SUMMARY. Serum levels of interleukin- 16 (IL- 1ß), interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) were assayed in 31 bum patients and 12 controls. A study was made of the correlation between cytokine levels and time post-burn, burn area, and mortality. The early IL-1ß, IL-6 and TNF-a systemic responses following thermal injury decreased with time post-burn but did not reach control levels. With increased burn area a significant elevation of IL-6 and TNF-a, but not of IL-1ß, was detected in the systemic circulation. All non-surviving burn patients had detectable levels of IL-1ß, IL-6 and TNF-oc; these were significantly higher than those of surviving patients. The results suggest that IL-1ß, IL-6 and TNF-a may play a role in the pathogenesis of septic shock or multiple organ failure and are therefore to be considered bad clinical omens.

Infection is the primary or major contributory cause in 75% of deaths from thermal injury. Thermal injury induces a dose-related suppression of the immune response which correlates significantly with the survival of the patient? Cytokines modulate a number of immunological functions following thermal injury and may influence the resistance of burn patients to infection.
Interleukin-I (IL-1) displays multiple biological activities of which activation of T-lymphocytes is one of the first. IL-1 serves to increase the adherence of neutrophils and lymphocytes to the endothelial cells. This is due to an increase in adhesion molecules on both the neutrophils and the endothelial cells. IL-1 also has the ability to induce fever. In addition to elevating serum copper and depressing serum zinc and iron concentrations, IL-I also causes the synthesis of hepatic acute phase proteins and induces production of interleukin-6 (IL-6).11 IL-1 causes neutrophilia by inducing colony stimulating factors, and it has been implicated in the pathogenesis of various diseases; septic shock and sepsis syndrome,  rheumatoid arthritis,  inflammatory bowel disease, and leukaemia.
Several biological activities have been ascribed to IL6. It is thought to play a protective role in inflammation. This is done by stimulating the host defence mechanisms to limit the injury (synthesis of hepatic acute-phase proteins) and to augment the clearance of pathogens (increased immumoglobulin production by the induction of maturation of B-cells to plasma cells). IL-6 possesses the additional property of inhibiting TNF production in vitro and in vivo and of decreasiing acute inflammation. IL-6 induces the formation of multipotential hernatopoictic colonies. The injection of recombinant IL-6 can cause fever.
Tumour necrosis factor (TNF) performs numerous biological activities, many of which overlap with those of IL- 1. It can induce the synthesis of other cytokines, including IL-6 and IL-8 Endothelial cells may be altered by TNF to assume a procoagulant profile 26 and increase adherence for inflammatory cells. TNF plays a role in transplantations, lysis of tumour cells 21 cachexiall and septic shock. It also suppresses lipoprotein lipase activity and stimulates lipolysis.
The aim of this study was to evaluate serum cytokine concentrations (IL-Iß, IL-6 and TNF-a) in patients with thermal injury and their relationship with clinical outcome.

The patients
Over an eight-month period (October 1994 - May 1995) 31 burn patients (14 male and 17 female; mean age 26.3 years; mean burn size 44.2% TBSA) were evaluated in a prospective randomized fashion for serum IL-1ß, IL-6 and TNF. The patients, normotensive and haemodynamically stable after uneventful resuscitation, were divided into three groups according to the percentage TBSA burned: Group 1 (0-25%), Group 11 (26.50%) and Group III (51-100%). There were 19 survivors and 12 non-survivors. The characteristics of the patients are shown in Table I.

Collection and processing of specimens
Peripheral blood samples (5 cc) were collected from each patient on admission, in the second and third weeks post-bum and after complete healing, either spontaneously or after surgical excision and skin grafting. The blood was drawn between 5.00 and 6.00 a.m. into blood collection tubes. The specimens were centrifuged at 750 g for 20 minutes, and the serum was removed and stored in aliquots at -70 °C until assay. Serum IL-1ß concentrations were measured in 81 samples from all 31 patients. Serum IL-6 concentrations were measured in 75 samples from the 31 patients. Serum TFN-a concentrations were measured in 83 samples from 31 patients. Twelve serum samples from 12 healthy laboratory persons were used as controls.

