Treatment of patients with multiple injuries has a tremendous socioeconomic impact (1). Despite improvements in technology and treatment of critically ill patients, sepsis and multiple organ dysfunction syndrome (MODS) still remain the most common causes for late post-traumatic mortality (2, 3). Treatment of infectious complications and sepsis is also known to increase the length of hospital stay and increase the cost of trauma (4, 5). A number of studies have shown that cell-mediated and humoral immune functions are depressed after trauma (6, 7). This depression seems to be a compensatory anti-inflammatory response to prevent possible harmful hyperinflammation. In the last decade, more and more evidence has been accumulated for a sex dimorphism in host defense after trauma, hemorrhage, and sepsis in experimental animals. Furthermore, human studies have shown that males have increased risk of infection after major trauma (8). In contrast, after certain elective or emergency surgical procedures, female sex has been correlated with higher mortality (9). Thus, it remains unclear whether there are sex-dimorphic responses to trauma and sepsis in humans.
Whereas androgenic hormones seem to have an immunosuppressive effect leading to increased susceptibility to and higher mortality after sepsis, estrogen has been shown to have beneficial effects in different trauma models (10-12). In this regard, sex hormones seem to alter immune response by influencing the synthesis and release of cytokines (11, 13). Lower levels of proinflammatory cytokines prevent infiltration of immunocompetent cells, an important step in the development of organ dysfunction. As a therapeutic approach, estrogen administration in males prevents immunosuppression in various experimental animal models (14). Other studies have shown salutary effects of estrogen and flutamide on cardiac function, which are mediated via a direct effect on intrinsic cells (15, 16). Furthermore, salutary effects of estradiol and flutamide on immune cell functions after trauma have been reported (10-12).
Although the beneficial effects of female sex and younger age have been demonstrated in various animal models, it remains unclear if such findings are also evident in humans. The aim of this study therefore was to investigate the effect of sex and age on the development of sepsis and organ dysfunction as well as plasma cytokine levels in patients with multiple injuries.
MATERIALS AND METHODS
Inclusion and exclusion criteria
Polytraumatized patients between the ages of 16 and 65 years who were admitted to Hannover Medical School Level 1 trauma center between January 1997 and December 2001 were included in this study. Patients with an injury severity score (ISS) less than 16 points were excluded. In addition, patients with a history of steroid use, anti-inflammatory treatment, or hormone replacement therapy were excluded. Patients with malignancies or chronic diseases of the liver, kidneys, or lung were also excluded (Table 1).
Ethical approval and informed consent
The Ethical Committee of the Hannover Medical School, Hannover, Germany, approved the study. Informed consent was obtained from all patients (or their relatives) included in this study.
Clinical parameter and outcome evaluation
Patients were carefully examined at 7 am, and blood (10 mL) was collected for routine analysis and cytokine measurement (TNF-α, IL-1β, IL-6, IL-8, IL-10). Plasma cytokines were measured using a commercially available kit (Immulite System: Random Access Immunoassay Analyser; DPC-Biermann, Bad Nauheim, Germany) following the manufacturer's instructions. The results of clinical examination and blood chemistry were recorded up to 14 days after admission (Table 2).
Diagnosis of sepsis was made according to the criteria of the consensus conference of the American College of Chest Physicians and the Society of Critical Care Medicine (17) on at least 2 consecutive days (18). MODS was diagnosed using the score of Marshall et al. (19). This score has been shown to be the most reliable score for diagnosis of MODS (20). As previously described, a manifest MODS was considered when the score was greater than 12 points on 2 consecutive days or at least 3 days during the observed period (19).
In-patient hospital stay, ventilator days, and length of stay in ICU were recorded, as well as results of microbiological testing and transfusion requirement. The number and kind of operative procedures were documented.
Patient management and treatment
After admission, all patients received an arterial and a central venous line. A standardized clinical examination, a focused assessment with sonography for trauma, and at least chest and pelvic x-rays were performed. After diagnostics in the emergency room, a trauma scan (computed tomographic scan of head, cervical spine, chest, abdomen, and pelvis) was performed. Results were analyzed by a radiologist and an attending trauma surgeon. At the time of admission to the ICU, the clinical examination and the focused assessment with sonography for trauma were repeated.
At the Hannover Medical School Level 1 trauma center, fractures were stabilized as soon as possible either by definitive internal fixation or temporary external stabilization. If the patient required laparotomy or craniotomy, stabilization of fractures was performed afterward. In unstable patients, a combined operation was accomplished.
Depending on the fulfillment of the MODS criteria, patients were divided into 2 groups, those with and without MODS (+MODS, no MODS), to analyze the effect of organ dysfunction on clinical course. Investigating the potential salutary effects of estrogen, premenopausal (≤50 years) and postmenopausal (>50 years) females with an ISS >25 and ≤25 were compared with age-matched males.
