The biology of inflammatory cytokines and related products such as NO is highly complex 
, and especially so in very acute processes such as the response to T/HS 
. This complexity is not surprising. Inflammation may be considered as a communication network that must integrate various stimuli and orchestrate an appropriate set of responses to these stimuli, in a manner that depends on the nature of the initiating stimulus and host-related factors (i.e. gender, age, and genetics). The acute inflammatory response is integrated with various physiological systems, an interaction that in the setting of T/HS acts both as a means of relaying information about the nature of the insult and as an outcome (e.g. degree of decompensation). Due to this complexity, extreme care must be taken when attempting to use cytokines as diagnostic and prognostic markers of outcome following T/HS 
, especially so if cytokines are to be future therapeutic alternatives 
Our primary goal in the present study was to examine a central question relating to the role of cytokines in post-T/HS inflammation: is early, robust inflammation–whose hallmark is the elevation of inflammatory cytokines such as TNF-α–associated with benefit or detriment? More specifically, we sought to associate the early inflammatory response of swine to T/HS with 1) the need for resuscitation and 2) response to later resuscitation (survival). In parallel, we sought to determine if the phenomena observed in the setting of experimental T/HS also held true in human T/HS.
Our studies suggest that a robust, early TNF-α response is associated with survival in trauma victims, a finding supported by data in large experimental animals subjected to T/HS. Indeed, the trauma patient cohort examined, both survivors and non-survivors, exhibited an inverse correlation between TNF-α production and organ damage/dysfunction. Moreover, this inverse correlation was observed even within the survivor sub-group, suggesting that early TNF-α serve either to limit organ damage or to induce reparative processes. While elevated plasma levels of TNF-α have been found in both hemorrhagic shock patients 
and in experimental animal models 
, our results suggest that this early, alarm-phase cytokine may need to be recast as being beneficial when indicating a self-limiting form of inflammation that signals for healing of injury.
The concept that inflammation is beneficial post-trauma may, at first glance, appear to contradict a large body of literature that points to morbidity and mortality associated with elevated inflammatory cytokines post-T/HS. However, attempts at modulating the canonical early pro-inflammatory cytokine TNF-α in the setting of T/HS have had mixed results. Bemelmans et al.
found that administering anti-TNF-α antibodies to jaundiced mice subjected to surgical trauma was not associated with improvement in survival 
. Similarly, mortality of wild-type mice subjected to hemorrhagic shock was unaffected by pre-treatment with anti-TNF-α antibodies 
. In contrast, Zingarelli et al.
found that ant-TNF-α antibodies improved survival in an extremely severe paradigm of hemorrhagic shock in rats (death by 30 minutes post-hemorrhage) 
. Various studies suggested improvements in histological parameters following treatment with anti-TNF-α in the settings of T/HS, but did not document effects on survival. For example, Marzi et al.
found that anti-TNF-α antibodies attenuated leukocyte adhesion in the livers of rats subjected to HS 
, and Abraham et al.
found evidence of reduced lung inflammation 
Indeed, a closer look suggests that the primary elevated inflammatory cytokine is IL-6, which we have suggested through computational studies may be indicative of a positive feedback loop of inflammation→tissue damage/dysfunction→inflammation 
Interleukin-6 is arguably the best biomarker of outcome of trauma patients with Systemic Inflammatory Response Syndrome, sepsis, and Multiple Organ Failure 
. Though we observed a weak, positive correlation between circulating IL-6 and Marshall Score, we did not observe any significantly elevated levels of IL-6 in either human or porcine T/HS. Ayala et al.
found that IL-6 increased continuously post-hemorrhage and was already increased after midline laparotomy and before initiation of hemorrhage compared with non-manipulated animals, while TNF-α was only detected once hemorrhage was initiated 
. These studies suggested that soft tissue trauma might be a potent stimulus to the production of IL-6 
. Our experiments comparing hemorrhaged animals to their surgery-only controls support these studies, but suggest that there is a threshold for overall injury that must be exceeded before TNF-α elevations are observed in the circulation.
An evolving literature points to a central role for the release of “alarm/danger” signals (also known as “Damage-Associated Molecular Pattern” molecules) which may damage tissues and cause the dysfunction of organs, and re-induce the release of TNF-α in a vicious cycle 
. In the present study, we found a positive association between IL-6 and organ dysfunction in trauma patients, as well as elevated plasma IL-6 levels after 60 min of hemorrhage in swine, consistent with this notion. The levels of IL-6 have been repeatedly reported to be elevated in both animal and clinical studies of T/HS 
. We note that in our relatively short-term animal study, there was no difference in IL-6 levels between survivors and non-survivors; additionally, the relationship between early IL-6 levels and late complications after trauma and hemorrhage was not studied. Our results therefore suggest that, as in contrast to sepsis 
, early elevations of IL-6 may play a prominent role in the response to T/HS. Later elevations in IL-6 are also associated with morbidity 
, and thus persistent elevations in IL-6 may be indicative of self-sustaining, tissue-damaging inflammation.
2 cytokines, central among them IL-10, are thought to contribute to immunosuppression and the development of sepsis 
. IL-10, which is characterized as an anti-inflammatory cytokine 
was assessed in T/HS patients 
and found to be a potent down-regulator of cell-mediated immune and pro-inflammatory responses 
. Experimental studies have demonstrated that IL-10 inhibits the production of pro-inflammatory cytokines, such as TNF-α and IL-6, by activated macrophages 
. Moreover, it has been demonstrated that IL-10 is an immunosuppressant in animal models of T/HS 
. We suggest that at early time points following trauma, circulatory and the neuro-endocrine derangements lead to the production of catecholamines, which in turn induce this later production of IL-10 
. Overly elevated IL-10 could suppress TNF-α produced by monocyte/macrophages in multiple tissues 
, perhaps accounting for the cytokine phenotype we have observed in non-survivor pigs. Investigators have suggested the possibility of gene therapy with IL-10 for acute inflammatory syndromes such as T/HS 
; our studies suggest that caution should be exercised when considering intervention using this cytokine.
