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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Shock. Author manuscript; available in PMC 2012 April 1.
Published in final edited form as:
PMCID: PMC3059368
NIHMSID: NIHMS272803

Proteomic Analysis of Human Mesenteric Lymph

Abstract

Extensive animal work has established mesenteric lymph as the mechanistic link between gut ischemia/reperfusion (I/R) and distant organ injury. Our trauma and transplant services provide a unique opportunity to assess the relevance of our animal data to human mesenteric lymph under conditions that simulate those used in the laboratory. Mesenteric lymph was collected from eleven patients; with lymphatic injuries, during semi-elective spine reconstruction, or immediately before organ donation. The lymph was tested for its ability to activate human neutrophils in vitro, and was analyzed by label-free proteomic analysis. Human mesenteric lymph primed human PMNs in a pattern similar to that observed in previous rodent, swine, and primate studies. A total of 477 proteins were identified from the 11 subject’s lymph samples with greater than 99% confidence. In addition to classical serum proteins, markers of hemolysis, extracellular matrix components, and general tissue damage were identified. Both tissue injury and shock correlate strongly with production of bioactive lymph. Products of red blood cell hemolysis correlate strongly with human lymph bioactivity and immunoglobulins have a negative correlation with the pro-inflammatory lymph. These human data corroborate the current body of research implicating post shock mesenteric lymph in the development of systemic inflammation and multiple organ failure. Further studies will be required to determine if the proteins identified participate in the pathogenesis of multiple organ failure and if they can be used as diagnostic markers.

Keywords: mesenteric lymph, neutrophil, superoxide, proteomics, trauma, hemorrhagic shock, multiple organ failure, acute lung injury

INTRODUCTION

Post injury multiple organ failure (MOF) is the net result of a dysfunctional immune response to injury characterized by a hyperactive innate system and a suppressed adaptive system. Acute lung injury (ALI) is the first clinical manifestation of organ failure, followed by renal and hepatic dysfunction. Circulatory shock is integral in the early pathogenesis of MOF, and the gut has been invoked as the “motor of MOF” (1). Mesenteric ischemia reperfusion (IR), subsequent to trauma/hemorrhagic shock (T/HS), is central in the pathogenesis of post-injury organ dysfunction (2). Previous work in our own laboratory has documented PMN priming occurring in the early post-injury period contributes to MOF (3).

The lymphatic system was first described during ancient times, but its significance remained obscure until the Renaissance period with the realization that lymphatics were different than veins (4). The lymphatic system collects extravasated fluid, proteins, and lipids from the interstitial space and returns them to the blood circulation via the subclavian vein. An important difference is mesenteric lymph contains a large amount of fat and other molecules resorbed from the intestine, giving it a unique descriptor, chyle. Lymph is formed due to a filtration process of the plasma in the capillaries. It contains a large number of lymphocytes, macrophages, and number of plasma proteins including coagulation factors and albumin. In one of the first studies to use modern approaches to characterize lymph, Leak et al. examined the protein expression profiles of normal ovine lymph (5).

Post shock mesenteric lymph (PSML) serves as the conduit by which exudates from the ischemic gut are delivered into the circulation, and lymphatic diversion prior to T/HS attenuates the severity of organ injury (68). Investigations to identify the specific agents contained in PSML have implicated both proteins and lipids (910) The physiologic response to trauma has been inferred by a number of specific proteins, including acute phase components, cytokines and contents of cellular lysates, yet the proteome of human PSML has not been characterized. A prior biochemical study by Kaiser et al. reported that the level of a 24 amino acid, N-terminal fragment of serum albumin was significantly increased in lymph collected from rat subjected to trauma with hemorrhagic-shock (10). Initial studies in our laboratory characterized the protein fraction of rodent mesenteric lymph in the context of an experimental trauma/hemorrhagic shock model using differential gel electrophoresis and mass spectrometry analysis (11). In addition, Mittel et al. as well as Fang et al. have published proteomic studies of rodent mesenteric lymph during fasted and fed states, acute pancreatitis and hemorrhagic shock models (1215). At the present time, our active trauma and transplant surgical services provide a unique opportunity to assess the relevance of the animal models to the human condition. The aim of this study is to provide the first proteomic description of human mesenteric lymph collected from critically ill or injured patients using a label-free semi-quantitative mass spectrometry based approach.