Statistical analysis was performed using SigmaStat (SPSS, Chicago, Ill, USA). Results are expressed as mean ± standard error of the mean. Statistical significance was assumed where probability values of less than 0.05 were obtained. Comparison between groups was performed using one-way ANOVA followed by Student t test or the rank-sum test (Mann-Whitney U test).
A total of 143 patients (106 males and 37 females) were included in this study. Age and ISS were comparable in all analyzed subgroups. Additional characteristics are shown in Table 3.
Pattern and severity of injury
Pattern and severity of injury were comparable in all analyzed groups. Furthermore, injury severity and pattern described by the Abbreviated Injury Scale (AIS) and ISS showed no significant difference in all analyzed subgroups (Table 4).
MODS influence on clinical course
Patients in the +MODS group had higher values using the score previously described by Marshall et al. (19). Patients with MODS had an increased mortality during in-patient time compared with patients not fulfilling the MODS criteria. Furthermore, sepsis was more frequent in the +MODS group. Apparent organ dysfunction led to more days in the ICU and more ventilator days. Length of hospital stay was not changed by development of MODS in all patients (Table 3). After elimination of fatalities, length of hospital stay was 35.4 ± 1.5 days for patients who did not develop MODS and 47.3 ± 3.8 days in patients meeting MODS criteria (P > 0.05). During the first 24 h after admission, patients with MODS required more transfusions such as RBC and FFP (Table 3). However, the number of operative procedures was not affected by MODS.
Young females develop less frequent MODS
Male patients had more frequent MODS in the observed period (Table 5). In the subgroup analysis of females and males not older than 50 years with an ISS greater than 25 points, males developed more frequent MODS and sepsis, although age, severity, and pattern of injury were comparable (Tables 4 and 6). No differences regarding sex difference were detected in patients with age older than 50 years or ISS not exceeding 25 (data not shown).
MODS influence on plasma cytokines
Tumor necrosis factor α-
Although plasma levels of TNF-α were comparable on day 1 in patients with and without MODS, a steady increase was observed in the MODS group (Fig. 1).
During the observed period, MODS did not influence plasma levels of IL-1β (data not shown).
During the entire observation period, patients with MODS had higher plasma levels of IL-6 than patients without MODS. Differences were more distinct during the first week (Fig. 2).
Plasma levels of IL-8 were significantly higher in MODS patients than in patients who did not have MODS, with distinct difference at day 1 (Fig. 3).
MODS patients had increased plasma levels of IL-10 on days 1 to 3 and days 9 to 14 compared with patients without MODS (Fig. 4).
Sex and age affect plasma cytokine levels
In the subgroup analysis of females and males not older than 50 years with an ISS greater than 25 points, males showed higher plasma levels of IL-6 and IL-8 during the first 4 and 2 days, respectively (Figs. 5 and 6). Premenopausal females with major injuries showed lower plasma levels of IL-10 on the second and third days as well as on days 8 to 12 than in the age- and injury-matched males (Fig. 7).
The present study indicates that MODS was associated with an increased incidence of sepsis and mortality. Additionally, organ dysfunction influenced the clinical course including the required transfusions, time in the ICU, and ventilator days. Although severity of injury was not a predictor for MODS, males had more frequent MODS. In contrast, premenopausal females with an ISS greater than 25 had lower incidence of MODS and sepsis compared with age-matched males. In addition, we could not identify any differences in pattern or severity of injury between the 2 groups. Nonetheless, during the early post-traumatic course, premenopausal females with an ISS greater than 25 had decreased plasma levels of cytokines compared with age-matched males.
The fact that younger males had a higher incidence of MODS is consistent with the findings of another study (21). In the present and other studies, an increased incidence of sepsis or MODS was not observed in patients with lower ISS (22, 23). Other authors have shown that this sex-related effect is dependent on age and severity of injury (22, 24). We believe that the critical stage for the separation of the age groups should be 50 years, a time when most females would have undergone changes consistent with menopause. Based on this assumption, we analyzed subgroups with an ISS greater than 25 and an age not older than 50 years. Inasmuch as any significant differences were not observed in less injured patients, one can assume that less severe trauma does not induce an over-reaching immune response. Thus, the benefit of female sex seems to be apparent only after severe injuries with a distinct immune response.
Whereas MODS was associated with higher mortality, sex did not influence survival rate in this study. This is most likely due to the low number of patients included in this study. In fact, sex has not been identified as a predictor of mortality after sepsis, major infection, or thermal injury (8, 25, 26). However, contrary to our results, Wohltmann et al. showed that females younger than 50 years had a decreased mortality, especially after severe injuries (ISS >25) (22). Although males tend to sustain more penetrating injuries, these could not be identified as a risk factor in the study of Wohltmann et al. (22). However, in another study, penetrating injuries were shown to be associated with increased mortality in males younger than 50 years (27).
