key: cord-0005980-suviqy15
authors: Stegmaier, J. C.; Kirchhoff, C.; Bogner, V.; Matz, M.; Kanz, K.-G.; Mutschler, W.; Biberthaler, P.
title: Dynamics of neutrophilic NF-kB translocation in relation to IL-8 mRNA expression after major trauma
date: 2008-11-13
journal: Inflamm Res
DOI: 10.1007/s00011-008-7207-6
sha: c09da5b3dd2cd1b88b796ec73c7e3b6013d3ad98
doc_id: 5980
cord_uid: suviqy15

OBJECTIVE: Systemic inflammation subsequent to polytrauma is connected to neutrophil (PMN) dysregulation characterized by reduced NF-kB-translocation and cytokine expression. The dynamics of NF-kB-activation as well as its down-stream regulation of IL-8-expression in PMN following major trauma remain unclear. The aim of this pilot study was to analyse NF-kB nuclear translocation in relation to IL- 8-mRNA-expression in PMN after major trauma. PATIENTS AND METHODS: PMN were isolated from blood samples of 15 major trauma patients (New Injury Severity Score, NISS > 16) drawn within 90 min and subsequently 6, 12, 24, 48, 72 h after trauma. NF-kB-translocation was analysed by Electrophoretic Mobility Shift Assay, EMSA and quantified by densitometry [arbitrary units], IL-8-mRNA-expression by RT-PCR, [copies/50 ng RNA]. Additionally, NF-kB-translocation and IL-8-expression in PMN of healthy volunteers were analysed natively (-control) and after LPS stimulation (+control). RESULTS: NF-kB-translocation and IL-8-mRNA-expression was significantly increased in polytrauma patients (n=15; NISS: 34 ± 8 [mean ± SEM]) initially. In non-survivors, NFkB- translocation was significantly increased on admission and subsequently reduced within 6 h, while it increased in the survivors group. After 24 h, a second significant increase in NF-kB-activity and IL-8-expression was found in survivors that was subsequently reduced in both groups. CONCLUSION: This pilot study has shown that a concomitant initial increase in transcriptional NF-kB-activity and IL-8 mRNA expression was observed in the early posttraumatic period which preceded the down-regulation of the innate immune system.

Mortality after major trauma is associated with insufficiency of the innate immune system [36] . Early destabilisation of the innate immune system after trauma has been described as Systemic Inflammatory Response Syndrome, SIRS and can provoke a posttraumatic transition to Multiple Organ Dysfunction Syndrome, MODS which can be followed by complete decompensation of organ functions as Multiple Organ Failure, MOF [25] . In this context, the macrophage/ leukocyte-system was identified to play a pivotal role in the development of the early MODS [12, 23] . Patients suffering from blunt multiple injuries exhibit an increase of proinflammatory mediators in the early posttraumatic phase [8, 11] .

The synthesis and release of these inflammatory cytokines and chemokines represent only the terminal step of inflammatory pathways and is regulated by nuclear factors such as NF-kB [17] . NF-kB is an inducible cytoplasmatic transcriptional factor which is enabled to translocate into the nucleus upon activation and bind to promoter regions of proinflammtory genes such as IL-1, IL-6, IL-8 and TNF-α. In contrast, in posttraumatic immunoparalysis, the capacity of de novo cytokine release in immunocompetent cells as well as the amounts of circulating cells could be shown to be diminished after major trauma [25] . Within in vitro experiments, diminished apoptosis rate with constitutively increased I-kB phosphorylation could be demonstrated after traumatic challenge.

However, the role of NF-kB in PMN as well as its downstream regulation of IL-8 expression in major trauma patients remains unclear, so far. Therefore, the aim of this study was to investigate NF-kB nuclear translocation by electrophoretic mobility shift assay, EMSA, and the mRNA expression of IL-8 by RT-PCR in PMN obtained from major trauma patients in the very early posttraumatic period as well as to correlate these results to systemic parameters and to clinical outcome after trauma.

