While our previous studies indicated a role of IL-6 in exacerbating myocardial dysfunction [10
], current findings in different age groups are somewhat surprising, and possibly contradictory to our hypothesis: 1) Despite increased pro-inflammatory cytokine mRNA expression, 14 MO mice showed no dysfunction until they are injured; 2) IL-6 deficiency protects cardiac function in 2 MO mice but worsens it in 14 MO mice following B+S injury; 3) total body IL-6 deficiency is not necessarily associated with a low STAT-3 or p-STAT-3 level, and phosphorylation of STAT-3 takes place after B+S independent of the IL-6 gene; and 4) IL-6 deficiency is associated with increases in TNF-α and IL-1β mRNA but not protein production.
The fact that non-injured 14 MO mice exhibit normal cardiac function despite abnormal mRNA production of IL-6, TNF-α, and IL-1β may indicate existence of compensatory mechanisms to overcome potential detrimental effects of these cytokine transcription abnormalities at this age. In addition, the lack of differences in protein concentrations of these cytokines may indicate possible post-transcriptional regulation that equalizes protein levels despite differences in transcriptional activities. We have previously shown that even in transgenic mice over-producing myocardial IL-6 and increased basal inflammation, cardiac dysfunction did not exist as long as these mice were not injured [11
]. It appeared that baseline mRNA transcription abnormalities in 14 MO mice were not associated with cardiac dysfunction. Older mice may ultimately show age-related functional differences as demonstrated by Yang et al using the same method of functional analysis as our study [24
In contrast to the non-injured state, B+S injury evoked worsened cardiac dysfunction, and such dysfunction was more pronounced in the 14 MO mice than the 2 MO mice. This phenomenon may reflect an additive effect of injury-elicited cytokine activation and the existing altered gene expression in older mice. The functional difference is in accordance with the clinical finding that outcomes after burns are worse in persons older than 50 years of age [9
] despite overtly normal functionality without the injury in most people at this age.
The previously demonstrated beneficial effect of IL-6 deficiency on myocardial function in 2 MO mice [11
] was not observed in the 14 MO mice. In fact, IL-6 deficiency worsens cardiac dysfunction in older mice after B+S injury. This finding contrasts with the demonstration of attenuated acute phase reactions and improved survival in 18–20 MO mice after sepsis induced by endotoxin injection [25
]. While such a difference in outcome could be attributed to the different injury models and ages used, our data with elevated myocardial levels of p-STAT-3 in IL-6 KO mice after B+S injury suggest that certain functions of IL-6 may be shared by other substances, allowing the gp130-STAT-3 pathway to be activated in the absence of IL-6. The ability of STAT-3 to be phosphorylated and the increase in TNF-α and IL-1β mRNA production in IL-6 KO mice may serve as evidence of the functional redundancy both within and outside the IL-6 family of cytokines. It is possible that a compensatory mechanism exists in the absence of IL-6, due to the functional redundancy of IL-6 family of cytokines. Such a postulation is in agreement with the fact that IL-6 KO mice manifest no significant structural or functional limitations despite the role of IL-6 in a wide range of biological functions including tissue and organ growth.
Despite IL-6 deficiency, STAT-3 phosphorylation remained intact after B+S injury at either age. In addition, IL-6 deficiency was associated with different mRNA production patterns of pro-inflammatory cytokines at different ages: TNF-α and IL-1β mRNA levels were higher in 14 MO mice than 2 MO mice, and TNF-α mRNA level is higher in KO than WT mice at this age. The increase in TNF-α and IL-1β mRNA may reflect a pro-inflammatory change which may play a role in the poor cardiac function in 14 MO KO mice.
Currently, we have no satisfactory explanation for the discrepancy between cytokine mRNA and protein levels. As pointed out in a survey [26
], high throughput mRNA assays have different sensitivities and noise levels from the traditional protein assays, and protein measurements are subject to post-transcriptional regulation and varying half-lives of proteins. The Western blot technique may have obscured some potential differences in protein production. Other investigators have also highlighted the importance of post-transcriptional cytokine regulation [27
]. The present study design prevented us from taking serial blood samples or applying more sensitive protein assay measurements, such as ELISA of supernatant of cultured myocardial cells, making it possible for us to have “missed” certain significant windows of protein production.
In summary, burn plus sepsis injury causes marked cardiac dysfunction. The role of IL-6 after such an injury appears to be complex and varies with age. IL-6 deficiency is partially protective of cardiac function in 2 MO mice, but is harmful in 14 MO mice. Aging itself is associated with baseline changes in cytokine mRNA levels, and IL-6 deficiency further accentuates these changes. In addition, the STAT-3 activation pathway remains intact after the complex injury even in the absence of IL-6. Cardiac function after burn and sepsis injuries may represent a complex interplay of aging-related cytokine interactions both within and outside the IL-6 family.