The higher incidence of sepsis seen in severely injured males not older than 50 years compared with matched females is in agreement with the findings of higher frequency of post-traumatic or postoperative infections (8, 23, 28, 29). As with MODS, this could be due to the immunodepressive effect of normal levels of 5α-dihydrotestosterone (30). A depression of immunocompetent cells such as natural killer cells, T cells, or B lymphocytes was detected in males until 5 days after abdominal surgery (31). In contrast, only a short insignificant depression in the function of these cells was seen in women (31). Among infections in severely ill patients, pneumonia is described to be the most common serious infection after severe trauma (8). Males have been reported to have higher incidence of pneumonia (32), whereas established diagnosis of pneumonia was associated with higher mortality only in females (29). In another study, female sex predicted increased mortality in critically ill surgical patients with documented infection (33).
Because patients with multiple injuries benefit from specific therapeutic concepts (e.g., damage control orthopedics), it is important to identify patients who are at risk for post-traumatic complications during the early clinical course (34, 35). Clinical evaluation and parameters (e.g., blood pressure, heart rate, blood pH) have not proven to be reliable prognosis markers (36). A detailed knowledge of post-traumatic immune response could lead to new diagnostic and therapeutic approaches for better management of severely injured patients.
The cytokines that were analyzed in the present study play a pivotal role in post-traumatic inflammation (37). The correlation between IL-6 and severity of injury has been demonstrated in different studies (38-40). Our present study indicates that IL-6 not only is a parameter for severity of trauma but also presents as a marker for a patient's potential risk of developing organ dysfunction. This suggestion is supported by the fact that patients with MODS had higher plasma levels of IL-6 than patients without organ dysfunction when the severity of injury was comparable. In another study, plasma IL-6 plasma levels predict outcome in septic patients (41). However, other investigators found no association between plasma levels of IL-6 and development of organ failure (42, 43). Cytokine levels are influenced by various events during the clinical course such as operative procedures and transfusions (40). However, both of these aspects were comparable in all analyzed groups. Nonetheless, experimental studies of sepsis have shown that higher IL-6 was associated with poor prognosis (44). The results of the present and other studies underline the importance of examining IL-6 during post-traumatic course. However, additional parameters besides IL-6 seem to be necessary for identifying patients at risk.
Studies have also correlated increased plasma levels of IL-8 with severity of injury and with development of complications during early post-traumatic course (45, 46). The present study not only confirmed those observations but also showed elevated levels during the entire observation period.
Besides the correlation with the severity of injury, patients with worse outcome presented higher levels of IL-10 during early post-traumatic course than patients with uncomplicated course (47, 48). A possible reason for this could be the immunosuppressive effects of this IL. It has been shown that plasma IL-10 levels increase after trauma (47, 48). The decrease in plasma IL-10 levels at day 4 was also seen in other studies and could be considered as a temporarily loss of anti-inflammatory immune response (40) at that time.
TNF-α and IL-1β play a central role during post-traumatic immune response (37). However, our data did not show any influence of MODS on plasma levels of these 2 cytokines, which could be due to the short half-life of both of these mediators (TNF-α, 12-16 min; IL-1β, 6 min) (49, 50).
It should be pointed out that with the exception of a single study (27), there is no other information concerning sex dimorphism and plasma cytokine levels during post-traumatic clinical course. The present study clearly showed lower cytokine levels in females during the early post-traumatic course, which were more distinct in the subgroup analysis of younger patients with an ISS greater than 25. However, the severity and pattern of injury were comparable. In a number of experimental studies, a strong correlation between the endocrine system and the post-traumatic immune response was shown (30). High plasma levels of testosterone and low levels of estrogen were associated with the depression of splenic T lymphocyte and splenic macrophage functions. Treatment with testosterone receptor antagonists or estrogen reversed those impaired functions in these cells (30). Thus, the difference in post-traumatic immune response could be due to sex-specific differences in plasma levels of sex hormones. This suggestion is supported by the fact that these differences were not seen in older male and female patients in whom the incidence of MODS and that of sepsis were not different. At an older age, females are in menopause, which is associated with a reduction in estrogen plasma levels. In addition, differences in plasma cytokine levels were predominantly apparent in premenopausal females.
It should be noted that the immunoprotection after experimental trauma was only detected in proestrus animals (51). Inasmuch as a menstrual history is not taken from patients with multiple injuries in our trauma center, this study does not contain information about menstrual state or the plasma sex steroid levels at the time of injury. Thus, although we do not know the hormonal status and we also do not know how many females were in the cycle with high estrogen versus those with lower estrogen, the fact remains that differences in immune response were clearly evident between males and females. Thus, sex difference was confirmed in MODS and sepsis, with a benefit in females that may be due to effects of estrogen on immune response. A sex-dimorphic response was also seen in plasma cytokine levels, which indicates the influence of estrogen on immune response by modulating synthesis and release of various proinflammatory and anti-inflammatory cytokines.
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