up to 11-fold increases (IL- 6) could be observed (IL- 1b: up to 3.5-fold; TNF- a: up to 8.5-fold) On the whole, the levels of proinflammatory cytokines increased with higher wound age In comparison with normal skin significant differences, however, were only found for IL- 1b in the early post-traumatic interval (#30 min) and for TNF- a in the wound age class from 1 to 2 h On an... Burleson, A Bruccoleri, Role of keratinocyte-derived cytokines in chemical toxicity, Toxicol Lett 82 / 83 (1995) 471–476 [23] I Tsuchiya, T Kasahara, K Yamashita, Y.C Ko, K Kanazawa, K Matsushima, N Mukaida, Induction of inflammatory cytokines in the pleural effusion of cancer patients after the administration of an immunomodulator, OK-432: role of IL- 8 for neutrophil infiltration, Cytokine 5 (1993)... methodological procedure and the type of evaluation with the formation of quotients between wound samples and control skin avoids some of the possible pitfalls and provides a good basis for a usable cytokine quantitation Considering the prerequisites mentioned, the quantitative analysis of proinflammatory cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular... endotoxic shock TNF- a reached a maximum after 90 min and fell back to basal levels after 210 min [ 26,2 7] Generally, it must be considered that plenty of natural diseases may influence the (mainly systemic) levels of proinflammatory cytokines (see reviews in Refs [11,13,58]) The local determination of the mediators in the skin wound and, in particular, the relation of the results to normal skin of the same... Brett, L May, D Stern, Induction of interleukin 6 (IL- 6) by hypoxia in vascular cells, J Biol Chem 270 (1995) 11463– 11471 [44] G.Z Feuerstein, T Liu, F.C Barone, Cytokines, inflammation, and brain injury: role of tumor necrosis factor-a, Cerebrovasc Brain Metab Rev 6 (1994) 341–360 [45] S Bailly, M Fay, B Ferrua, M.A Gougerot-Pocidalo, Comparative production of IL- 1b and IL- 1a by LPS-stimulated human... experiments [21–34] To our knowledge extracts of human organs have only 261 been used for the determination of IL- 8 (skin) [15] and stem cell factor (placenta) [14] Furthermore, Kondo and Ohshima [20] presented the single study under medicolegal aspects: in a murine animal model experimentally-incised wounds were examined quantitatively for IL- 1a, IL- 1b, IL- 6 and TNF- a Their data from a controlled animal... sepsis II: IL- 1 and IL- 6, in: G Schlag, H Redl (Eds.), Pathophysiology of Shock, Sepsis, and Organ Failure, Springer, Berlin, Heidelberg, 1993 [10] K Arai, F Lee, A Miyajima, S Miyatake, N Arai, T Yokota, Cytokines: coordinators of immune and inflammatory responses, Annu Rev Biochem 59 (1990) 783–836 ¨ [11] H Ibelgaufts, Lexikon Zytokine, Medikon, Munchen, 1992 [12] S.F Lowry, Cytokine mediators of immunity... supernatants) They clearly show up-regulation of the blood levels of various factors, especially of IL- 6, following inflammatory or traumatic stimulation [21–34] The temporal occurrence of the various mediators is almost invariably protracted compared to our results (dimension of hours) It is revealing that also in the clinical literature a close relation between the severity of trauma or disease course and the