After approval by the local ethics committee [Munich, reference number 012/00] this prospective pilot study was conducted at our university level I trauma center including major trauma patients suffering from multiple injuries according to a new injury severity score, (NISS), [4, 31] of at least 16 points. The major cause for blunt multiple injuries were high velocity traffic accidents as well as falls from a height. Signed informed consent was obtained from all patients or in case of unconsciousness from a legal representative. Excluding criteria was isolated traumatic brain injury, anamnestic malignancies and autoimmune dysfunctions. After initial resuscitation and subsequent surgical interventions patients were admitted to the intensive care unit.

In order to establish positive and negative controls, two blood samples each were taken from eight healthy volunteers. One sample was processed natively without stimulation as described below which served as negative control. As positive control, from the second blood sample PMN were separated after incubation with 10 µl of a 100 ng/ml lipopolysaccharide, LPS-solution (Sigma-Aldrich, Munich, Germany) for 2 h for inflammatory stimulation and NF-kB activation [34] .

A total volume of 30 ml whole blood was drawn into ethylenediaminetetraacetic acid (EDTA) on admission within 90 minutes and 6 h, 12 h, 24 h, 48 h and 72 h after the traumatic event. 42.5 ml red blood cell lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM ETDA, pH 7.2) was added. After lysis time of 10 minutes the samples were centrifuged for 10 minutes at 450xg at 20 °C. Two additional lysis steps were performed with the cell pellet using 5 ml lysis buffer to achieve quantitative lysis of the red blood cells. Thereafter, cells (1×10 8 in 80 µl) were incubated with 200 µl CD15 antibody coated micromagnetic beads (Miltenyi Biotech, Auburn, CA, USA) for 15 min at 4 °C for magnetic cell sorting (MACS). After incubation the volume was adjusted to 15 ml using PBS/ EDTA buffer and cells were sedimented by a centrifugation step (450xg, 10 min, 20 °C). The cell pellet was resuspended in 500 µl PBS/EDTA buffer and added to a high gradient magnetic separation column using the magnetic separator MiniMACS system (Miltenyi Biotech, Auburn, CA, USA). The column was washed three times with PBS/EDTA buffer. After removing the column from the magnetic field cell elution was performed by addition of 1 ml PBS/EDTA buffer. To save patients material, the purity of the PMN preparation was controlled by Fluorescence Activated Cell Sorting, FACS analysis of samples from three healthy controls using anti-CD15 antibodies (Beckman Coulter EPICS.XL.MCL., Krefeld, Germany).

Nuclear protein was extracted as described before [2] . Briefly, 0.5x 10 8 PMN were lysed in ice-cold lysis buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, DTT, 1 mM phenylmethylsulfonyl fluoride, PMSF and 1 % protease inhibitor cocktail which contains per ml the following ingredients: 500 µg antipain, 500 µg aprotinin, 500 µg leupeptin, 50 µg pepstatin, 750 µg bestatin, 400 µg phosphoramidone, and 500 µg trypsin inhibitor (ROCHE, Mannheim, Germany). The cell suspension was incubated on ice for 10 minutes in order to induce PMN lysis, and centrifuged at 12000xg for 5 minutes (4 °C). From the supernatant the cytosolic fraction was care-fully extracted. Then the nuclear pellet was resuspended in ice cold lysis buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 1 % protease inhibitor cocktail, see above) and rocked vigorously for 20 minutes on a shaking platform. The nuclear protein fraction was isolated via centrifugation and total protein concentration was determined by Bradford assay (Coomassie Plus, Pierce, Rockford, IL, USA).

