INTERLEUKIN-6, CD4, AND CD8 SUBSETS OF T-LYMPHOCYTES IN A POPULATION OF EGYPTIAN BURN PATIENTS

Mabrouk A.,* Mabrouk R.R., Sabry M., Fedawy S.F.

* Plastic Surgery and Clinical Pathology Departments, Ain Shams University, Egypt

SUMMARY. The defects causing immunosuppression after burn injury are still very much under consideration. IL-6, CD3, and its subsets CD4 and CD8 were determined in 40 patients on the fourth day after burn injury. The results were compared with those of 20 normal healthy controls and correlated with total body surface area (TBSA), sepsis, mortality, and liver and kidney function tests. The results demonstrated I lie elevation of IL-6 compared with controls and its significant correlation with TBSA, mortality, sepsis, and liver function tests. CD3 and C D4 were significantly decreased compared with controls. CD3 was significantly correlated with T13SA, death, ALT, TP, alburnin, BUN, and creatinine. CD4 was significantly correlated with T13SA, AST, and alburnin. CD8 not significantly different from the control group and was correlated only with mortality, ALT, and BUN. It may thus be concluded that IL-6 and to some extent CD4 play an important role in the development of the immune response and clinical manifestations after burn injury.

Burn patients often experience a devastating inflammatory response to burn injury.' Extensive thermal trauma results in impaired immune function that is largely attributed to altered cytokine synthesis' and to numerical and functional changes in T lymphocytes?
Some reports have suggested the involvement of IL-6 in morbid conditions and burn patient mortality. Studies related to post-burn changes in T-lymphocyte subsets (CD4 and CD8) have been reported in an attempt to reveal their role in immunosuppression. A better understanding of how these agents behave during the post-burn period could lead to procedures capable of reducing post-burn morbidity and mortality.
The aim of this work was to determine the levels of IL6 and CD3 and its subsets, C134 and CD8, on day 4 postburn. The purpose was also to investigate the relationship between these parameters and percentages of total body surface area (TBSA) burned, mortality, sepsis, kidney function tests (including BUN and creatinine), and liver function tests (including total protein, alburnin, alanine aminotransferase [ALT], and aspartate ammotransferase [AST]).

This study was one of the first immunological studies of burn patients admitted to the Burn Unit at Ain Shams University in Cairo, Egypt, after its inauguration in 1995. It was performed with the informed consent of the patients or family members.
The study concerned 40 patients (24 female, 16 male) with a burned TBSA of at least 10% (range, 10-75%; mean 34 ± 16%). Ages ranged from 16 to 55 yr (mean, 25.7 ± 9.5 yr).
A group of 20 healthy individuals matched in age and sex served as control in this study. Eighteen (45%) of the patients died. Burn injury was associated with sepsis in fourteen (35%) out of the 40 patients.
The patients were divided into three groups according to percentage T13SA burned: group A 10-19%, n = 6, mean = 13 ± 2.7%); group B 20-39%, n = 18, mean = 25 ± 3.5%; group C 40% or more, n = 16, mean = 51 ± 11.2%). There was no significant age difference between these groups.
When three conditions (Tab. I) were met simultaneously, the patient was diagnosed as having sepsis.

Samples were collected on day 4 post-burn. Blood samples were collected by means of an endotoxin-free syringe. This blood was divided into two tubes. In the first set of tubes the blood was left to clot and then centrifuged at 3000 rpm for 1 min. The sera were separated. Renal and hepatic function tests were assayed immediately. The remaining sera were collected in aliquots and stored at -80 'C until they were assayed for IL-6. The second set of tubes consisted of lyophilized ethylene diamine tetfacetate (EDTA) 1.5 mg/ml as an anticoagulant. These samples were suitable for performing double dual colour flow cytometric immunophenotyping of CD4 and CD8 as well as the monoclonal determination of CD3. This was performed on mononuclear cells separated by Ficoll Hypaque density gradient centrifugation using a matched combination of rhodamine and fluorescin isothiocynate conjugated murine monoclonal antibodies for CD3 and by double colour fluorescence flow cytometry with anti-CD8 and anti-CD4. These monoclonal antibodies were obtained from Coulter Electronics. Negative controls (IgG) conjugated to appropriate fluorochrome isotype matches were used with each sample. These controls were used to set markers and to distinguish negative from positive fluorescence.
IL-6 was determined by enzyme-linked immunosorbent assay (ELISA) (Immunogenetics, Zurjnaarde, Belgium). The limit of determination of the assay was 8 pg/ml. The liver function tests and the renal enzyme were determined by Synchron CX5.

