CYTOKINE EXPRESSION PROFILE OVER TIME IN SEVERELY BURNED PEDIATRIC PATIENTS

Abstract

A severe burn leads to hypermetabolism and catabolism resulting in compromised function and structure of essential organs. The massive release of cytokines is implicated in this hypermetabolic response. The aim of the present study was to compare cytokine expression profiles from severely burned children without signs of infections or inhalation injury (n = 19) to the cytokine profiles from normal, noninfected, nonburned children (n = 14). The Bio-Plex suspension array system was used to measure the concentration of 17 cytokines. The expression of proinflammatory and anti-inflammatory cytokines was maximal during the first week after thermal injury. Significant increases were measured for 15 mediators during the first week after thermal injury: interleukin (IL) 1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p70, IL-13, IL-17, interferon γ, monocyte chemoattractant protein 1, macrophage inflammatory protein 1β, and granulocyte colony-stimulating factor (P < 0.05). Granulocyte-macrophage colony-stimulating factor was significantly increased during the second week after burn (P < 0.05). Within 5 weeks, the serum concentrations of most cytokines decreased, approaching normal levels. When compared with the cytokine levels measured in normal children, a total of 16 cytokines were significantly altered (P < 0.05). After severe burn, a specific cytokine expression profile is observed in patients without complications such as inhalation injury or sepsis. The cytokine concentrations decrease during 5 weeks after burn but remain elevated over nonburned values. Furthermore, the elevation in most serum cytokine levels during the first week after burn may indicate a potential window of opportunity for therapeutic intervention.

The systemic inflammatory response to insults such as burns trauma, surgery, critical illness, or infection encompasses the release of large quantities of proinflammatory cytokines such as interleukin (IL) 1β, IL-6, IL-8, or tumor necrosis factor (TNF) (^1-4). Anti-inflammatory cytokines, such as IL-2, IL-4, or IL-10, are then released in an attempt to counterregulate the effects of the proinflammatory cytokines (⁴). Perturbations of proinflammatory and anti-inflammatory cytokine expression result in altered immune function and protein metabolism, potentially leading to compromised structure and function of multiple organ systems (e.g., the immune system, liver, skin, or muscle) (^{3, 5-8}). In addition, hypermetabolism leads to futile protein utilization resulting in induction of a dynamic hypercatabolic state concurrent with altered cytokine expression (^{3, 8}). Because the changes in cytokine levels occur before the changes in metabolism, it may be possible to modulate the hypermetabolic response after burn by altering cytokine response. These findings delineate the importance of cytokines as proinflammatory mediators and indicate that modulation of cytokine expression may represent a potential therapeutic approach to improve hypermetabolism and catabolism. To determine the time of occurrence and duration of the therapeutic window in severely burned children, elucidation of the cytokine expression profile induced by thermal injury is necessary. As a time course analysis of concerted changes in cytokine expression has not been previously performed, the aim of the present study was to determine the cytokine expression profile in severely burned pediatric patients and compare the profile to normal unburned children, similar in age.

MATERIALS AND METHODS

Patients

Nineteen severely burned children were enrolled in this prospective study. Patients were included if they were 0 to 16 years of age, admitted within 7 days after injury to the Shriners Hospital for Children, Galveston, Tex, had burns covering more than 40% of total body surface area (TBSA) with a third-degree component of more than 24%, and required at least one surgical intervention for escharectomy and skin grafting. Patients were excluded if there was any sign of infection (defined as >10⁵ organisms present) or sepsis at admission or during hospital stay, presence of inhalation injury, or concomitant major injuries or complications throughout hospital stay. Patients were critically observed for the development of sepsis using criteria based on the guidelines of the Society of Critical Care Medicine (Table 1).

Table 1: Criteria for definition of burn sepsis

After admission, patients were treated according to the standard of burn care at our institute, including early excision and grafting of the burn wound and fluid and caloric resuscitation according to the Galveston formulas (⁷). Patients were fed enterally because of our findings that total parenteral nutrition is associated with higher mortality (⁹).

Normal children

Fourteen nonburned, noninfected, noninjured children, 2 to 15 years of age, from South America were used as the normal cohort. Blood was collected once from these subjects at the Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Serum cytokines

Blood was collected from the burn patients at the time of admission and then weekly for 5 weeks for serum cytokine analysis (Fig. 1). The number of total samples per week after burn was as follows: 8 (first half of week 1), 19 (second half of week 1), 10 (week 2), 9 (week 3), 14 (week 4), and 7 (week 5). Blood was drawn in a serum-separator collection tube and centrifuged for 10 min at 1320 rpm, the serum was removed and stored at −70°C until assayed. The Bio-Plex Human Cytokine 17-Plex panel was used with the Bio-Plex Suspension Array System (Bio-Rad, Hercules, Calif) to profile expression of 17 inflammatory mediators (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p70, IL-13, IL-17, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFN-γ], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1β [MIP-1β], and TNF). The assay was performed according to the manufacturer's instructions. Briefly, serum samples were thawed and then centrifuged at 4500 rpm for 3 min at 4°C. Serum samples were then incubated with microbeads labeled with specific antibodies to one of the aforementioned cytokines for 30 min. After a wash step, the beads were incubated with the detection antibody cocktail, each antibody specific to a single cytokine. After another wash step, the beads were incubated with streptavidin-phycoerythrin for 10 min and washed, and the concentrations of each cytokine were determined using the array reader.

