multiple trauma is the third most abundant cause of death for all ages , following heart disease and cancer . various immunological alterations known as posttraumatic inflammatory responses depend on the individual injury pattern and the time elapsed after injury . during the posttraumatic inflammatory reaction il-6 has recently proven to be of outstanding clinical interest and can now be used as a prognostic clinical marker in sepsis . with respect to open wounds and fractures after multiple trauma , one could regard the overall infection rate as remarkably low . it seems that there are mechanisms in the immune system preventing trauma patients from infections . antimicrobial peptides ( amp ) belong to the host defense peptides ( hdp ) and are key elements of the innate immunity , providing the first barrier in many epithelial cells against invading microbes . although open fractures as well as wounds can give rise to local infections and sepsis , amp regulation after trauma has barely been examined at all . in previous investigations we verified that serum of multiply injured patients has a significantly higher antimicrobial capacity than serum of healthy donors . further observations supported the hypothesis that small hdp were responsible for the antimicrobial effect . as possible mediators we detected an induction of the human beta - defensins 2 and 3 ( hbd-2 and hbd-3 ) and the cathelicidin ll-37 in serum of multiply injured patients . although these hdp were significantly upregulated after trauma , the source of the high antimicrobial capacity of trauma serum could not be clarified completely . lysozyme is the first described host defense peptide   and operates synergistically with hbd-2 , hbd-3 , and ll-37 . it is a key component of the innate immune system and kills bacteria by catalytic hydrolysis of cell wall peptidoglycan , but it also exhibits catalysis - independent antimicrobial properties [ 8 , 9 ] . belonging to host defense peptides , it also controls host physiological functions such as inflammation , angiogenesis , and wound healing . additionally the evaluation of lysozyme blood levels has clinical significance in the diagnostic of chronic diseases like sarcoidosis , rheumatoid arthritis , or crohn 's disease . nevertheless host defense peptide expression , especially of lysozyme , has not been fully elucidated yet with regard to inflammatory reaction . in this study we analyzed serum of trauma patients for lysozyme induction with regard to the antimicrobial effect and investigated possible cellular sources . inclusion criteria were an injury severity score ( iss ) of 16 points or greater without the presence of severe traumatic brain injury ( though abbreviated injury scales for head injury were included in the iss ) and primary admission to our institution . ais for head injuries with a score of three or more were considered to be severe traumatic brain injury . exclusion criteria were penetrating trauma , severe traumatic brain injury , and patients not directly admitted to our institution following the traumatic event . all procedures were in concordance with the revised version of the declaration of helsinki ( 41st general meeting in 1989 in hong kong ) . first - degree relatives signed the informed consent for inclusion of the patient in the study , as the patients were intubated and anaesthetized . a standardized clinical examination was followed by a focused sonography of the abdomen and pelvis . all patients received a trauma ct - scan of head , chest , abdomen , pelvis , and spine . the results were analyzed by a radiologist and an attending trauma surgeon . at the time of admission to the intensive care unit ( icu ) , the clinical examination and the focused assessment including sonography were repeated . fractures were stabilized as soon as possible either by definitive internal fixation or by temporary external stabilization . a group of 6 healthy volunteers ( 3 women and 3 men ) at the age of 18 to 65 years were randomly selected for determination of the normal baseline values of all parameters evaluated . venous edta - blood ( 9  ml monovette , sarstedt , germany ) was collected daily at 7 a.m. from traumatized patients over a 14-day period for plasma preparation . the blood was centrifuged for 10  min at 2000  g at room temperature within 30 minutes . the lysozyme capture antibody and detection antibody were provided by nordic immunological lab , the netherlands . for real - time polymerase chain reaction ( pcr ) , rna was isolated from the cultured human hepatocytes ( hepg2 ) with the rneasy - total rna kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . rna purification of whole blood samples of multiply injured patients and healthy volunteers was performed with qiaamp rna blood mini kit ( qiagen , hilden , germany ) . total rna ( 1,000  ng ) was used for reverse transcription and subsequent real - time pcr with gene - specific primers . the gene of interest was detected using taqman probes : hs01548808_m1 ( applied biosystems , foster city , ca , usa ) . after skin incision a 50  2  mm piece of epidermis was cut for further examinations . all operations were performed at the departments for trauma surgery and visceral surgery at the university medical center of schleswig - holstein , kiel campus . venous blood samples of 9  ml were taken from healthy volunteers by the use of edta monovette . pmn were isolated from whole blood using polymorph - prep ( axis - shield , norway ) according to the manufacturer 's instructions . 5 ml of blood was carefully layered on 5  ml of polymorph - prep . after centrifuging at 500  g for 30  min at room temperature mononuclear cells and polymorphonuclear cells were separated and resuspended in 0.45% nacl . for experiments isolated cells were ingested in rpmi 1640-cell culture medium with 7% fetal bovine serum , 1% l - glutamine ( 2  mm ) , and 1% penicillin / streptomycin ( 5000  u / ml/5000  g / ml ) . pmn were applied on 6-well plates with 1.510 cells in each well and stimulated with 50  ng / ml interleukin ( il-6 ) ( tebu , offenbach , germany ) and sa ( supernatant 1  :  100 ; assembled as described by glser et al . ) at 37c for 24  h. concentration of lysozyme in supernatant was measured by elisa . venous blood samples of 9  ml were taken from healthy volunteers by the use of edta monovette . mononuclear cells located in the interphase after use of polymorph - prep were ingested in 30  ml iced phosphate buffer saline and washed 5 times with 30  ml phosphate buffer saline at 300  g . mononuclear cells were ingested in iced medium free of serum and counted in a counting chamber for adjustment of cell concentration of 10  cells / ml . meanwhile autologous serum gained from density gradient centrifugation was centrifuged , respectively , at 720  g and 1600  g . petri dishes were incubated with serum - free thrombocytes for 15  min . after washing the petri dishes with 5  ml phosphate buffer , saline fetal calf serum was added to mononuclear cells so that a concentration of 3% fetal calf serum in medium was generated . all the dishes were filled with 5  ml of cell suspension and incubated for 45  min under standard conditions ( 37c , 5% co2 , 21% o2 , and 98% humidity ) . dishes with remaining adherent cells were covered with 5  ml iced serum - free medium and incubated on ice for 1  h. after 1  h a sphering of monocytes caused by cold stimulus could be seen through a transmitted light microscope so that these cells could be rinsed with iced phosphate buffer saline by the use of a 1000  l pipette . monocytes were collected in a 10  ml tube and washed in iced phosphate buffer saline at 600  g . cells were counted by the use of a counting chamber and carefully examined for vitality by suspension in trypan blue . the experiments were performed with immortalized liver cells , hepg2 ( cell lines service , germany ) . 800,000 cells were filled into a 25  cm cell culture bottle with 2  ml of cell medium . cells were stimulated with 50  ng / ml il-6 and 1  :  100 supernatant of sa . for blocking experiments , cells were incubated with 10  g / ml tlr2 antibody ( ebioscience , san diego , usa ) for 1  h and stimulated afterwards . to investigate whether lysozyme is induced in serum after trauma , we analyzed sera of trauma patients ( iss > 16 ) over a period of 14 days and compared them to a healthy control group . maximum levels were measured on day 6 ( 6.5  2.0  g / ml ) . significant elevations of lysozyme concentrations could be demonstrated between day four and day seven in comparison to the healthy control group ( 3.15  0.1  g / ml ) . after detecting lysozyme in serum of multiply injured patients an upregulation of host defense peptides after infection has been shown before for skin , liver , and blood cells , especially for monocytes and polymorphonuclear leukocytes ( pmn ) . therefore we focus on these cell lines in the present study . as a