Keratinocyte growth factor, interleukins (1 beta, 6, 8, 10, 12), and tumor necrosis factor alpha in culture medium of dermal fibroblast of burned patients

PURPOSE
To evaluate the level of cytokines and keratinocyte growth factor (KGF) or Fibroblast Growth Factor 7 (FGF-7) in the culture medium of cultured human dermal fibroblasts from patients with large burn in comparison to small burn.


METHODS
Fibroblasts of 10 patients (four large burns, four small burns and two controls) were initiated by the enzymatic method using collagenase. Cytokines and KGF in the supernatant of the culture medium was measured by, respectively, flow cytometry using Cytometric Bead Array Human Inflammation kit (CBA, BD Biosciences, USA) and the enzyme immunoassay method using the Quantikine (r) Human KGF. The experiments were performed in triplicate.


RESULTS
The expression of IL-12 protein in patients with large burns showed a tendency to increase. IL- 6, IL- 10, and IL- 1beta were observed no difference. For IL - 8, TNF - alpha and KGF was observed a significant difference between the expression in large and small burned patient.


CONCLUSION
That IL-8, TNF-alpha and KGF showed higher expression in cultured fibroblasts of large burned patients.


Introduction
Burn is a major public health problem for the world.
Occur per year, more than 300,000 deaths caused by accidents with fire, without considering the scald burns, electrical burns, chemical burns, and others forms that do not have their statistics available on global levels. In addition, millions cases occur resulting in permanent disabilities and aesthetic problems that often cause social rejection and depressive disorders 1 .
Agay et al. 2 evaluated the interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 beta (IL-1beta) in the blood, lung, liver, and brain of rats subjected to full thickness burns on the dorsal region between 20 and 40% bovine serum albumin (BSA). They found no changes in plasma levels of IL-1beta and TNF -alpha in groups 20 and 40% TBSA 2 . IL-6 showed no detectable levels in tissues within the first five days.
In the lung, an increase was detected in the synthesis of IL-1beta in the first six hours after injury, regardless of the size of the burn and the presence of lung infection; this level remained high in the first five days, probably indicating that this cytokine may be involved in the pathophysiology of systemic inflammatory response syndrome (SIRS). Mikhal'chik et al. 3 compared the cytokine profile in plasma and wound exudates in children with severe burns during treatment surgical. The results indicate that during the acute phase treatment, the wound serves as a source of inflammatory cytokines 3 .
A variety of growth factors and cytokines can influence the production of keratinocyte growth factor (KGF) or Fibroblast Growth Factor 7 (FGF-7) 4 . When analyzed cytokines and growth factors that influence the production of KGF by fibroblasts, the results suggested that other growth factors such as PDGF -beta, IL-6 and TGF -alpha moderately stimulate KGF RNA levels.
The aim of the present study as to evaluate the level of KGF and IL-1beta, IL -6, IL -8, IL -10, IL -12, and TNF -alpha produced in the culture medium of dermal fibroblasts from patients with burns.

Methods
The project was approved by the Ethics Committee of Federal University of Sao Paulo (UNIFESP) (0842/11).
The present study design is experimental, in vitro, using donated tissues from burned patients. It is observational, analytic, controlled, and conduced in a single center. All the patients included in this study read and signed the Free and Clarified Consentment Term.
The patients recruited to this study were burn victims (

Inclusion, exclusion and non-inclusion criteria
Inclusion criteria for the study were patients of both genders, over 18 years old, who agreed to participate and signed a consent form, being hospitalized in Burns Unit, and requiring surgery. A criterion was added to the group with large burns: having deep partial thickness or full thickness burns affecting between 25% and 50% of total body area surface (TBSA) or which require partial skin graft in 10% TBSA. For the small burns group, the criterion added was that the affected TBSA should be 5% or less, for deep partial thickness or full thickness burns and the need of partial skin graft. To the control group was included the criterion of not having previous diseases, not smoking, and performing aesthetic surgery.
Patients who had previous skin diseases, such as psoriasis and similar, superficial skin lesions or illnesses that might interfere directly in the inflammatory process, as rheumatologic diseases in general were not included.
Exclusion criteria were contamination of the culture flasks, low proliferation rate without achieving confluence of 80% of the cells in the culture flasks, insufficient quantity of extracted RNA that prevents the evaluation of patient data or non-viability of the extracted material.

Surgery
The debridement of burned tissue with deep partial thickness or full thickness burns occurred four days after injury.
Normal skin around the lesion is removed because of the surgical procedure itself.

Fibroblasts culture
The

Statistical analysis
Was used for comparisons between patients with burn and control Student's t unpaired test and p values less than 0.05 was considered significant difference between patients and controls.  As for IL -1 beta, it was not observed difference between groups (p= 0.28) and the mean and standard deviation was 17.14 ± 0.93 and 15.85 ± 0.45 to large and small burn patients, respectively ( Figure 5).  Analyzing IL -6 was noted that there is no difference between large and small burn (p = 0.14) and the mean expression and standard deviation was, respectively, 17.47 ± 0.56 and 16.43 ± 0.26 (Figure 4).  TNF -alpha protein expression presented difference between large and small burn patients (p = 0.0054*). We observed a pattern of 17.13 ± 0.19 for large increase in patients with large burns comparing those who had small burn and mean and standard deviation and 16.16 ± 0.12 for patients with small burn (Figure 6).

KGF expression
Analyzing the concentrations of KGF, there is difference in protein expression to large compared to small burn (p = 0.0378 *). Comparing and interpolating the data between the three groups came to the following result seen in Figures 7 and 8.

Discussion
Studies have shown that the stimulatory effects of cytokines promote wound healing by promoting the recruitment and maturation of neutrophils, increase in collagen synthesis, proliferation of keratinocytes and fibroblasts, increased chemotaxis of neutrophils and monocytes, increased neutrophil degranulation, and expression of cell adhesion molecules [6][7][8][9] .
Although Singer and Clark 10 show that pro-inflammatory cytokines, particularly IL-1, IL -6 and TNF -alpha are increased during the inflammatory phase of healing, the present study showed no significant difference in the concentration of IL -6 between the groups of large and small burn, whose cells were obtained immediately following an injury suffered in the skin by burning phase, unlike a single healing process analyzed by the authors.
Rumalla and Borah 7 showed that the concentration of IL -10 was taken as detectable on the first day and may remain so until ten days after the trauma. In the present study the difference between large and small burn did not show small differences, although present in both groups as the aforementioned study.
This may suggest standard concentrations of IL-1, IL-6, IL-10, and IL-12 independent of the extent of injury increase. As for IL -8 significant changes was observed.
It is known that IL -12 is produced by activated macrophages and antigen presenting cells and induces differentiation of helper T lymphocytes and thereby of cellular immunity. It is considered a pro-inflammatory cytokine, together with TNF-alpha. The increased expression in body fluids is detected in the first twelve hours after injury, and is primarily   KGF showed a significant difference when compared between the two study groups. In the group of large burns the expression of this protein was in greater concentration than in the group of small burned.
Gauglitz et al. 12 conducted a study in which they compared the morphology and cytokine expression profile of keratinocytes in the skin following acute burn and unburned skin.
It was observed that skin allograft in the concentration of unburned keratinocytes was higher when compared with the proliferation of these substances on the edges of burn injuries.
In the present study we noted that the control group

Conclusion
The IL-8, TNF-alpha, and KGF showed higher expression in large burned patients.