Fig. 1: Timeline for obtaining serum samples from burn patients.

Ethics and statistics

The study was reviewed and approved by the institutional review board of the University of Texas-Medical Branch, Galveston, Tex. Before the study, each subject, parent, or child's legal guardian signed a written informed consent form. Approval was also obtained from the institutional review board of Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, for the collection and use of serum from nonburned children.

One-way analysis of variance with Bonferroni post hoc correction, one-way analysis of variance on ranks with Dunn test, and paired and unpaired Student t tests were used to compare differences in cytokine expressed. Data are expressed as percentages or means ± SEM, where appropriate. Significance was accepted at P value of less than 0.05.

RESULTS

The demographic data for the two cohorts are shown in Table 2. The nonburned normal children were similar for sex but were significantly younger when compared with the burned patients (Table 2; P < 0.05).

Table 2: Demographics of burned and nonburned groups

Over the 5-week study period, significant increases were measured in serum IL-6, IL-8, IL-1β, MCP-1, MIP-1β, IL-13, GM-CSF, IL-5, and IL-7 (Fig. 2, A-I; P < 0.05). All of these proinflammatory cytokines, with the exception of GM-CSF (Fig. 2I), were significantly increased during the first week after burn injury; levels of IL-6, IL-8, IL-2, IL-7, and GM-CSF were significantly elevated at specific intervals before the fifth week after burn as well. Thermal injury increased serum IL-6 and IL-8 concentrations 465-fold and 24-fold, respectively, and IL-1β, MCP-1, MIP-1β, IL-13, IL-5, and IL-7 concentrations 3- to 9-fold over normal values.

Fig. 2: A-I, Serum IL-6 (A), IL-8 (B), IL-1β (C), MCP-1 (D), MIP-1β (E), IL-13 (F), and IL-5 (G) were all significantly increased during the first week after burn. GM-CSF (H) was significantly increased 2 weeks after burn. *Significant difference between burn and nonburned normal, P < 0.05 by Dunn test. ^#Significant difference between burn and normal, P < 0.05 by analysis of variance. Data are presented as mean ± SEM.

Anti-inflammatory cytokines, as well as those that play a role in the immune response, such as IL-10, G-CSF, IL-17, IFN-γ, IL-12 p70, and IL-4, were also found to be significantly increased after burn when compared with the concentrations detected in nonburned normal children (Fig. 3, A-F; P < 0.05). Similarly to the proinflammatory cytokines, the anti-inflammatory mediators were significantly increased during the first week after burn. Only serum G-CSF and IL-12 p70 increased significantly at later time points (Fig. 3, B and E). Of the anti-inflammatory/immune responsive cytokines, IL-10 and IFN-γ were increased most significantly over normal values (77- and 35-fold, respectively). The other anti-inflammatory cytokines were increased when compared with expression in normal serum, but because the normal levels were "not detected," calculation of fold increase was not possible.

Fig. 3: A-G, Serum IL-10 (A), G-CSF (B), IL-17 (C), IFN-γ (D), IL-12 p70 (E), IL-4 (F), and IL-2 (G) were significantly increased during the first week after burn. G-CSF (B), IL-12 p70 (E), IL-4 (F), and IL-2 (G) were also significantly increased at other periods during the 5-week postburn recovery period when compared with nonburned normal. No significant differences were found beginning on the second week after burn for all other cytokines. *Significant difference between burn and nonburned normal, P < 0.05 by Dunn test. ^#Significant difference between burn and normal, P < 0.05 by analysis of variance. Data are presented as mean ± SEM.

Despite apparent increases in serum levels of TNF, these elevated levels were not statistically different from nonburned normal values (Fig. 4).

Fig. 4: Serum TNF concentrations were not significantly different between burned patients and nonburned normal children. Data are presented as mean ± SEM.

Cytokine expression profiles were generated for the normal and the weekly burn serum cytokine concentrations (Fig. 5).

Fig. 5: Heat map comparing normal and burn serum cytokine protein expression profiles. Values are log₁₀ (average cytokine concentration, pg/mL), with blue indicating lowest levels, yellow indicating highest levels, and black in the middle. Gray squares indicate that no expression was detected.