The nuclear protein fraction was subjected to electrophoretic mobility shift assay, EMSA, as described before [16] . Briefly, oligonucleotides were end-labeled using a double-stranded consensus sequence of NF-kB (sense: 5´-AGT TGA GGG GAC TTT CCC AGG C-3´, antisense: 3`-TCA ACT CCC CTG AAA GGG TCC G-5´, Promega, Madison, WI, USA) in the presence of [g-32 P]-ATP. The labeling reaction was catalyzed by T 4 -polynucleotide kinase and incubated in 10x kinase buffer at 37 °C for 30 minutes. Nuclear extracts (0.8 µg each) were incubated in binding buffer (20 mM HEPES, 1 mM EDTA, glycerol 40 %, 2 mM DTT, 0,5 mM PMSF, 10 mM MgCl, 5 mM EDTA, pH 7 500 mM NaCl, 100 mM TrisHCL, pH 7.5, poly(dI-dC), (ROCHE, Mannheim, Germany) for 15 minutes at room temperature. Thereafter, the radiolabeled nucleotide was added (approx. 50,000 cpm), and the mixture was again incubated for 15 minutes at room temperature. EMSA was performed in a 5 % acrylamide gel in 50 mM Tris-45 mM boric acid-0.5 mM EDTA, pH 8.4. For specific competition, a 50 fold excess of unlabeled, 'cold' oligonucleotides was added, as nonspecific competitor SP1 (sense: 5'-ATT CGA TGC GGG CGG GGC GAG C-3', antisense: 3' -TAA GCT AGC CCC GCC CCG CTC G-5', Promega Madison, WI, USA) was used. For supershift experiments, each sample was incubated with anti-p50 antibody (Santa Cruz, CA, USA). EMSA was performed for 2 h with 100V using 5 % polyacrylamid gels in 0.5 % TBE, [9] (Tris base, boric acid, EDTA), buffer. Gels were dried and exposed to X-ray film (Kodak BioMax MR) for up to 3 days at -80 °C to avoid artifacts. After development of the autoradiographs the intensity of the blackening according to radioactive labeled NF-kB-DNA complexes was quantified by densitometry.

IL-8 mRNA expression was analysed by quantitative RT-PCR as described before [22] . Total RNA preparation from lysed PMN was performed using RNeasy midi kit according to the suppliers instructions. Nucleic acids were digested by DNase (RNase-Free DNase Set; Qiagen). Concentration of isolated RNA was determined by spectophotometry (260nm; Biophotometer, Eppendorf, Hamburg, Germany). Extracted RNA from all samples was stored at -80 °C.

An aliquot of 8.2 µl standardized to 1 µg of RNA was reverse transcribed using avian myeloblastosis virus-reverse transcriptase (RT) and oligo(dT) as primer (First Strand cDNA synthesis kit; Roche, Mannheim, Germany). The obtained cDNA was diluted 1/25 with water and 10 µl were used for amplification. The quantitative analysis of target gene expression was performed on a LightCycler by real time-PCR using the LightCycler FastStart DNA SYBR Green I kit (Roche, Mannheim, Germany) according to the instructions of the supplier. RT-PCR was performed by denaturation and polymerase activation (95 °C, 10 min), amplification of RT-PCR products in a 45 cycle one-step PCR including denaturation (95 °C,10 sec)/annealing (68°-58 °C, -0.5 °C/cycle)/extension (72 °C, 16 sec) for each cycle. The results of negative control samples were set as baseline level. PCR results were standardized to this baseline level and given as [copies/50 ng RNA]. Specificity of the amplification products was verified by melting curve analysis combined with agarose gel electrophoresis. For calculation, data processing and documentation the LightCycler analysis software (Version 2.0) was used.

Dynamics of neutrophilic NF-kB translocation in relation to IL-8 mRNA expression after major trauma 549

Clinical data concerning age, gender, as well as the New Injury Severity Score, NISS, was recorded on admission. Vital parameters, such as blood pressure, BP, heart rate, HR, ventilation parameters, systemic concentration of C-reactive protein, CRP, procalcitonin, PCT, amounts of white blood cells, WBC, differential cell counts, therapeutic intensive care regime including catecholamines demand, amounts of transfused packed red blood cell units (PRBC-U), fresh frozen plasma (FFP) and platelets (PU) were recorded according to the detection time points during the initial 72 h after trauma.