possible source for serum levels , we analyzed leukocytes of whole blood drawn from multiply injured patients . corroborating the protein levels in serum , real - time pcr revealed a significant increase in lysozyme gene expression on day 7 ( 2.1  0.4 compared to control ( figure 2 ) ) . a great amount of hdp is stored in granules of pmn and makes up to 50% of their granule content . we tested pmn and monocytes as important cellular mediators of posttraumatic inflammatory serum response examining whether they express comparable concentrations of lysozyme ( figure 3(a ) ) . pmn contained a higher concentration of lysozyme ( 7.4  1.1  g / ml in 100,000 cells ) than monocytes ( 5.1  0.7  g / ml in 100,000 cells ) . il-6 is a well - known cytokine , which is upregulated after multiple trauma and correlates with the risk of multiple organ dysfunction syndrome ( mods ) after multiple trauma . major causes for hyperinflammation and systemic inflammatory response syndrome ( sirs ) which may lead to mods are invading staphylococcus aureus ( s. aureus ) in the context of extensive soft tissue damage . to examine whether lysozyme is induced by typical stimulants of posttraumatic inflammation , pmn were incubated with il-6 and s. aureus . both stimulants increased the concentrations of lysozyme significantly as shown in figure 3(b ) : il-6 ( 14.1  1.5  g / ml in 100,000 cells ) , s. aureus ( 14.1  1.9  g / ml in 100,000 cells ) . with regard to further sources of lysozyme expression , we analyzed samples of liver without severe injuries and compared them to epidermis , a well investigated source of lysozyme . in relation to the wet weight , the highest amount of intracellularly stored lysozyme was measured in pmn ( 7.4  1.1  g / mg wet weight ) . samples of liver contained similar amounts of lysozyme as epidermis samples ( 2.0  0.2  g / mg and 2.5  0.2  g / mg wet weight ) ( figure 4 ) . the high amounts of lysozyme in liver tissue led us to the idea to further analyze the induction . we cultivated immortalized hepatocytes ( hepg2 ) and stimulated them with il-6 and s. aureus ( figure 5(a ) ) . lysozyme expression was increased after stimulation with both stimulants but only stimulation with s. aureus ( 8.0  g / ml in 100,000 cells  0.3 ) revealed a significant increase compared to controls ( 4.1  g / ml in 100,000 cells  0.4 ) . as expected from protein assay , both enhance gene expression of lysozyme in hepg2 , but only s. aureus showed significant induction compared to control ( figure 5(b ) ) . to examine whether tlr2 plays a role in lysozyme expression , we stimulated hepg2 cells with s. aureus and in combination with the blocking antibody for tlr2 ( figure 6 ) . treatment of hepg2 cells with tlr2 blocking antibody alone did not lead to an alteration of lysozyme expression . stimulation of hepg2 cells with s. aureus induced an upregulation of lysozyme concentration ( 8.0  g/100  000 cells  1.4 ) . compared to stimulation of hepg2 cells with s. aureus alone , stimulation with s. aureus in combination with tlr2 blocking antibody showed a significant decrease of lysozyme concentration ( 5.9  g/100  000 cells  1.4 ) , suggesting a major role of tlr2 in the regulatory process . amp are effector molecules of the innate immunity and act as endogenous antibiotics by directly killing bacteria , virus , and fungi . due to rising numbers of multiresistant infections , research on these peptides has increased rapidly during the last decade . particularly multiply injured patients with a combination of wounds and open fractures are faced by a multiplicity of invading gram - positive and gram - negative bacteria . in previous investigations we showed that serum of trauma patients reveals an enhanced antimicrobial effect in comparison to healthy volunteers serum . in this study in contrast to hbd-2 and hbd-3 , lysozyme expression was upregulated up to 8 times compared to its minimal effective dose of antibacterial protein expression in serum ( 1  g / ml ) . additionally it is known to act synergistically with further amp like hbd-3 , which is also induced in trauma serum [ 5 , 7 ] . lysozyme cracks the polysaccharide chain of the bacteria wall and therefore hbd can infiltrate easily . to extend the comprehension of lysozyme regulation in patient 's serum , we investigated possible sources of lysozyme . particularly neutrophils play an important role in the defense of the injured tissue from the first 10 minutes until 3 days after trauma . inflammatory mediators like host defense peptides are induced all along [ 19 , 20 ] . severe injury generates activation of different inflammatory cells such as granulocytes , monocytes , and macrophages   whereas especially monocytes and neutrophils are well known for lysozyme expression . lysozyme is stored in granules of human neutrophils   that are activated immediately after trauma . in this study we were able to document the induction of lysozyme in leucocytes of multiple trauma patients with comparable progression to the serum concentration . lysozyme induction in different cell lines has been reported for s. aureus and numerous cytokines like il-6 , ifn- , and il-17 [ 16 , 25 , 26 ] . particularly il-6 plays a major role in posttraumatic inflammatory reaction ; therefore , we tested the key player for its effect on lysozyme expression in pmn . as shown before bacteria seem to be a strong inducer for lysozyme . with regard to open fracture , wounds , and deep tissue contusion , bacteria constitute a major cause for systemic infection after trauma . our in vitro data revealed that both il-6 and s. aureus induce lysozyme in pmn . relating to systemic inflammation and shock syndrome , liver constitutes a key figure in terms of the regulation of mediators . markers of acute - phase reactants are usually of hepatic origin , are activated by cytokines , and regulate innate defense after severe injury . interestingly we detected the same amounts of lysozyme in liver tissue and in skin , which is known for huge lysozyme expression . therefore we investigated hepatocytes for regulation of lysozyme and ascertained upregulation provoked by il-6 and gram - positive bacteria . in multiply injured patients kupffer cells remove pathogens which invaded in case of open wounds through phagocytosis . the role of lysozyme in killing bacteria in these cells has not fully been elucidated yet . additionally the liver resembles a central organ of cytokine activity ; particularly kupffer cells of the liver release a variety of cytokines . these cytokines might stimulate hepatocytes to express lysozyme . to get one step forward to comprehend the regulation of lysozyme as shown before , induction of amp is regulated by toll - like receptors [ 31 , 32 ] which are activated by cytokines and bacterial fragments [ 25 , 32 ] . these receptors are associated with the induction of nuclear factor - kappa b ( nf-b ) and the induction of antimicrobial peptides . nf-b was defined to bind to nf-b - like sequence of the lysozyme promoter and induce lysozyme expression in macrophages and pmn . therefore we focused on the lysozyme induction in hepatocytes in the present study and were able to document that the blockade of tlr2 in stimulated hepatocytes showed a downregulating effect on lysozyme expression . we assume that tlr2 is involved in lysozyme regulation not only in pmn , but also in hepatocytes . with this study we showed high antimicrobial activity and significant upregulation of lysozyme induced by trauma relevant mediators for the first time . with regard to the application of amp in context of systemic infection after multiple trauma , these results bring us one step closer to the comprehension of the immune system of multiply injured patients . considering the complexity of this issue , lysozyme will require further attention .
the antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity . in the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways . 
 expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by elisa . to investigate major sources of lysozyme 
 , its expression and regulation in serum samples , different blood cells , and tissue samples were analysed by elisa and real - time pcr . 
 neutrophils and hepatocytes were stimulated with cytokines and supernatant of staphylococcus aureus . 
 the present study demonstrates the induction and release of lysozyme in serum of multiple injured patients . 
 the highest lysozyme expression of all tested cells and tissues was detected in neutrophils . 
 stimulation with trauma - related factors such as interleukin-6 and s. aureus induced lysozyme expression . 
 liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils . 
 after stimulation with bacterial fragments , lysozyme expression of hepatocytes is upregulated significantly . 
 toll - like receptor 2 , a classic receptor of gram - positive bacterial protein , was detected as a possible target for lysozyme induction .