MATERIALS AND METHODS

Study population and data collection

Injured patients admitted to the Rocky Mountain Regional Trauma Center surgical ICU (SICU) at Denver Health Medical Center (DHMC) and patients undergoing brain-dead organ donation at Colorado hospitals were evaluated for inclusion into a study from 2008 through 2010. DHMC is a state-designated level I trauma center verified by the American College of Surgeons Committee on Trauma and the academic trauma center for the University of Colorado Denver. The data collection and storage processes are in compliance with Health Insurance Portability and Accountability Act regulations. The local institutional review board approved the study. Patients with various injury mechanisms were included in the study: trauma/hemorrhagic shock, brain death, mesenteric lymphatic injury, and elective spine surgery.

Mesenteric lymph collection

After obtaining informed consent from patients undergoing semi-elective spine injury reconstruction, the exposure of the patient’s distal thoracic and proximal lumbar vertebral bodies is achieved via a left thoraco-abdominal incision. The cisterna chyli is visualized between the aorta and spine and lymph aspirated using a 27-gauge needle. During the donor operation, before cold preservation, a right medial visceral rotation is performed to expose the vena cava and the aorta just inferiorly to the take off of the superior mesenteric artery. At this level, the left renal vein is easily identified anteriorly crossing the aorta. Running along the retroperitoneal small bowel mesentery and crossing anterior and perpendicular to the left renal vein are typically large distended lymphatic vessels. These are cannulated with a 21-gauge angiocatheter to procure the mesenteric lymph. Patients are NPO (nil per os) overnight in preparation for semi-elective surgery and all nutrition (enteral and parenteral) is discontinued at the time of consent for organ donation. Consent for organ donation as well as lymph collection is obtained by the organ procurement organization, from the deceased (based on a document of gift: donor card, living will, or driver’s license) or from the next of kin.

Mesenteric lymph (100µl − 1 mL) was collected and placed into an EDTA containing tube. Samples were centrifuged at 3500 × g for 10 min to remove cellular components, and stored in a freezer at −80 °C. Protein concentration was quantified using the Bradford assay, and lymph bioactivity measured by superoxide dismutase-inhibitable cytochrome c reduction in human neutrophils. Patients were categorized based on the predominant illness or mechanism of injury. The Injury Severity Score (ISS), a numerical method to describe the overall magnitude of injury, was calculated for trauma patients.

Human PMN Isolation

Human polymorphonuclear neutrophils (PMNs) were isolated from the heparinized blood of healthy donors with the use of dextran sedimentation followed by Ficoll density gradient, as previously described (8). Briefly, 10 mL from the samples was centrifuged at 200 × g for 15 min. The upper layer was removed and the buffy coat was mixed with phosphate-buffered saline (PBS). This mixture was then placed on 5 mL of Ficoll and was centrifuged at 400 × g for 30 min. Hypotonic lysis was performed twice at 4 °C to eliminate contaminating red blood cells. The resulting PMNs were washed once and resuspended in Kreb’s Ringer phosphate with dextrose buffer (KRP-d) at pH 7.35 to a final concentration of 2.5 × 107 cells/mL. The final cell population was more than 95% PMNs as determined by cell sorting.

Superoxide Production Assay

Lymph was tested for bioactivity using the spectrophotometric assay for superoxide dismutase (SOD) as described previously (8). Briefly, isolated PMNs (3.75 × 105) were incubated with either Kreb-Ringer phosphodextrose buffer (control), PAF (1 µM) or lymph mesenteric (10% vol/vol) for 5 minutes at 37 °C then centrifuged for 3 minutes at 400 × g and resuspended in fresh Kreb-Ringer phosphodextrose at 37 °C. All priming assays were completed at 37 °C in duplicate with a separate superoxide dismutase blank and an unstimulated control, to assure that the resting cells were not primed before beginning the experiment. The respiratory burst was initiated with the addition of 1 mM N-formylmethionyl- leucyl-phenylalanine (fMLP) to experimental wells. The fMLP, a known chemoattractant and granulocyte activator, was used as an internal standard. The maximal rate of superoxide dismutase inhibitable superoxide anion production (Vmax) was determined by the slope of the absorbance curve using Softmax software (Molecular Devices, Silicon Valley, CA). The data were recorded as the maximal rate of superoxide anion production (nM O2/3.75 × 105 cells per minute).