Finally, since NO contributes to the host's inflammatory defense and can cause circulatory disorders, it may be an important mediator in the setting of inflammation and organ failure, possibly by altering outcome after T/HS 
. Endothelial cells produce NO from a largely constitutive isoform of NO synthase (NOS), whose expression and activity is known to be reduced in hemorrhagic shock 
. Most cells also produce NO via an inducible form of NOS (iNOS), the expression of which is induced by cytokines 
levels in trauma patients have been previously reported both immediately after trauma 
and at later time points 
. This elevated NO production reflected severity of injury during the first two hours after the traumatic insult, suggesting that increased NO production might play a role in the very early post-injury period 
. Other studies have focused on NO as a possible mediator of decompensation, with increases in iNOS activity being reported in several organs after prolonged hemorrhagic shock 
In the present study, we observed significant differences between survivors and non-survivors only in Group A swine only when the NO2-
data were taken as a whole for each outcome group, but not in trauma patients and not as a function of time in injured swine. These results suggest that iNOS is probably not involved in the phenomena studied herein, though they may suggest that eNOS activity is altered in some settings (consistent with prior findings 
). The lack of a role for iNOS in swine may reflect the early time points studied. Alternatively, the activity of eNOS might be affected differentially in T/HS survivors vs. non-survivors, but the relative insensitivity of NO2-
as a measurement outcome may necessitate alternative methods (e.g. directly measuring NO by a NO-sensitive electrode or other means 
) to address this point.
A general limitation of our study centers on the fact that the overall number of subjects and inflammatory analytes studied was relatively low in both humans and swine. With regard to patient number, we believe that our data (17% mortality rate) are representative of the type of outcomes seen in patients presenting with blunt trauma (e.g. the stud of Sperry et al. 
, in which only 5% mortality is reported in trauma patients). Given this limitation, we augmented our study by assaying inflammatory analytes in serial samples from trauma patients. We also carried out a clinically realistic animal model of trauma/hemorrhage, in which defined alarms triggered resuscitation and led to a low mortality that is concordant with that seen in trauma patients. We believe that our results are valid because even when examining all samples from all patients (both survivors and non-survivors) we found a negative correlation between early circulating TNF-α levels and organ damage, suggesting that an early pro-inflammatory response is associated with a positive outcome. In the same cohort, circulating IL-6 correlated positively with organ damage, as would be expected from a large number of studies that have examined circulating IL-6 in trauma patients (thereby helping validate, at least in part, the cohort of patients studied herein). The choice of cytokines utilized in the analysis described in the present manuscript was based on a defined number of cytokines that have been well-vetted with regard to their role in trauma/hemorrhage (as described above) and that could be measured in both humans and swine. We hope that as the number of available pig-specific cytokine assay kits increases, we will be able to expand the present study to a broader panel of inflammatory mediators.
Another caveat that needs to be considered with regard to our studies in swine concerns the experimental protocol used. Our model of severe hemorrhagic shock allows for a wide range of hemodynamic fluctuations in the course of the experiment and reflects the animals' compensatory responses. Not surprisingly, this experimental paradigm was associated with significant inter-animal variability in both time course and outcome. Furthermore, analysis was carried out up only to the pre-resuscitation time point due to the fact that resuscitation has been shown to influence the inflammatory response to trauma and hemorrhage 
. Ideally, swine should be subjected to a combination of soft tissue injury and bone fracture, in combination with mild to moderate hemorrhagic shock, in order to simulate the types of blunt injury seen in trauma patients. However, due to limitations placed on us by animal use regulations, we cannot easily carry out such studies, and moreover could not carry out such studies and then recover the animals and follow them for 1–2 days. Nonetheless, we suggest that the characteristics of our experimental preparation render it particularly suitable for the practical assessment of dynamic response characteristics, and especially since one of our central goals was to compare the responses of large experimental animals with those of human trauma victims.
Alternative hypotheses may be raised with regard to our findings. Higher circulating levels of TNF, IL-6, IL-10, and NO2-
are found in septic patients 
. In one study, higher levels of plasma cytokines were reported in non-survivors of sepsis, and in this study fluid resuscitation was associated with lower mean cytokine levels 
. Rivers et al.
have hypothesized that better perfused organs suffer less damage/dysfunction, and thus are less inflamed 
. Thus, it may be argued that trauma results in such profound hypoperfusion that cytokines are not flushed out of damaged organs, contrary to sepsis, where many organs remain perfused. This alternative hypothesis could be tested (at least in experimental animals) by quantifying tissue levels of cytokines.
Another limitation of our comparative study in humans and swine involves a somewhat different sampling methodology in these two species; clearly, we were able to sample blood in experimental animals more frequently than in trauma patients. Despite the fact the time points in both settings fell within a 6-h range, it may be argued that we are observing different kinetics of cytokine production and therefore different phenomena.
In conclusion, our studies suggest that the role of TNF-α in T/HS may need to be re-evaluated in light of our findings. On a broader level, there may be a need to distinguish between early and late inflammation induced by injury. Our studies suggest that early, adequately robust production of TNF-α following injury is a hallmark of a proper response, while unchanging, low-levels of this cytokine may reflect pathology. This situation may be analogous to that observed when studying physiologically variable responses such as heart rate 
. Though further study is warranted, our findings raise the possibility of re-interpreting the role of TNF-α post-T/HS, and suggest that caution should exercised when thinking of TNF-α antagonism in this setting.