The present study provides a description of the human mesenteric lymph proteome from eleven patients. We applied a strategy that combines immuno-affinity depletion, 1D SDS-PAGE separation and LC-MS/MS to increase the dynamic range of detection. We have identified, with greater than 99% confidence, a total of 477 proteins. Several of the proteins identified in our study are markers of hemolysis and oxidative stress, matrix degradation, and general tissue damage. Our lab and others have demonstrated the pro-inflammatory activity of post shock mesenteric lymph in a variety of animal models. While the detailed proteome for rodent mesenteric lymph has been reported to date (11
) studies using human samples have been limited.
Out of the top 200 proteins in our dataset, several are not found in the validated 889 human plasma proteome project (HPPP) dataset (17
). This finding is consistent with work by Leak et al.
that showed ovine lymph contains plasma proteins and additional metabolic products of connective tissue cells and proteins released from lymphatic endothelium (5
Our previous proteomic analysis of mesenteric lymph was from a well-studied rodent model of shock. We used the differential gel electrophoresis (DIGE) technique and MS to identify proteins that change in relative abundance. Not surprisingly, most of the identified proteins in rodent lymph where also identified in human mesenteric lymph. However, the rodent lymph proteome contains a population of proteins such as major urinary protein, alpha-1-inhibitor 3, murinoglobulin-1 that are not presented in the human lymph dataset.
We have demonstrated that many of the proteins amongst the 477 identified appear to correlate with the PMN priming activity of mesenteric lymph and therefore may correlate with MOF and critical illness. While the polymetric analysis used here did not reveal a clear relationship between patient samples when using the entire dataset, analysis on subsets of related proteins resulted in patient sample clustering that correlated with bioactivity. Interestingly, both tissue injury and shock correlate strongly with production of bioactive lymph. While the two patients that generated lymph with the highest bioactivity were not in shock, each patient with a vasopressor requirement generated bioactive lymph samples, and all of the trauma patients and brain dead organ donors generated lymph that primed PMNs greater than fMLP. This indicates that lymph from critically ill patients has the capacity to initiate a strong inflammatory response, and that shock is sufficient but not a necessary condition for neutrophil priming. Because the lymphatic system is permeable, there is little, if any, exclusion of interstitial molecules (39
). In addition, the increased microvascular permeability following severe injury, suggests that markers of MOF identified in mesenteric lymph could lead to the identification of plasma biomarkers.
While the methods employed in this study allowed for the identification of several hundred proteins, our dataset is not a comprehensive list of all proteins in human mesenteric lymph. The limited sample, high dynamic range of protein concentrations in lymph, and inherent sensitivity of the analytical platform used precludes the identification of many bioactive components such as cytokines. Due to the heterogeneous patient population in this study, it is not possible to conclude that the correlations observed are a direct consequence of trauma, shock, or brain death. It is likely that the priming of neutrophils observed is mediated, at least in part, by additional factors not identified in our dataset i.e. endotoxin, cytokines, etc. However, the diverse collection of immunomodulatory proteins identified in this paper provides insight and offer direction for future studies.
Our human findings substantiate the current body of research implicating post shock mesenteric lymph in the development of systemic inflammation and MOF. Further studies are required to determine if, and how, the proteins identified here provide a mechanistic link between the ischemic gut and remote organ injury, serving as potential biomarkers for early identification of patients at risk for the development of multiple organ failure.