DISCUSSION

The systemic inflammatory response after a severe burn injury leads to hypermetabolism and thus to protein degradation and catabolism. Consequently, the structure and function of essential organs, such as the muscle, skin, heart, immune system, and liver, are compromised, contributing to multiple organ failure and mortality (^{10, 11}). Uncontrolled release of proinflammatory mediators exacerbates protein wasting and organ dysfunction (^{12, 17}). Breakdown of organ function can then lead to increased incidence of infection and sepsis, ultimately resulting in multiple organ failure and death. This circulus vitiosus is very difficult to break, and successful therapy has yet to be defined. To improve patient outcome, modulation of cytokine expression should be attempted(^{15, 16}). However, little is known about the duration or magnitude of the cytokine cascade after a severe thermal injury. The aim of the present study, therefore, was to measure cytokine expression in severely burned patients during a 5-week recovery period and compare it to the serum cytokine profile in normal, nonburned, noninfected children (Table 3). Our results indicate that proinflammatory cytokines, such as IL-6, IL-8, IL-1β, MCP-1, MIP-1β, IL-13, IL-2, IL-7, and GM-CSF, are significantly increased after a severe burn when compared with normal pediatric levels. Immunomodulatory cytokines, such as IL-10, G-CSF, IL-17, IFN-γ, IL-4, and IL-12 p70, are significantly elevated when compared with nonburned normal levels. No difference was detected for TNF.

Table 3: Serum Cytokine Levels for Normal, Nonburned, Noninfected Children

Although these cytokines are typically involved in angiogenesis, cell proliferation, apoptosis, and the recruitment and activation of immune cells, conclusions concerning the functional significance or biological meaning of these findings cannot be made; however, this was not the aim of the study. Although the elevated levels of the measured cytokines are extremely high, it is not known which mediators are induced directly by burn, which are induced as secondary mediators and which are merely markers of systemic inflammation. We rather suggest that these data represent the cytokine profile in severely burned children without inhalation injury, sepsis, infection, or other serious concomitant injuries. The concurrent elevation of the expression profiles of proinflammatory and anti-inflammatory mediators may indicate the severity of burn injury or the presence of concomitant conditions such as infections, sepsis, or inhalation injury. Severe injuries, infections, or inhalation injury lead to increased cytokine release, resulting in altered resistance to infections. The cytokine expression profile described here can be used to compare the cytokine profile to burned children with infections, sepsis, or inhalation injury, potentially allowing improved identification and treatment of these conditions. We further propose that it is not the expression of an individual cytokine that indicates clinical outcome, but rather the expression profile showing the balance of proinflammatory and anti-inflammatory cytokines that may be indicative of clinical outcome.

A study of serum cytokines from 275 normal children found that IL-8 and IFN-γ are rarely measured, TNF expression is age-dependent and is present in higher levels in children than in adults, and IL-6 is detected at low levels (¹⁸). Despite the weakness that our unburned normal group was significantly younger compared with the burned group by approximately 2 years, two factors make it unlikely that this age difference is clinically or biologically important. The serum cytokine concentrations of IL-8, TNF, IFN-γ, and IL-6 detected in our normal cohort fall within the reference range reported for children (¹⁸). In addition, the average age of our normal and burned groups does not encompass the ages typically associated with onset of puberty, and the majority of patients in both study populations were prepubertal.

Modulation of cytokine expression after burn is of great interest as these mediators may be therapeutic targets. After major thermal injury, IL-6 has been implicated in protein metabolism-both turnover and catabolism (³)-whereas TNF and IL-1 are important for muscle protein degradation (^{3, 8}). These three cytokines have been measured frequently after burn; IL-6 is elevated after burn (^{3, 13, 14, 19-21}) with the highest levels detected at the time of admission (^{3, 12, 13}). The findings regarding postburn TNF levels are not as clear-cut; several studies measuring serum TNF levels in burned adults did not demonstrate a temporal relationship (^{3, 13, 14}), whereas other studies found increased TNF after burn (^{12, 19}). In adult burn populations, IL-1β has been found to be highest at the time of admission (^{14, 19}). Multicytokine analysis via concurrent enzyme-linked immunosorbent assays revealed that, in addition to IL-6, IL-8, IL-10, and TNF are also increased at the time of admission and peaked 4 days after burn in patients who were admitted on the day of burn (¹²). Several other serum cytokine levels have been reported for burn patients. IL-12 underproduced in adult burn patients for the first 14 days after injury as compared with serum levels in nonburned adults (²²). IL-13 was not found to be different during 28 days after burn (²¹). IL-2 and IL-8 are elevated in burn patients (^{14, 21}). IL-10 increases immediately after burn (^{12, 14, 21}). IL-1 and IL-8 peaked at time of admission (¹⁴). IFN-γ, when detected, is found at low levels at the time of admission and increases 10 days after the first operation (¹⁴). Our study shows, for the first time, the temporal relationship of the expression pattern of 17 cytokines-including those mentioned above-after major thermal injury.