All results are summarized in Tables II-VI.
Table II presents a statistical comparison between patients and control groups. IL-6 was the only parameter that was significantly increased in the patient group compared with control. CD3, CD4, TP, and alburnin were significantly decreased in the patient group compared with control. There was no significant difference in CD8 between control and the patient group.

Table III shows a statistical comparison between surviving and deceased patients with regard to the main parameters studied. TBSA and IL-6 were significantly higher in patients who died than in those who survived. CD4 and CD3 were significantly lower in patients who died than in survivors. CD8 was lower in patients who died than in survivors, but without any significant difference.

Table IV gives a statistical comparison of groups A, B, and C with reference to the main parameters studied. IL-6 was significantly higher in group C than in groups B and A, but without any significant difference in group A compared with group B. CD3 and CD4 were significantly lower in group B compared with group A and in group C compared with group A but without significant differences between groups B and C. CD8 showed no significant differences in the three groups. The CD4/CD8 ratio was signifi~ cantly lower in groups B and C than in A, but without any significant difference between groups C and B.

Table V presents a statistical comparison between the septic and the nonseptic group with reference to the main parameters studied. TBSA and IL-6 were the only parameters that were significantly higher in the septic group than in the nonseptic group.

Table V1 provides a statistical correlation between different parameters. IL-6 was significantly correlated with TBSA, CD3, C134, C138, death, sepsis, liver enzymes, total proteins, and alburnin. CD3 was significantly correlated with all parameters and studied except sepsis and AST. CD4 was significantly correlated with IL-6, T13SA, CD3, liver enzymes, and alburnin. CD8 showed a significant correlation with CD3, death, ALT, and BUN. The CD4/CD8 ratio showed no significant correlation with any of the parameters except with AST and total proteins.

