The development of a trauma-induced exaggerated inflammatory response is recognized as a part of the physiologic reaction after trauma and infection (1). An overwhelming systemic inflammation (systemic inflammatory response syndrome, SIRS) has been attributed to a state that predisposes to further posttraumatic complications (2).
Among the proinflammatory cytokines, IL-6 is regarded as one of the most important mediators of early response to trauma (3). IL-6 has been clearly shown to represent one of the best markers for outcome in patients with severe blunt trauma (4). Permanently increased plasma levels of IL-6 were associated with an adverse outcome after trauma, whereas significantly lower IL-6 plasma concentrations were seen in patients who survived complications in the posttraumatic course (4). Likewise, systemic IL-8 concentrations were reported to correlate with injury severity and were found to be an early predictor of survival after trauma (5).
Despite a similar injury severity and injury distribution, the intensity of inflammation varies between polytraumatized patients. Clinical and experimental studies have suggested that the inflammatory response is regulated at the genetic level (6-14). Certain cytokine gene polymorphisms, including those in loci such as TNF-α, IL-1β, and IL-6, have been associated with a predisposition to numbers of inflammatory diseases such as arthritis, pneumonia, and sepsis. Variable associations between cytokine production and polymorphisms have been reported. It was suggested that polymorphisms in regulatory regions may affect the transcriptional regulation of cytokines. A single nucleotide polymorphism (SNP) within the human IL-6 gene (G/C) was demonstrated to exhibit an enhanced spontaneous and inducible production of IL-6. Furthermore, a SNP in the TNF-α promotor was shown to increase TNF-α synthesis (6, 8-11).
In the present study it was hypothesized that genetic polymorphisms may also influence the systemic inflammatory response in the traumatic setting. Therefore, the distribution of TNF-α, IL-1β, IL-6, and IL-8 genotypes in patients with severe trauma were determined and it was investigated whether an association with the systemic inflammatory response and clinical outcome occurs.
PATIENTS AND METHODS
The study followed the guidelines of the revised UN declaration of Helsinki in 1975 and its latest amendment of 1996 (42. general meeting). After approval by the ethical committee of Hanover Medical School, informed consent was obtained from patients or their closest relatives for blood sampling and genotyping.
Between March 2001 and December 2002, patients sustaining blunt trauma were consecutively enrolled. Injuries of the various body regions were classified by the Abbreviated Injury Scale and the Injury Severity Score (ISS) was calculated (15).
Inclusion criteria included an initially estimated ISS ≥ 16, patients age of ≥16 years and ≤65 years, a survival of more than 24 h in the intensive care unit (ICU), and primary admittance to our institution within 1 h after injury.
Patients were excluded in case of steroid and nonsteroid anti-inflammatory medication, any hormone therapy, and vascular obstruction (such as cardiac coronary disease, renal dysfunction, and diabetes).
General therapeutic characteristics
In all trauma patients, resuscitation was started by an emergency physician once seen. After resuscitation, patients were admitted to the trauma center. At the time of admission, further stabilization of cardiovascular and pulmonary function was performed using mechanical ventilation, placement of a central venous line and an arterial line for monitoring, and i.v. infusion via peripheral venous lines. All patients had a standardized examination, including all x-rays, abdominal ultrasound, and cerebral CT scan, on admission to the hospital and surgical ICU. The trauma protocol foresees that major fractures are stabilized acutely by internal operations or by external fixation. Patients in a critical condition because of another associated injury may undergo laparotomy or craniotomy first and then be submitted to stabilization of their fractures. A patient who is highly unstable during this procedure may undergo external fixation parallel to the abdominal/cranial operation or may be transferred to the ICU, where an external fixateur is placed.
Clinical parameters and outcome evaluation
Clinical data, including demographics, source of injury, and operative interventions, as well as the time period of intensive care and mortality, were abstracted from the patients' chart review. Furthermore, laboratory, hemodynamic, and respiratory parameters were monitored for the length of stay in the ICU.
The Glasgow Coma Scale (GCS) was determined daily until a significant change in the GCS could be noted. Diagnosis of acute lung injury, and Adult Respiratory Distress Syndrome (ARDS) was made according to the criteria of the American-European Consensus Conference on ARDS (16). Sepsis was defined according to the American College of Chest Physicians and Society of Critical Care Medicine Consensus conference criteria (17).
SIRS was defined according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine consensus conference. The diagnosis of SIRS requires that two or more of the criteria are met (17). SIRS severity was classified as follows: no SIRS, meeting none or one of the SIRS criteria; SIRS 2, meeting two criteria; SIRS 3, meeting three criteria; and SIRS 4, meeting four criteria.