Protein Sample preparation

The two most abundant proteins, albumin and IgG, were removed from the lymph samples using ProteoPrep® spin columns (Sigma-Aldrich) according to manufacturer’s protocol. Total protein content was determined by the bicinchoninic acid assay (BCA) and absorbance read at 595 nm. A portion of the sample (30 µg) was diluted into SDS-PAGE sample buffer, heated at 70 °C for 10 min and loaded in a single lane on a 1-mm-thick 4–12% Bis-Tris gel (Invitrogen). After separation, the gel was stained with Coomassie Blue stain (Invitrogen). Each lane of the gel was divided into 10 equal-sized bands and proteins in the gel were digested as follows. Bands were destained in 200 µl of 25 mM ammonium bicarbonate in 50 % v/v ACN for 15 min, and then 200 µl of 100% ACN was applied for 15 min at room temperature. Dithiothreitol (DTT) was added to a final concentration of 10 mM and incubated at 65 °C for 30 min to reduce the disulfide bonds. The reduced cysteines were then alkylated with the addition of iodoacetamide (IAM) at a final concentration of 20 mM and incubated at room temperature in the dark for 30 min. The iodoacetamide was then removed, and washes were performed with 200 µl of distilled water followed by addition of 100 µl of ACN. Then ACN was removed, and 50 µl of the 0.01 µg/µl trypsin solution was added to each plug and allowed to rehydrate the gel plugs at 4 °C for 30 min and then incubated at 37 °C overnight. The tryptic mixtures were acidified with formic acid up to a final concentration of 1%. Peptides were extracted three times from the gel plugs using 50% ACN, 1% FA, concentrated by SpeedVac to a desired volume (~18 µl), and subjected to LC-MS/MS analysis.

Liquid Chromatography-Tandem Mass Spectrometry

Nano-flow reverse phase LC-MS/MS was performed using a capillary HPLC system (Agilent 1200, Palo Alto, CA) coupled with a linear ion trap mass spectrometer LTQ-FT Ultra Hybrid ion cyclotron resonance mass spectrometer (Thermo Fisher; San Jose, CA) through an in-house built nanoelectrospray ionization source. Eight microliters of the tryptic peptides were preconcentrated and desalted onto a C18 trap column ZORBAX 300SB-C18, (5 µm i.d. × 5 mm, Agilent Technologies, Santa Clara, CA) with 5% ACN, 0.1% FA at a flow rate of 15 µL/min for 5 min. The separation of the tryptic peptides was performed on a C18 reverse phase column (75 µm ID × 360 µm OD × 100 mm length) packed in-house with 4 µm 100 Å pore size C18 reversed-phase stationary phase (Synergy, Phenomenex, Torrance, CA) kept at a constant 40 °C using an in-house built column heater at a flow rate of 380 nl/min. The mobile phases consisted of 5% acetonitrile with 0.1% formic acid (A) and 95% acetonitrile with 0.1% formic acid (B), respectively. A 90-min linear gradient from 5 to 50% B was typically used. Data acquisition was performed using the instrument supplied Xcalibur (version 2.0.6) software. The LC runs were monitored in positive ion mode by sequentially recording survey MS scans (m/z 400–2000), in the ICR cell, while three MS2 were obtained in the ion trap via CID for the most intense ions.

Mass Spectrometry Data Analysis

MS/MS spectra were extracted from raw data files and converted into mascot generic files (mgf) using an in-house script. Mascot (version 2.2; Matrix Science Inc., London, UK) was used to perform database searches against the human subset SwissProt database of the extracted MS/MS data. Peptide tolerance was set at ± 10 ppm with MS/MS tolerance set at ± 0.6 Da. Trypsin specificity was used allowing for 1 missed cleavage. The modifications of Met oxidation, protein N-terminal acetylation, and peptide N-terminal pyroglutamic acid formation were allowed for, and Cys carbamindomethylation was set as a fixed modification.

Scaffold (version 2, Proteome Software, Portland, OR, USA) was used to validate MS/MS based peptide and protein identifications. All mascot. DAT files, for each subject’s lymph (10 bands each) were loaded together as one “biological sample” within Scaffold. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified unique peptides.

Both unique peptides and total peptides per protein were calculated for each patient and normalized to total unique and total peptides per sample. These values were used for a semi-quantitative comparison between subjects. Linear regression (R-squared method) was used to identify proteins with abundance patterns that correlated with lymph bioactivity as measured by PNM priming. The correlation coefficients and p values are uncorrected for multiple comparisons and represent nominal values.

Polymetric clustering analysis

PermutMatrix (version 1.9.3, Montpellier, FR) was used for clustering analysis for proteins that were identified in seven or more of the eleven patient’s samples. The multiple-fragment heuristic (MF) seriation rule was used for the ordering of individual proteins. The sum of all pairwise distances of neighboring rows was S=13.294 and R=0.487 for the objective function. Patients were clustered using McQuitty’s criteria for the linkage rule (path length S=10.198, objective function R=0.546). The Euclidean distance method was used to calculate dissimilarity (16).