Assay procedure
The cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The IL-1ß, IL-6 and TFN-a ELISA kits were obtained from Medgenix Diagnostics SA, B-6220 Fleurus, Belgium.

* Principle of the test
Medgenix IL-1ß, IL-6 or TFN-a is an enzyme-amplified sensitivity immunoassay (EASIA) performed on a microtitre plate. It is based on the oligoclonal system in which several monoclonal antibodies (Mabs) directed against distinct epitopes of IL-1ß, IL-6 or TFN-a are used. The use of several distinct Mabs avoids hyperspecificity and allows highly sensitive assays with extended standard range and short incubation time. The assay is performed directly on serum, plasma or culture media samples, without any treatment or extraction. Mabs I - the capture antibodies - are attached to the lower and inner surface of the plastic well. Standards or samples are added to the well. After incubation, washing removes the occasional excess of antigen. Mabs 2-HRP (horseradish peroxidase) labelled antibody is added. After an incubation period, to allow the formation of a sandwich, and washing, the microtitre plate is washed to remove unbound enzyme-labelled antibodies. Th~ revelation solution tetramethylbenzidine (TMB) - (H2 2) is added and incubated. The reaction is stopped with H2SO4 and the microtitre plate is read at the appropriate wavelength. ermined coloriich is proportiorations. A standard curve is plotted and the cytokine concentrations in the samples are determined by interpolati~n from the standard curve.

Statistical Design

Data were collected, tabulated and statistically analysed using chi-square for comparison of frequency of occurrence and Z-test for comparison percentages. The difference is considered significant if P <0.05.

Serum cytokines following thermal injury at presentation
Of the serum samples taken from b admission, 89.7%, 64.5% and 32.1% con amounts resnectivelv of IL-Iß IL-6 and burn patients on ained detectable TNF-a. The percentage of samples with detectable amounts of IL-1ß and IL-6 was significantly higher in the burn patients than in controls (P <0.001 and <0.01 respectively) (Table II).

Serum cytokines and time post-burn
Fig. 1 shows the percentage of patient samples containing detectable amounts of cytokines irl the first three weeks post-burn. Patient serum samples c ntained detectable levels of IL-1ß, decreasing from 89.7ck d ri h fi week post-burn to 68.2% during the sec 18.2% during the third week. This was stat cant (P <0.001). IL-6 was detectable in 64.5% serum samples during the first week post-b to 26.7 % during the second week and risir g the third week. This was statistically signifi TNF in patient samples during the first, u ng c e rst ond week and istically signifi4.5% of patient urn, decreasing to 30% during cant (P <0.05). week post-burn was 32.1%, decreasing to 27.8% during both the second and the third weeks. This was statistically non-significant (P >0.05).

The relationship between cytokine levels and the percentage of TBSA burned was examined (Table III). Fig. 2 shows that the percentage of patients with positive IL-6 and TNF-a levels increased with increasing burn size (P <0.001 and <0.05 respectively). There was no apparent relationship between bum size and positive IL-113 patient samples.

Table IV shows'that 83.3%, 36.8% and 17.6% of serum samples from the 191~surviving burn patients contained detectable amounts respectively of IL-1ß, IL-6 and TNF-a. Serum samples from the 12 non-surviving burn patients contained detectable amounts of IL-18 (100%), IL-6 (91.7%) and TNF-a (54.5%), which were significantly higher than in the surviving burn patients (P <0.05, <0.01 and <0.05 respectively).

The relationship between cytokine levels on admission and after complete healing of the burn patients, either spontaneously or after surgical excision and skin autografting, was examined (Table V). Percentages of detectable serum IL-1ß levels show a highly significant decrease from admission to healing, while the percentage of detectable IL-6 and TNF-a levels shows a non-significant decrease.