Thermal trauma results in impaired immune function. The defects causing immunosuppression are still very much under consideration. The immunological deficit has been attributed over years the to changes in T lymphocytes and their subsets.' Also, the increasing interest in the control of inflammatory reactions by cytokines may be of great importance in the near future.
The inflammatory response to the burn injury is always experienced within the first two weeks.' In our study, day 4 post-burn was chosen for specimen collection on the basis of reports by De Bandt" and Yamada et al.1 that IL-6 increased and reached its maximum concentrations on the third to fourth day post-burn. T-lymphocyte changes were also reported to be mainly affected within the first 48 h after the burn injury." In our study, IL-6 was significantly higher in the patient group compared with control, presenting a significant correlation with TBSA. The level of IL-6 was significantly higher in group C than in groups B and A.
In the study perfonned by Yamada et al. 6 levels of IL-6 at the first post-burn visit were detected in only 70% of the patients, increasing slightly with TBSA. However, over the whole course of the research, their results were consistent with ours. The results of our study were also consistent with other reports.',' The differences in the detected levels of IL-6 and its statistical differences compared with control are due to the timing of the sampling, since in Yamada's study' the undetected levels of IL-6 occurred during the first visit post-burn, gradually increasing during the whole course. It is thus concluded on the basis of our results that IL-6 reflects the severity of inflammation on day 4 post-burn.
In our study IL-6 was significantly higher in the group that later developed sepsis than in the nonseptic group. This result was consistent with the reports of Yamada et al .6 IL-6 in our study was not only an indication of sepsis but also acted as a sepsis prognosis factor because sepsis developed later, after sample collection and determination of IL-6. This was also the case when IL-6 was determined in surviving patients and in deceased patients, with a significant correlation to the death rate. These results are consistent with those reported by Ueyama et aL' and Yamada et al.1 IL-6 thus acts as a prognostic factor for mortality.
Although the liver functions (ALT, AST, TP, alburnin) and kidney functions (BUN, creatinine) studied were not significantly higher in the patient group than in controls, IL-6 was significantly correlated with these enzymes and may thus be related to the organ affection during the postburn course.
In the burn injury IL-6 is not only produced by T and B lymphocytes but is also by stimulated Kupffer cells, enterocytes, and macrophages.1 The stimulus for the production of IL-6 might be endotoxin and/or gram-negative bacteria." However it has been reported that there is no translocation of endotoxins or gram-negative bacteria into the blood stream in the early stages of burn injury." It has also been claimed that the stimulus for production might be the burn injury itself. IL-6 could also be produced at local sites of the burn, entering the blood and triggering the various inflammatory responses.
In burn injury the action of IL-6 includes the enhancement of the effect of IL- I and TNF, which in turn produce other inflammatory reactions in the post-burn injury period. It also stimulates the production of acute phase protein from the liver and acts on the hypothalamus, causing fever and other manifestations associated with the burn injury. It also stimulates the proliferation of B lymphocytes .
Clearly much work is needed to reveal any interrelationships among IL-6 and other elements of the post-burn inflammatory response. T-lymphocyte changes have been considered important cause of immunosuppression after burn injury. These changes are numerical and/or functional. T-lymphocyte defects are characterized by decreased responsiveness to infection.2 In our study CD3, CD4, and C138 percentages were analysed using flow cytometry. CD3 and CD4 were significantly lower in the patient group than in controls and were correlated with TBSA percentage, being lower in group C than in B and A. However, CD8 showed no significant difference between the control group and the patient group and did not correlate with TBSA.
The numerical changes of C133 and CD4 in our study were consistent with other results observed in the early post-burn period.
CD3 was reported to be decreased owing to the decrease of CD4.9, 10 In our study group, in contrast, CD8 showed no significant difference in the patient group compared with controls. This result contradicts other results which reported a decrease in CD8. This controversy can be explained if one considers that in a study performed by Rollof" C138 was affected about three weeks after the burn injury.
In our results, CD3 and its subsets CD4 and CD8 were not significantly lower in the septic group than in the nonseptic group. These results differed from those reported by Rioja et al.11 The differences in results could also be accounted for by the timing of specimen sampling since Rioja et al. determined the parameters on the 7th and 60th day, while in our study results were determined earlier, most probably at a time when the decrease in T lymphocytes and its subsets cannot be regarded as indicators or prognostic evidence of sepsis.
C133 and C134 were significantly lower in patients who died than in patients who survived. CD8 was not different in either group.
The CD4/CD8 ratio could provide valuable information as to prognosis and the establishment of early treatment in order to avoid likely future complications. 10 However, in our study, the CD4/CD8 ratio was not significantly correlated with any parameters except alburnin and AST. This can be explained by the nonaffection of the C138 subset. There is a significant negative correlation between CD3 and C134 on the one hand and IL-6 on the other. This might be explained by the activation and potentiation of IL-6 to IL-1 and TNF which act as alami cytokines in the post-burn injury period, stimulating Tlymphocyte changes.' On the other hand, IL-6 also stimulates glycogenesis" which in turn mediates T-lymphocyte changes.
The decrease of C134 and CD8 subsets in the postburn injury period is related to immunosuppressive factors. One of these factors is a burn toxin that was characterized by Schoenerberger" as a polymerized complex of cell membrane lipid proteins (lipid protein complex, LPQ. This has been shown to inhibit the proliferation of normal T lymphocytes in response to stimulation.
The decline in CD4 was also explained as being mediated by corticosteroids" on the basis of elevated levels of endogenous glucocorticoids found in the first 48h after burn injury. The reduction of C134 cells could also be a consequence of the programmed or selective death of these cells in the post-burn period (a process known as apoptosis) The T-lymphocyte decrease in the post-burn period has profound implications for patient susceptibility to infection A correct understanding of the causes of T-lymphocyte decrease in the post-burn period is important for the development of therapies using immunomodulatory molecules to reverse inflammatory reactions.
In conclusion, IL-6 and T-lymphocyte subsets are considered to be important parameters in immunoaffection and modulation of inflammation in the post-burn period. IL-6 can be defined as the main mediator and prognostic factor for mortality, sepsis, and organ affection in the early period of burn injury. A good understanding of these immunodefects is essential for the development of new therapeutic approaches in the post-burn injury period.

RESUME. Les défauts qui causent l'immunosuppression chez les patients brûlés sont toujours à l'étude. UIL-6, la CD3, et ses sousensembles CD4 et CD8 ont été étudiés dans 40 patients quatre jours après la brûlure. Les résultats ont été comparés avec les résultats obtenus chez des sujets témoins sains et corrélés avec la surface corporelle totale brûlée, la sepsis, la mortalité, et les tests de la fonction hépatique et rénale. Les résultats ont démontré les valeurs hautes de FIL-6 comparées avec les témoins et une corrélation significative avec la surface corporelle totale brûlée, la mortalité, la sepsis, et les tests de fonction hépatique. Les valeurs de la CD3 et la CD4 étaient corrélées en manière significative avec la surface corporelle brûlée, les décès, ALAT (alanine amino transférase), TP (taux de prothrombine), l'alburnine, l'azote uréique sanguin (BUN), et la créatinine. La CD4 était corrêlée en manière significative avec la surface corporelle brûlée, ALAT, ASAT (aspertate amino transférase), et l'alburnine. Par contre, la CD8 ne présentait pas aucune différence significative par rapport au groupe témoin et démontrait une corrélation seulement avec la mortalité, FALT, et le BUN. Les Auteurs concluent que l'IL-6, en maniete particulière, et la CD4, en manière plus limitée, jouent un rôle important dans le développement de la réaction immunitaire et des manifestations cliniques après la brûlure.

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