RESULTS

Lymph samples were collected from eleven subjects in accordance with local and federal regulations. A summary of the patient profiles are listed in Table 1. The diverse group covers a wide range of ages and medical conditions, with recovery of lymph during semi-elective spine reconstruction, lymphatic injuries or organ donation (Figure 1). A neutrophil priming assay was used to evaluate each of the samples for bioactivity (Figure 2). The priming ranges from slightly greater than platelet-activating factor (PAF) for patient #1 to slightly less than the control (fMLP), for patient #11. Of the patients samples collected, two were classified as having high bioactivity (> 2 fold priming over fMLP), three medium (> 1.5 fold over fMLP) and six with low bioactivity (<1.5 fold over fMLP). All trauma patients and brain dead organ donors in this study generated lymph that had bioactivity in our PMN priming assay. Although two patients without shock had the highest lymph bioactivity, all of the samples from patients with a vasopressor requirement (5/5) at the time of collection had lymph with bioactivity of 1.4 fold higher priming over fMLP.

Figure 1
Mesenteric lymph was obtained from the efferent lymph ducts in patients undergoing organ donation. Typically, these ducts were found at the junction of the IMV (inferior mesenteric vein) and the LRV (left renal vein). Also pictured are the aorta and the ...
Figure 2
Parametric analysis of immunoglobulin entries.
Table 1
Patient profiles.

Proteomics analysis

Using a 1D-PAGE separation step prior to nanocapillary LC-MS/MS analysis, we analyzed the protein profiles of mesenteric lymph from the eleven human subjects. To improve proteome coverage, we performed single step depletion of the two most abundant serum proteins, albumin and IgG. While this approach is likely to also deplete specific proteins that are bound to albumin or possibly non-specifically to the depletion column, depletion of these highly abundant proteins has proven to be a particularly beneficial step in achieving greater proteome coverage. The depleted protein samples were separated by 1D SDS-PAGE followed by slicing gel lanes into 10 bands (gel fractions). Each band was subjected to tryptic digestion and the resulting peptides were analyzed by LC-MS/MS. A total of 477 proteins with 99% confidence (FDR<1%) were identified from the eleven lymph samples (30 µg of total protein each). The proteins identified in our study include extracellular matrix components, secreted proteases and proteases inhibitors, mitochondrial and cytoskeleton proteins, other intracellular proteins, in addition to classical blood proteins. Representative examples from several classes of proteins are listed in Table 2. Most abundant proteins, such as complement proteins and apolipoproteins, were identified in all patients. Rare proteins such as neutrophil serum deprivation-response protein and Xaa-Pro dipeptidase were only found in single patient’s samples. The protein results were ranked based on the total number of samples a protein was identified in and the average number of unique peptides per protein (complete results in Supplementary Table S1). This rank gives a crude evaluation of relative protein abundance in lymph and more importantly allows us to compare the proteins identified to other proteomic datasets, such as the Human Plasma Proteome dataset, which has been collected by 35 collaborating laboratories.

Table 2
Representative proteins identified in human lymph.

Bioactivity trends

We found that several proteins were strongly associated with bioactivity, as measured by the ability of lymph to stimulate superoxide production by human neutrophils. Several of the proteins that had high correlation with bioactivity were markers of hemolysis. We compared the proteomic analysis of erythrocytes by Kakhniashvili et al. to identify potential protein contributions to the lymph from hemolysis (18). Table 3 shows the top RBC proteins and their rank in our dataset. Of the top 25 proteins in the RBC dataset 21 were found in human lymph, 14 with a correlation coefficient of greater than 0.60, and 17 with a P value of less than 0.1 when comparing high and medium to low bioactivity samples.

Table 3
Comparison of lymph and erythrocyte proteomes (17).

Despite having used immuno-affinity depletion to remove IgG, roughly 75% of the immunoglobins found in serum, some IgG peptides were identified in addition to a large number of the additional proteins in this class of abundant serum proteins. It is not unexpected to find an abundant amount of IgGs in lymph fluid based on the immune-surveillance function of this organ system. Spectral counts of the IgG sequences identified were one of the best predictors of lymph having lower PMN priming activity as shown (Figure 3). Interestingly polymetric clustering shows that lymph from patient #11, that had the lowest bioactivity in the priming assay, was an outlier. It has been shown that several antibodies have immuno-modulatory properties such as those that can prime PMNs (19), to that those that neutralize pro-inflammatory cytokines, examples include autoantibodies that bind IL-8 (20), IL-6 (21), IFN-γ (22).

Figure 3
The bioactivity of human mesenteric lymph was assessed by superoxide production in human PMNs. PAF (platelet activating factor) was used as a PMN NADPH oxidase priming agent. FMLP (formyl-methionyl-leucyl-phenylalanine) was employed as an activator.