Interleukin-Iß, interleukin-6 and tumour necrosis factor-a are proinflammatory cytokines. Their role in thermal injury has been the objective of considerable researchl.Drost et al. reported that serum ILA activity is increased in burned rats compared with controls. Drost et al . showed increased plasma IL-1ß and IL-6 concentrations following thermal injury, while TNF-a was increased only in a subpopulation of patients .3 Our study found that acute thermal injury initiates an early IL-1ß, IL-6 and TNF-a systemic response. This may account, at least in part, for some of the physiological responses characteristically seen after injury, e.g. fever without infection, muscle wasting associated with negative nitrogen balance, and an acute phase response accompanied by attendant elevations in certain serum proteins and decreases in albumin and transferrin.
In this study, serum IL-1ß and IL-6 decreased in relation to time post-bum but did not reach the control level. This may coincide with the decrease in hypermetabolism, the return to normal hormone levels and the positive nitrogen balance occurring after the third week post-bum.
The initial elevation of cytokines observed in this study is consistent with the findings of Rodriguez et al., who reported that acute thermal injury initiates an early systemic, lung and skin response involving TNF, ILß and IL-8, which are generated locally and do not originate from the systemic cytokine pool. Kupper et al.11 also reported that the human bum wound is a primary source of IL-1 activity. On the basis of these findings, we would suggest that the initial elevation of cytokines in bum cases is of both systemic and local origin and that other events besides burn severity induce their production.
This study found a statistically significant positive correlation between bum size and the levels of both serum IL-6 and TNF-u.. IL-1ß did not show this correlation. This is in contrast with the results of Drost et al.,1 who observed a relation between increasing burn size and increased levels of IL-1ß levels, but not of IL-6 or TNF-a. Our findings are consistent with those of Schluter et al.,11 who reported an increased IL-6 production as a potential mediator of lethal sepsis after major thermal trauma. Marano et al." reported no correlation between TNF-(x and burn size.
However, in our study, we found a statistically significant correlation between mortality and serum cytokine IL-1ß, IL-6 and TNF-a. The highest incidence of mortality was in group 111 (100%), which presented a high percentage of TBSA burn, a high percentage of third-degree burn, inhalation injury, and a high incidence of septic episodes. Rodriguez et al.11 reported no association between mortality or local organ infection and TNF, IL-6 and IL-8. Hack et al.11 and Drost et al. found a relationship between IL-6 and mortality. Marano et al.11 reported a relationship between TNF-a and mortality. Several other reports correlated mortality with TNF, IL-6 and IL-8 systemic production. In the light of our study, we can postulate that serum cytokines play a role in multiple organ failure and septic shock. We therefore conclude that cytokine elevation may be a bad clinical omen.

RESUME. Après avoir essayé les niveaux sériques d'interleukine- 1 B (IL-1ß), d'interleukine-6 (IL-6) et du facteur de nécrose tumorale-a (TNF-a) dans 31 patients brûlés et 12 témoins, les auteurs ont corrélé les niveaux des cytokines avec le temps post-brûlure, l'extension de la brûlure, et la mortalité. A la suite des lésions thermales les réponses systémiques précoces de l'IL-1ß, IL-6 et TNF-a diminuaient en fonction du temps mais n'arrivaient pas aux niveaux des témoins. Avec une extension augmentée de la brûlure, des niveaux élevés l'IL-6 et de TNF-a, mais non d'IL-1ß, ont été observés dans la circulation systémique. Tous les patients non survécus présentaient des niveaux appréciables d'IL-1ß, d'IL-6 et de TNF-oc qui étaient supérieurs aux valeurs observées dans les patients survécus. Les auteurs concluent que l'IL- 1ß, l'IL-6 et le TNF-a peuvent jouer un rôle dans la pathogenèse du choc septique ou de l'insuffisance organique multiple et qu'il faut donc les considérer des présages cliniques néfastes.

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