For EMSA results, the densitometric data is given in [arbitrary units] representing the blackening area of the X-ray film, for RT-PCR, data is given as [copies/50 ng total RNA] as mean ± SEM. The Sigma Stat ® 3.0 software package (SPSS ® Inc., Chicago, USA) was employed for all statistical analysis. Statistical significance between groups was determined by independent two-tailed t-test, ANOVA on ranks and SNK-test. Clinical data according age, NISS, MOF, amounts of transfused red blood cell units and fresh frozen plasma units as well as EMSA results are given as mean ± SEM. P-values below 0.05 were considered significant. 

Multiple organ failure, MOF scores of the enrolled patients were recorded according to the sampling time points, respectively and were defined following the score by Goris in the modification of Lefering [15, 24] which scores 'O' for unaffected function of the vital organs such as lung, heart, kidney, liver and blood. One point is assigned to each organ system if the following functional parameters are presented by the patient: mechanical ventilation with a PEEP < 10 and FiO 2 < 0.4; blood pressure > 100 mmHg with low dose of vasoactive drugs, serum creatinine > 2mg/ dl; SGOT > 25U/l, bilirubin > 2mg/dl; leukocytes > 30,000/µl, platetes < 50,000/µl. The maximum of 2 points is scored if patients exhibit more severe organ dysfunction such as mechanical ventilation with a PEEP > 10 and FiO 2 > 0.4; blood pressure < 100 mmHg and/or high dose of vasoactive drugs, hemofiltration or hemodialysis; SGOT > 50U/l, bilirubin > 6mg/dl; leukocytes > 60,000 or < 2,500/µl. (18,700 ± 6,300) . In 76 % a significant left shift occurred between three and eight hours after trauma verified by differential blood count. At 24 h after trauma, 86 % of the surving but none of the deceased patients presented a left shift. After 12 h, CRP was elevated to 5.6 ± 1.5mg/ dl, PCT increased to 3.3 ± 0.8ng/ml. Analysis of WBC, CRP and PCT adduced no significant differences. In the higher MOF group, elastase was inconstantly increased to 151 ± 43 µg/l.

All patients required mechanical ventilation during at least 72 h. The heart rate on admission was 98 ± 25 and 96 ± 24 after 6 h. In ten patients, the mean arterial blood pressure was kept above 68 ± 21 mmHg by catecholamine therapy. The initial base excess [-4.3 ± 1.2] obtained from blood gas analysis provided no significant difference. Lactate concentration in survivors was significantly lower on admission (p = 0.02), after 12 h (p = 0.002) and 24 h (p = 0.002) as compared to non-survivors. In all trauma patients, creatinine kinase was increased to 1001 ± 256U/l. The MOF score was 3.8 ± 0.7 on admission, 4.0 ± 0.9 after 6 h, 3.9 ± 0.6 after 12 h, 4.1 ± 0.9 after 24 h, 3.6 ± 0.8 after 48 h and 3.6 ± 0.8 after 72 h. Amounts of transfused packed red blood cell units (PRBC-U; approx. volume 300 ml) and fresh frozen plasma units (FFP; approx. volume 250 ml) within 72 h after trauma as well as clinical outcome within 90 days are depicted in the Table 1 .

In control experiments, the cell isolation system using CD15 antibody coated micro magnetic beads enabled a PMN cell fractionation purity of 92 ± 3 % (n = 3, mean ± SEM) controlled by FACS. From a total number of 0.5x10 8 cells of each sample a variable total nuclear protein fraction ranging from 2 µg up to 8 µg was isolated.