For analysis of allele frequencies and genotype distribution, trauma patients were separated in two groups according to the severity of posttraumatic SIRS. One group included patients with no SIRS and SIRS 2 (group −SIRS), and the other group comprised individuals with SIRS 3 and SIRS 4 at 2 consecutive days or at 3 days of the observation period (group +SIRS). Patients were assigned to the +SIRS group as soon as they fulfilled these criteria during the observation period, whereas patients were included in group −SIRS at the end of the observation period. This group distribution was chosen based on the results of a previous study showing the most significant results for patients with a SIRS score of more than 2 (18). Each group was post hoc divided in two subgroups for confirmation of possibly positive findings of subgroup 1 with subgroup 2.
DNA isolation and genotyping
Once blood for the isolation of DNA was withdrawn into EDTA tubes (1.6 mg EDTA/mL blood, Monovette; Sarstedt, Numbrecht, Germany), each patient's genomic DNA was extracted by using a commercially available DNA isolation kit (QiAmp blood kit; Qiagen, Hilden, Germany) according to the instruction of the manufacture.
Restriction length polymorphisms: IL-1β-Taq1, TNF-Nco1, IL-6-174G/C, and IL-8-251 A/T-
The regions containing the polymorphic sites were amplified by PCR. Enzymatic digestion of the products was done with specific restriction endonuclease. After digestion, samples were electrophoresed in agarose gel and stained with ethidium bromide. The agarose concentration was chosen as suggested by Bowtell and Sambrook (19). Bands were visualized under ultraviolet light and were photo documented. Specific conditions for genotyping of the different polymorphisms are described in Table 1.
Parametric data were first observed by analysis of variance and thereafter were subjected to Student t test. The level of statistical significance was considered at P < 0.05. Nonparametric data as genotype distribution or mortality were subjected to the chi-square test or Fisher's exact test. Data are presented graphically as mean ± SEM. Odds ratios were calculated using the SPSS package, version 11.5 (SPSS Inc., Chicago, IL).
Group distribution according to SIRS severity
Patient demographics and clinical outcome-
Of 97 patients, 56 were assigned to group +SIRS and 41 patients were included in group −SIRS. Mean ISS was comparable in both groups. The mortality rate in group +SIRS was 16.1% and in group −SIRS, it was 4.9% (P < 0.05; Table 2).
Allele frequencies and genotype distribution according to SIRS severity-
The allele and genotype distribution of the observed polymorphisms are presented in Tables 3 and 4. Comparison of allele and genotype distribution of the IL-6 promoter polymorphism (−174G/C) between +SIRS and −SIRS trauma patients showed significant differences (P < 0.001).
Association between cytokine genotypes and other posttraumatic complications-
In addition to the described significant association between SIRS severity and the IL-6-174G/C polymorphism, no other significant association between a posttraumatic complication (acute lung injury, ARDS, sepsis, or mortality) and a cytokine polymorphism was found.
Systemic inflammation is supposed to play the decisive role in the development of posttraumatic complications (20). Clinical and experimental studies have suggested that the intensity of the inflammatory response in severe illness of different genesis is regulated at the genetic level (6-14). Therefore, cytokine gene polymorphisms may be associated with susceptibility to adverse outcome. In the traumatic setting, studies mainly focus on the association between genetic polymorphisms and susceptibility to sepsis (8, 10). It remains unclear whether genetic polymorphisms are also associated with the severity of the posttraumatic systemic inflammation. This could be important for determining operative strategies such as damage control or early total care. It is known that this also has an impact on the posttraumatic inflammation (23). In this study, a significantly different distribution between the genotypes of the biallelic IL-6 promoter polymorphism (−174 G/C) was observed between groups +SIRS and −SIRS. In contrast, all other cytokine polymorphisms (TNF-Nco1, IL-1β-Taq1, and IL-8-251A/T) did not show a significantly different distribution.
The development of the systemic inflammatory response with liberation of proinflammatory cytokines is recognized as a part of the physiologic response to trauma. IL-6 was shown to be one of the most reliable markers among these cytokines used to identify polytraumatized patients at high risk for adverse outcome (3, 21, 22). Likewise, a correlation between early increased IL-6 plasma concentrations, high ISS, severity of posttraumatic SIRS, and severe posttraumatic metabolic dysfunctions is well established (22, 23). However, IL-6 was not predictive for septic complications after blunt multiple trauma (24), whereas in cases of sepsis, an association between IL-6 plasma concentrations and the severity of illness, but not with outcome, was found (25).
