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 se
Trang 1113 (2000) 251–264 www.elsevier.com / locate / forsciint
Quantitative analysis of proinflammatory cytokines
(IL-1b, IL-6, TNF-a) in human skin wounds
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 9 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 5 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
1 Introduction the close relationship between various cytokines and
their pathways Minute concentrations, rapid
biological processes such as inflammation, sepsis and They are produced by a variety of cell types such as
medi-ators IL-1b, IL-6 and TNF-a were determined
*Corresponding author Fax: 149-6841-166-314. quantitatively in extracts of human skin wounds and
0379-0738 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd All rights reserved.
P I I : S 0 3 7 9 - 0 7 3 8 ( 0 0 ) 0 0 2 1 8 - 8
Trang 2tested for their usefulness in the estimation of vitality vessels the above mentioned time interval of ‘‘up to
prob-able wound age In this group, there were also cases (railway suicides, emergency measurements in the
2 Material and methods agonal or early supravital period) with extremely
short survival times or possibly supravital injury
evalu-ated separately
Individuals with known immunodeficiencies or
up to 3 h (average: 73656 min) Four wound age
classes were formed: #30 min (N523), 31–60 min
19) Most of the skin samples originated from the
cooling conditions (#48C, in liquid nitrogen, on ice)
Trang 348C The resulting solution (soluble cytokine frac- P,0.05 were regarded as statistically significant.
and finally centrifuged again The resulting solution
contained the matrix associated cytokine fraction
3 Results
2.3 Determination of protein content
3.1 General remarks
In some samples the protein content (mg / ml) was
separately (Sections 3.2 and 3.3)
follows: the decisive variable was the content of
by quantitative ELISA analysis (kits and protocols of associated cytokines as extracted by guanidinium It
pg / ml The detection limits were 1 pg / ml for IL-1b, other correlations or quotients did not give useful
possible The results were statistically independent of
The partially performed determination of the
ed: pg of cytokine / mg of protein, pg of cytokine / g
Table 1
of tissue and pg of cytokine / mg of protein / g of
Cytokine levels in normal human skin
tissue
(vital, pg / g) (postmortem, pg / g)
PBS extracts in pg / ml were finally related to the wet
IL-6 205.16159.0 190.26384.3
independent of artificial changes Statistical
evalua-TNF-a 1470.863186.6 627.161503.6
tion was performed by the t-test using the quotients
a
Trang 4• autopsy material: 1.9560.60 mg / ml in skin demonstrated a considerably greater variability with
The quotients amounted to approximately 1.0 (i.e.,
and doubled in the following wound age classes
increase was not significant (Table 2)
3.2.1 Interleukin-1b
the TNF-a quotients elevated to 1.6161.20 already
Fig 1 Distribution of IL-1b quotients in human skin wounds (surgical material).
Trang 5Fig 2 IL-1b in human skin wounds (surgical material) Mean quotients and standard deviations.
values in normal skin in the medium wound age
disclosed the highest levels in the later wound age
excessive elevation of cytokine levels was frequently standard deviation statistical significance could not
by far IL-6 proved to be the ‘‘key marker’’
3.3.2 Interleukin-6
.24 h (Fig 7, Table 3) The rapid increase to an average IL-6 levels were detectable in the medium
Table 2
a
Proinflammatory cytokines: statistical significances for surgical samples
a
P-values of quotients, n.s.5not significant.
Trang 6Fig 3 Distribution of IL-6 quotients in human skin wounds (surgical material).
Fig 4 IL-6 in human skin wounds (surgical material) Mean quotients and standard deviations.
Trang 7Fig 5 Distribution of TNF-a quotients in human skin wounds (surgical material).
Fig 6 TNF-a in human skin wounds (surgical material) Mean quotients and standard deviations.
Trang 8Fig 7 IL-1b in human skin wounds (autopsy material) Mean quotients and standard deviations.
quotients (9.21611.42) in the wound age class from elevated in the earliest post-traumatic interval
significance However, statistically significant results absolute increases, but also by higher variations, so
levels similar to the control skin in the late wound age group (quotient: 1.0460.33)
Table 3
Proinflammatory cytokines: statistical significances for autopsy
a
samples
3.3.4 Final summary of postmortem investigations
Wound age #5 min 1–24 h 24 h
The quantitative analysis of proinflammatory
very early post-traumatic interval of less than 5 min
IL-6 ,0.05 32 n.s 5 ,0.05 10 (vitality) (Fig 10, Table 3) All three parameters
TNF-a ,0.01 29 n.s 6 n.s 9 IL-1b, IL-6 and TNF-a showed significant increases
a
P-values of quotients, n.s.5not significant. (P,0.001–0.05) in stab and incised wounds despite
Trang 9Fig 8 IL-6 in human skin wounds (autopsy material) Mean quotients and standard deviations.
Fig 9 TNF-a in human skin wounds (autopsy material) Mean quotients and standard deviations.
Trang 10Fig 10 Summary of proinflammatory cytokines in human skin wounds (autopsy material) Mean quotients and significances.
a: 1.5761.04) Statistically significant differences to so that it may be difficult to get quantities above the
The medium post-traumatic interval from 1 to 24 h especially resistant material
relation between the soluble and the matrix associ-ated cytokine pool, we firstly also used a re-ex-traction by guanidinium The results, however,
most of the cases (relation: 0.6–5.8) and that the
synthesized and / or released at the site of tissue variations in the extraction yield did not influence the destruction (autocrine / paracrine effect) Systemic final result, as the control skin of the same individual
Trang 11quotients between wound levels and levels in control been used for the determination of IL-8 (skin) [15]
an additional consideration of the protein content like were examined quantitatively for IL-1a, IL-1b, IL-6
Trang 12an earlier positive reaction in a quantitative bio- Further influencing factors for systemic cytokine
Our findings are in concordance with the results of this direction In any case, it is evident that numerous the clinical and experimental literature on the role of factors influence the complex cytokine network and
culture supernatants) They clearly show up-regula- vitality and wound age, in particular in the very early tion of the blood levels of various factors, especially post-traumatic interval (classic stab wounds, vitality),
various mediators is almost invariably protracted
compared to our results (dimension of hours)
It is revealing that also in the clinical literature a
Acknowledgements
close relation between the severity of trauma or
disease course and the cytokine concentrations is
The experimental work presented in this paper was stated Similar to our study the most significant
performed at the Institute of Forensic Medicine of increases were described for the stimulator of the
the University of Cologne, Cologne, Germany It acute phase reaction, IL-6 [30–32] IL-6 can be
was supported by grants from the ‘‘Maria-Pesch-regarded as a key mediator for trauma, injuries and
¨ Stiftung’’ and the ‘‘Verein der Freunde und Forderer operations [33,50–53] Systemic levels follow
der Universitat zu Koln’’ The authors are very
ly elevated levels in wound fluids [32]
grateful to Prof Dr M Paulsson, director of the
In sepsis, excessively elevated IL-6 levels were
Institute of Biochemistry II, University of Cologne, observed persisting partially for days [6–9,28,54–
for inestimable support in the development of bio-57] On the other hand, a rapid down-regulation after
chemical techniques and advice in the selection of stimulation is also well-known: in experimental
parameters for a useful evaluation
animals in endotoxic shock TNF-a reached a
maxi-mum after 90 min and fell back to basal levels after
210 min [26,27]
Generally, it must be considered that plenty of
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