NF-kB translocation increased significantly to 476 ± 148 [AU] after LPS-stimulation (positive control) (p = 0.028). Mean transcriptional NF-kB activity in non-survivors was elevated significantly on admission (378 ± 189 vs. 46 ± 21; p = 0.002) and (132 ± 66 vs. 107 ± 32) after 6 h as compared to surviving patients. After 12 h, the transcriptional level was diminished fast in both groups (4 ± 2 vs. 6 ± 2) to increase again in the survivors group at 24 h (65 ± 18 vs. 8 ± 5

[AU]; p = 0.004) and to be further on reduced in both groups (48h: 6 ± 2 vs. 2 ± 1; 72h: 2 ± 1 vs. 3 ± 1 [AU]) ( Figure 1 ). NF-kB nuclear translocation was reduced in patients with higher MOF scores during the whole analyzed time. After 6 h, patients who presented a MOF score over 4 presented significantly reduced NF-kB activity as compared to the lower MOF group (55 ± 18 vs. 134 ± 46 [AU]; p = 0.029). While NF-kB was reduced in both groups in 12 h, a second activity peak was present after 24 h in the lower MOF group.

Patients presenting a MOF score less than 4 points revealed increased neutrophilic NF-kB activity on admission and after 6 h (276 ± 104 vs. 53 ± 18 [AU]; p = 0.029) as compared to the higher MOF group (Figure 2 ).

Patients presenting low and moderate NISS values (NISS < 24; > 24< 34) presented relatively constant NF-kB activity for the first 6 h (Figure 3) . In all groups, NF-kB activity decreased at 12 h after trauma to increase again significantly in the lower NISS group after 24 h (NISS < 24: 107 ± 6 vs. NISS > 34: 14 ± 3 [AU]; p = 0.014). (Figure 6 ).

PMN play a pivotal role in the first response to injury. Additionally to superoxide and elastase synthesis [7, 8) , PMN are enabled to synthesize inflammatory mediators such as IL-8. PMN are characterized by a high turn-over which is especially regulated by apoptosis. Physiologically, PMN having fulfilled their function in the tissue, undergo apoptosis. Within the posttraumatic period a delayed apoptosis was demonstrated which can last up to 3 weeks after trauma [30] . NF-kB translocation was shown after various artificial stimulation such as hypoxia, LPS, TNFa and IL-6 [27, 32, 33] . While TNFa as well as IL-1ß were found elevated at the beginning of the posttraumatic inflammatory cascade, they both induce augmented secretion of IL-8 which leads to leukocyte recruitment and activation [18] . Cytokine synthesis is increased in the early posttraumatic period followed by hyporesponsiveness. Neutrophilic NF-kB induction with consecutive cytokine synthesis was shown after external stimulation [32] . NK-kB dependent inhibition of posttraumatic apoptosis was shown after in vitro stimulation by sera of major trauma patients [27] . Differential blood analysis provided a significant left shift in surviving patients. Such a left shift was not found in non-survivors later than 24 h post trauma. Surviving patients might exhibit lower apoptotic effects with consecutive stimulation of bone marrow towards increased granulopoesis [20] . Further more RelA was shown to play a key role in apoptotic regulation. Rel A deficient mice exhibited significantly increased apoptosis rate and embryonic lethality [20] . Apoptosis leads to diminished phagocytic capacity and synthesis of pro-inflammatory mediators while pro-inflammatory reaction followed by reduced apoptosis rate in PMN and consecutive phosphorylation of I-kB was demonstrated [29] . Following the transcriptional NF-kB signalling, IL-8 expression was increased on admission to be rapidly reduced. IL-8 expression was shown to be mainly induced by NF-kB [3, 14] . Over the whole analysed period, significantly increased neutrophilic IL-8 mRNA expression was found after major trauma as compared to healthy volunteers. The initial increase of IL-8 mRNA expression over all patients appeared stronger than the up-stream NF-kB translocation. This phenomenon might be due to initial posttraumatic priming effects as well as to alternative transcriptional pathways responsible for IL-8 induction such as AP1 [38] . Further more, NF-kB undergoes degradation within 90 minutes after trauma. Reduction of pro-inflammatory mediators release due to I-kB stabilization following major trauma has been previously described [40] . While post mortem analysis of plasmatic IL-8 concentration revealed significantly higher values in traumatically deceased patients [19] , significantly elevated plasma IL-8 level was recorded in respect of developing MODS after major trauma [13] . Following our results, IL-8 mRNA expression was significantly reduced in patients exhibiting highly affected organ functions (MOF > 4). In accordance, significant induction of IL-8 mRNA expression was found in surving patients as well as in patients with moderate MOF.