In addition to plasma concentrations of cytokines, a genetic high-risk marker (i.e., genotype) for developing posttraumatic complications may represent an important diagnostic tool. However, associations of different cytokine polymorphisms with the severity of posttraumatic SIRS, as well as systemic cytokine concentrations, have not been observed in a homogeneous group of multiple trauma patients. Previous studies have investigated the association between cytokine polymorphisms in diverse clinical settings (appendicitis, coronary revascularization, etc.) or heterogeneous populations (critically ill patients) (26-30). In these studies, a genetic basis for differences in the production of cytokines has been well established. Variations in the regulatory regions of the cytokine genes are associated with higher or lower cytokine production. Stimulation of human blood cultures with bacterial lipopolysaccharide (LPS) shows large interindividual variations in cytokine secretion, which has been demonstrated to have a genetic component of over 70% (6). Specific polymorphisms were shown to be associated with the synthesis of several cytokines. The TNF-Nco1 polymorphism and a polymorphism at position −308 within the TNF locus correlated with increased TNF-α plasma concentrations (8-10). Furthermore, similar associations were found for polymorphisms within the IL-1, IL-6, and IL-8 locus (26-30). Interestingly, it was also shown that polymorphism within the TNF (TNF-308G/A) and the IL-1 β (IL-1β-511C/T) gene were associated with systemic IL-6 release in critically ill patients (29).
The results of the present study correspond with the findings of clinical studies demonstrating that the G allele is associated with a greater risk for complications after appendicitis (perforation, gangrene, and necrosis) (30) and a higher mortality rate in patients with ARDS compared with patients with the C allele (31). Likewise, a significantly prolonged hospital stay has been observed in patients with the G allele after surgical coronary revascularization (32).
According to our data and the results of other studies, genotyping for the IL-6 -174G/C polymorphism did not allow an early identification of patients with sepsis (11, 18, 33). These conflicting results between posttraumatic SIRS and infectious complications might be explained by the fact that the pathogenetic relevance of IL-6 and the IL-6-174G/C polymorphism may be relevant for SIRS, but not for sepsis and pneumonia (11, 33). Accordingly, different other clinical studies previously demonstrated that IL-6 was not predictive of infectious complications in multiply injured patients, whereas a marked elevation of IL-6 concentrations was able to discriminate trauma patients who develop most severe SIRS in the clinical course (23, 24). A varying significance of polymorphisms for different posttraumatic complications was already shown for the TNF gene in trauma patients (8-10).
In one of our subgroups, the -251A allele was significantly associated with an enhanced systemic inflammation. This finding was not supported by the second subgroup. Therefore, we were not able to corroborate a possible relation between this polymorphism and the inflammatory response. Furthermore, a trend toward an association of the -251A allele and an increased incidence of posttraumatic ARDS was found, without reaching statistical significance (P = 0.08). As IL-8 was reported to have a central role for the activation of neutrophils with subsequent development of ARDS, and because of its predictive value regarding survival after trauma (5), this polymorphism might also be of major importance in the posttraumatic course of the polytraumatized patient. Further studies are warranted to clarify the role of this polymorphism in the development of ARDS or other posttraumatic complications.
The Taq-1 polymorphism of the IL-1β gene as well as the TNF-Nco1 polymorphism were not associated with the severity of SIRS or with the development of posttraumatic complications. These results are in line with different other studies showing that outcome after sepsis and inflammation was not associated with the IL-1β-Taq-1 and TNF-Nco1 polymorphisms (7, 34-36). However, results have been conflicting, as other in vitro and in vivo data has given evidence for a genetic influence on susceptibility to severity of SIRS and sepsis (9, 28, 37, 38).
Numerous studies have explored the genetic basis of inflammatory and infectious diseases (18, 39, 40). As described in our study in polytraumatized patients, the significance of genetic variations on disease severity, i.e., severity of SIRS, is also well established in sepsis and other inflammatory diseases (8, 10, 30).
However, discordant results with no functional effects of polymorphisms on the clinical course have also been observed in septic and critically ill patients (7, 11). Likewise, we did not find an association between cytokine polymorphisms and posttraumatic complications (sepsis, ARDS, and mortality). It has to be considered that all genes and their multiple polymorphisms encoding proteins involved in the posttraumatic inflammatory process may influence the genetic predisposition for adverse outcome after trauma and infection. In this context, an interaction of different IL-6 polymorphisms on outcome has already been demonstrated in critically ill patients (41). Furthermore, an influence with other risk factors (gender, age, underlying disease, etc.) and alterations in the transcriptional regulation of cytokine expression by trauma and critical illness (42, 43) have to be taken into account.
In this study, an association between the IL-6-174G/C polymorphism and the severity of posttraumatic SIRS has been identified that has not been described for trauma patients before. Thus, it may be assumed that genetic variations may determine the further clinical course of the polytraumatized patient. This polymorphism might be useful as an early marker for trauma patients at high risk for developing posttraumatic complications. Further studies are needed to investigate the mechanisms of enhanced risk for severe systemic inflammation after trauma and its association to the IL-6 promoter polymorphism (−174 G/C).
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