Our data show that both types of thermal injury inflicted a clearly demarcated full-thickness burn. We also observed that the scald burn method as compared with the flame method resulted in increased mortality, hematocrit, systemic IL-6, subdermal temperatures ( and ), and lymphocyte depletion (). These data strongly suggest that the scald model produced a greater severity of injury. We suggest that the scald method transfers more thermal energy, and this results in an increased amount of tissue damage. This difference in the severity of injury alters the immune response as previously shown (17
). Altogether, we believe this is the first study to directly compare the injury severity and immunological response to two distinct methods of inflicting thermal injury.
On postburn day 1, the spleens from scalded mice had decreased mass as compared with spleens from sham- and flame-burned mice (). Additionally, splenic cell numbers were significantly decreased in the scalded mouse spleen as compared with sham and flamed mouse spleens. Further examination of purified T cells taken from scald-burn spleens showed that these T cells expressed increased activated caspase 3 as compared with T cells purified from sham-burned mice, demonstrating that these cells were undergoing apoptosis. Our data showed that lymphocytes were depleted after the scald burn as compared with the flame burn. We suggest that this depletion contributed to the immunosuppression after trauma in two distinct ways. First, the elimination of naive lymphocytes limits the adaptive immune system's ability to respond to antigens. Second, tens of millions of apoptotic cells appearing within 24 h likely results in immunosuppression as previously reported (18
). In this report, the presence of apoptotic cells was linked with a decrease in IFN-γ production after ex vivo
stimulation of splenic T cells. On postburn day 1, a similar reduction in IFN-γ production was observed in scald-burned splenic T cells, but not in the flame- or sham-burned splenic T cells. Thus, we suggest that the suppressed IFN-γ production was at least partly associated with apoptosis-driven lymphocyte depletion.
We observed that splenic T cells from scalded mice produced significantly less IFN-γ as compared with sham- and flame-burned mice 1 day after thermal injury (). Intracellular staining showed little differences in the percentage of cells producing IFN-γ from all three groups of mice. However, the intensity of IFN-γ expression () and T-bet expression () was decreased in the CD8 T cells from scalded mice. These decreases, coupled with the significant depletion of naive T cells (), is likely partly responsible for the decreased IFN-γ production. Interestingly, this is not the case for T cells isolated from flame-burned mice. After a scald burn, it has been reported that mitogen-activated protein kinases and nuclear factor–κB and activator protein 1 activity are altered in T cells (7
). Inhibition of these proteins is known to result in decreased IFN-γ production. Thus, the combination of decreased numbers, inhibition of key transduction molecules, and decreased T-bet expression are all likely playing a key role in the reduced IFN-γ accumulation from scalded mice. This is significant in that it has been shown that IFN-γ plays a pivotal role in combating bacterial infections (18
Previous studies have shown that after burn injury, tissue edema becomes evident (21
). The amount of tissue edema is a result of a combination of events to include the size and location of the burn, the type burn, and the amount of fluid resuscitation (23
). At 6, 24, and 48 h after the sham or burn injury, we isolated the lungs, kidneys, intestine, and heart. We then conducted wet and dry weight measurements on these tissues to determine edema. Although we observed increases in tissue edema in the tissues tested, we observed no significant differences between the two models or between the flame and scald model. We speculate that the lack of differences seen are due to differences in burn size, rodent used, anesthesia, and resuscitation amounts.
We and others have demonstrated that burn injury can cause an enhanced Toll-like receptor–mediated proinflammatory response by cells of the innate immune system (24
). Further studies have shown that treating C57BL/6 mice with 50 µg of staphylococcal enterotoxin B demonstrated lethality in burn-injured, but not sham-injured, C57BL/6 mice (26
). Here, we showed that there was a significant increase in splenic CD11b+
macrophages 8 days after both the flame and scald burn, whereas there were no significant differences found in the absolute number of CD11b+
macrophages (). Thus, after both types of burns, there was an increase in hyperinflammatory macrophages. This is significant in that upon treatment with LPS, the CD11b+
macrophages were large producers of the proinflammatory cytokine TNF-α (). It is likely that both flame- or scald-burned mice would have a robust proinflammatory response similar to LPS. Interestingly, it has been recently shown (27
) that when scalded mice were challenged with Escherichia coli
7 days after a scald burn, the burned mice were less susceptible to the infection. Furthermore, the report demonstrated more F4/80-expressing cells at the site of infection. We think that the presence of these hyperinflammatory F4/80-expressing macrophages is beneficial when clearing an infection.
On postburn day 8, T-cell numbers in blood, thymus, and spleen were returning to those seen in the sham-burned mice (). In addition, on postburn day 8, IFN-γ production and T-bet expression in T cells from flame-burned mice were similar to that found in sham-burned mice. However, IFN-γ accumulation was approximately 3-fold higher in the T-cell samples from scalded mice. The percentage of T cells producing IFN-γ was generally higher in these cells (). In contrast, the amount of IFN-γ was somewhat lower (). In general, our data suggest that compared with the other conventional T cells tested, a higher percentage of nonnaive CD8 cells produced IFN-γ. Furthermore, T-bet expression was higher in these cells ( and ). We found that in the T cells isolated from scald mice, there was a 6-fold higher number of nonnaive CD8 T cells as compared with sham- and flame-burned mice. We speculate that it is this increased number in nonnaive T cells that is responsible for the observed increase in IFN-γ accumulation.
In summary, our findings indicate that the scald burn induces a greater severity of injury as compared with the flame burn. Associated with this increased severity is an increase in systemic IL-6, mortality, hematocrit, lymphocyte depletion, and functionality. However, another finding is that 8 days after trauma in scald-burned mice, both the innate and adaptive arms of immunity are primed more toward a proinflammatory phenotype. It is likely that this will allow for a better bacterial clearance if infected. Altogether, these findings show that depending on the severity of injury and the timing, the response to an infection can be different. Furthermore, the characterization of both models here will allow for better interpretation of previously reported results and the design of future experiments.