Increased neutrophilic ROS were investigated in septic patients as well as in septic shock due to higher apoptosis rate [25] . While elastase synthesis seems to be significantly influenced by trauma and hemorrhage (5, 35] , in our hands, systemic release of elastase was only inconstantly increased in the higher MOF group but not in non-survivors.

In vitro effects of IL-8 on PMN are increased chemotaxis, adherence and degranulation [39] . Within posttraumatic ARDS as well as within ischemia-reperfusion syndrome, IL- Dynamics of neutrophilic NF-kB translocation in relation to IL-8 mRNA expression after major trauma 553 8 was demonstrated to increase PMN migration in the tissue as well as to prolong inhibition of apoptosis by these tissue damaging effects [21] . No specific inhibitors of IL-8 have been identified up to now [26, 28] . IL-8 expression after elective surgery was shown to be constant [40] . Following our analysis, no change of cytokine expression could be investigated due to surgical interventions in the posttraumatic period In densitometric analysis of EMSA results, a significant increase of NF-kB translocation was found within the first 90 minutes after trauma and after 6 h. The second peak after 24 h fits well to the previously described biphasic immunological activation in monocytic cells after trauma [20] .

No NF-kB activity in monocytic cells was found by initial detection on day one after trauma [1] . NF-kB reduction was shown to be preceded by an initial significant posttraumatic increase as compared to healthy volunteers [6, 34] .

For validation of injury severity the NISS in the modification of Osler et al was used which reflects the sum of squares of the most severe traumatic lesions regardless of the anatomic region [4, 31] . In accordance to the valid paradigm [10] but in contrast to further investigation of I-kB in major trauma patients, significantly increased NF-kB activity as well as increased down-stream IL-8 expression was found in patients exhibiting a moderate injury severity (NISS < 24).

As including criteria a NISS over 16 points was chosen as a parameter of critical increased incidence for the development of posttraumatic MOF [37] . Within a retrospective analysis of 1,278 multiple trauma patients an 80 % incidence for SIRS development as well as for MODS (85 %) could be shown in correlation with high NISS.

In conclusion we investigated neutrophilic transcriptional activity of NF-kB in relation to IL-8 mRNA expression briefly after major trauma within this pilot study regarding the anatomical injury severity, the development of organ failure as well as the clinical outcome. Thereby, a concomitant initial increase of transcriptional activity and the IL-8 mRNA expression could be elucidated in the early posttraumatic period which precedes the down-regulation of the innate immune system. The demonstrated biphasic increase of the nuclear factor kB as well as of the down-stream IL-8 expression fits well to the previously described biphasic dynamic immune response to injury. We demonstrated a correlation of high NF-kB, IL-8 levels and increased mortality after trauma.

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Acknowledgements. The authors thank the physicians and nurses of the Intensive Care Unit and the Emergency Department (Department of Traumatology and Orthopedic Surgery, Ludwig-Maximilians-University Munich) for their permanent support. We thank Dieter Muehlbayer, Christine Bretz and Angelika Steinle (Institute of Clinical Chemistry and Laboratory Medicine, Ludwig-Maximilians University Munich) for logistic support and Claudia Geissler for her invaluable technical support.