Mesenteric lymph has been implicated as the mechanistic link by which bioactive mediators elaborated from the ischemic gut during hemorrhagic shock can provoke distant organ injury(1
). Collectively, basic bench work with rodent models of hemorrhagic shock from Deitch, et al and our lab have shown PSML to contain factors that can incite acute lung injury through mechanisms of endothelial inflammation and apoptosis as well as neutrophil priming(3
). Current research in this area has emphasized identifying the toxic bioactive mediators in PSML. Initial investigations eliminated the seemingly obvious molecules such as TNF, LPS and proinflammatory cytokines(17
). Therefore, given the enormous amount of possible biologic mediators in the lymph, we began our analysis with a global evaluation using a quantitative proteomics approach. This provided an armamentarium of information for further focused studies. Although the results of this current study are observational, we now have an initiating focus to delineate the causative agent(s) and specific components of lymph that contribute to the bioactivity. Previously, we have reported the identification of a neutral phospholipase A2-derived lipid as a potential mediator found in the PSML with proinflammatory activity(8
). Conceivably, this lipid mediator may be present in the inactive preshock lymph possibly bound to a carrier protein. The depletion of specific proteins exacerbated by dilution from resuscitation may allow the unbound lipid fraction to increase in effective concentration thereby becoming biologically active. This theory led us to employ proteomics to pursue the identity of lipid-binding proteins that change following hemorrhagic shock and resuscitation.
These results illustrate some of the strengths and disadvantages of proteomic analysis of lymph. Few proteins (albumin, globulins, etc) account for over 80% of the proteinaceous content of the lymph. Therefore, the enormous dynamic range poses a challenge to identifying less abundant proteins. Color differences of fluorescently tagged proteins in preshock vs post-shock lymph, though accurate, often cannot be appreciated with the naked eye. Thus, displays a section where the depletion or enrichment of specific proteins can be easily discerned on grayscale, outside the area of the tails of prominent proteins. Where consistent differences can be detected (by quantitative fluorescent contrasts), confident identification of a particular protein remains dependent on being able to generate sufficient fragments (tryptic digests), successful sequencing, and rigorous matching to known proteins within available databases. The identification of gelsolin met all the criteria above including a consistent 2-fold change.
Gelsolin, a known clinical marker(13
), was identified to have a significant change in PSML. It is an interesting molecule with several physiologic capabilities that may explain its contribution to lymph bioactivity. Intracellularly, gelsolin (MW 90kD) is regulated by calcium and functions to cleave, then cap actin filaments thereby aiding in chemotaxis and movement of intracellular structures. Plasma gelsolin (MW 93kD) contains an additional 25-amino acid residue at the amino terminus and is believed to be primarily secreted by muscle cells(29
). It functions as an actin scavenger by binding actin in the circulation(26
), then the complex is cleared by the reticular endothelial system(30
). Gelsolin also functions as a lipid carrier with strong binding affinity to several cystolic and plasma lipids such as phosphotidylinositol, lipopolysaccharide, and lysophosphatidic acid(11
). The biologic consequences of this particular action are still under investigation.
In our current investigation, gelsolin was found to be present in preshock mesenteric lymph at concentrations that mirror the plasma. Following hemorrhagic shock, gelsolin levels decrease significantly in the lymph. Our results support previous findings of the depletion of gelsolin following significant injury and seen in critically ill patients with increased mortality(13
). These clinical investigations have shown that plasma gelsolin concentrations continue to decrease 24hrs to as long as 5days following injury in human subjects(13
). This depletion correlates with the fall in other plasma proteins such as albumin; however, the recovery of other proteins is much quicker than that of gelsolin(13
) suggesting a specific depletion and lack of replenishment. It may be that the critical level of gelsolin has not yet been reached in our 3rd
hour lymph following resuscitation and the actin scavenging system has not been overwhelmed thereby leading to maintained “normal” levels of actin at this particular time point. The eventual consumption may prevent the secondary activity of gelsolin and increase lipid bioactivity possibly predisposing the lung and other organs to endothelial injury.
experiments have shown decreased inflammatory changes and cellular injury with the addition of gelsolin to samples(28
). This has been further supported by in vivo
experiments including rats sustaining significant burn injury that have been conducted with the addition of recombinant gelsolin and have shown attenuated lung injury in these subjects(15
The diverse activity of this particular molecule makes it an interesting culprit in the investigation of mesenteric lymph toxicity. It provides a possible explanation of the precise mechanism of how depletion of gelsolin is associated with a worsened clinical outcome either as a result of tissue injury secondary to impaired actin scavenging or a lack of toxic lipid modulation. More importantly, this provides a potential target to the development of a pharmacotherapy for the treatment or possibly prevention of post-injury multiple organ failure.
In summary, the results of the current study demonstrate the presence and reduction of gelsolin in the PSML. The depletion may be a result of a consumptive process due to actin scavenging to prevent capillary plugging and tissue ischemia. However, this consumptive process may prevent a secondary action of lipid binding and may account for the lipid bioactivity that contributes to the toxicity of PSML and post-injury multiple organ failure.