The interactions between proinflammatory and anti-inflammatory mediators are crucial. We found that IL-6 and IL-8 appear to be the main inflammatory cytokines that are up-regulated over a prolonged time. Surprisingly, IL-1β and TNF are not as important as previously thought. Anti-inflammatory cytokines are similar to proinflammatory cytokines significantly increased during the first week after burn, then decrease. Given the prolonged increase of IL-6 and IL-8, it may be that this imbalance causes immunocompromise and immunodysfunction. However, further studies with more patients have to be conducted to determine the exact role and interaction of the various cytokines.

Several aspects of these studies may account for seemingly contrary results in the literature. One important factor that may play a role is the measurement of one or a few cytokines that undergo major modulation after burn, such as IL-6 and IL-8. The large variability in the ages and burn sizes included in some of the studies may also confound the data as several studies encompassed patients between 12 and 77 years of age with 9% to 95% of TBSA burned. Cytokine expression is known to change with age (¹⁸) and burn size,(²¹) and only large burns with greater than 40% of TBSA induce the hypermetabolic response (²³). We have tried to limit the impact of confounding factors by comparing normal and burned cohorts with similar patient characteristics and demographic data. In addition, patients were only studied if they had greater than 40% of TBSA burned to monitor cytokine changes in patients who would become hypermetabolic.

An interesting finding of the present study is that nine of the cytokines (IL-1β, IL-4, IL-10, IL-13, IL-17, IFN-γ, G-CSF, MCP-1, and MIP-1β) were only significantly increased during the first week after burn and that the expression of these mediators decreased dramatically beginning on the second week after burn. In addition, G-CSF, IL-2, IL-7, GM-CSF, and IL-12 p70 were also expressed at increased levels 2 or 3 weeks after burn; these levels returned toward normal during the following weeks. IL-6 and IL-8 remain significantly elevated for 4 and 3 weeks, respectively. Taken together, these data indicate that a typical pediatric burn hospital course is characterized by a normalized cytokine profile beginning at 2 weeks after the burn, although wound closure has not occurred and the patient is still in the intensive care unit. We therefore suggest that the deviations from the cytokine profile presented here may indicate a pathological process, such as infection, sepsis, or a concomitant trauma. Cytokine profiling thus may have potential use as a diagnostic tool for monitoring the recovery of burned patients. As most increases in cytokine levels were only detected within the first week after burn, we suggest that initiation of treatment to attenuate inflammation, hypermetabolism, and cytokine expression during this time may have greater effectiveness.

The benefits of using multiplex bead technology to profile expression of multiple cytokines are myriad. Given the drastic alterations in cytokine expression after trauma or burns,(^{19, 24, 25}) the use of single cytokines may not be as insightful in other patient populations, as a single change, as opposed to a panel of changes, is more likely to be masked by the inflammatory response to burn. The ability to rapidly monitor and measure the changes in expression of multiple cytokines may prove to be more meaningful for diagnostic purposes than to measure a single cytokine. Although specific cytokines can be measured singly, the use of a multiplexed bead panel to profile cytokine expression will further expand the potential for early identification of patients at risk for development of infections and sepsis or immunocompromised patients.

The profiles presented here are representative of the cytokine changes attributed solely to the burn injury. None of the patients had an inhalation injury, sepsis, infection, or other concomitant injuries. Our inclusion criteria for the study only allowed burn injury with no other injury. In future studies, we would like to determine subclasses or different disease processes and compare their cytokine profiles. Our overall hypothesis is that the cytokine profile can be used to predict outcomes. Having said that, we believe that it is necessary to determine the cytokine profile of burned children with no concomitant injuries and complications to normal children with no underlying diseases or infections or trauma. The difference from these two groups can then be taken for further analysis. Future work will cover the changes in cytokine profiles associated with infection, sepsis, and inhalation injury.

In the present study, we showed that the expression of proinflammatory and anti-inflammatory cytokines was maximal during the first week after burn. By determining the cytokine expression pattern in burned children without any complications, we now have a tool to study whether differences in cytokine expression are associated with clinical outcome. Furthermore, we suggest that the elevation in serum cytokine levels during the first week after burn offers a potential window of opportunity for therapeutic intervention.""

ACKNOWLEDGMENTS

The authors thank Mary Kelly, Karen Henderson, and Amanda Sheaffer for technical assistance.