Hemolysis

A significant increase of hemolysis products such as hemoglobin alpha, beta, gamma and delta (Table 3 and and4)4) correlates with bioactivity from human mesenteric lymph samples. Released hemoglobin may represent a common factor resulting from multiple pathways such as oxidative stress, nitric oxide (NO) depletion and platelet activation and aggregation (23). These processes may aggravate ongoing inflammation to produce vascular damage and lung injury and also drive damage in other susceptible organs, such as the kidneys. Decreased levels of haptoglobin also correlated with bioactivity from human mesenteric lymph samples. Haptoglobin is an acute phase glycoprotein that binds free hemoglobin with high avidity during hemolysis, protecting organs from iron-generated reactive oxygen species. In addition to sequestration of hemoglobin, haptoglobin is an alpha-2-glycoprotein with anti-inflammatory properties (24). Thus, the depletion of haptoglobin and increase in free hemoglobin found in post shock mesenteric lymph may indicate a loss of protection from inflammation and free radical damage, ultimately contributing to remote organ injury.

Table 4
Additional proteins discussed.

An additional product of hemolysis, the cytosolic carbonic anhydrase (CA) isoenzymes 1, 2 and 3 abundant in the RBC proteome and in our dataset, increased proportionally to human mesenteric lymph bioactivity. Carbonic anhydrase, a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide, a product of cellular aerobic energy production. It participates in various biological processes including acid-base regulation, gas transport, bone resorption, ureagenesis, gluconeogenesis, cell proliferation, oncogenesis and lipogenesis. In addition to these physiological functions, CA hydrolyzes various esters (25).

Tissue damage

Several cellular proteins associated with tissue injury, such as creatine kinase (MB isoenzymes), myoglobin, cytoplasmatic malate dehydrogenase, and fatty acid binding protein, were identified in human mesenteric lymph. Liver type fatty acid-binding protein (L-FABP) correlates positively with bioactivity from human mesenteric lymph samples. This relatively small (14 kDa) cytoplasmic protein is abundantly expressed in the cytoplasm and the nucleus of hepatocytes. L-FABP has high affinity and capacity to bind long-chain fatty acid oxidative products. Additionally, L-FABP has been identified as a marker of acute liver injury (26).

Cytosolic malate dehydrogenase (MDH) is another protein correlating with human mesenteric lymph bioactivity. MDH was identified in all high and medium priming samples and conversely, was not presented in any of the samples with low bioactivity. This enzyme plays a crucial role in the malate–aspartate shuttle and the citric-acid cycle in all aerobic tissues of mammals. Previous work from our laboratory reported that MDH levels increased in rat post-shock mesenteric lymph as well (11). The levels of other glycolytic pathway proteins also correlate with the pro-inflammatory activity of mesenteric lymph. These proteins include fructose-1,6-bisphosphatase (FBP), gyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). A substantial increase of fructose 1,6-bisphosphatase has been observed in the early phase of hemorrhagic shock (27). FBP is a critical regulatory enzyme in glycolysis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate.

Cytoskeletal proteins

We identified several cytoskeletal proteins including actin, tubulin, cofilin-1, spectrin, gelsolin and profilin-1. Among these markers of tissue damage, cofilins are the principal actin filament depolymerizing proteins and cofilin-1 has been implicated in regulation of ischemia-induced endothelial cell alteration, leading to cell damage and microvascular dysfunction (28). A significant increase in the relative abundance of actin, cofilin-1 and profilin has been previously reported in rodent mesenteric lymph following hemorrhagic shock (12).

Mitochondrial proteins

Sixteen mitochondrial proteins were identified in the human mesenteric lymph samples. There have been reports showing that circulating mitochondrial DNA and formyl peptides can mediate organ injury through PMN activation (29). While we did not identify specific mitochondrial DNA binding proteins or formylated peptides that target the formyl peptide receptor (a GPCR) (30), our data suggests the presence of lysed mitochondria, presumably from cell death.

ECM components

The extracellular matrix (ECM) is a complex protein network composed of collagens, connective tissue glycoproteins, elastic fibers, hyaluronan and proteoglycans. The ECM provides a molecular scaffold and can serve as a repository for cytokines. Several identified ECM components such as fibrinogen, von Willebrand factor, and fibulin-1 are abundant in blood. Collagens, laminins and ECM-1 are structural matrix components found in human mesenteric lymph and known substrates for the gelatinases. The presence of these ECM components suggests tissue damage either by the trauma itself or by degradation of other matrix by activated proteases. One such protease identified in human mesenteric lymph was matrix metalloproteinase 9 (MMP-9). Several clinical studies have shown that neutrophil-derived MMPs (specifically MMP-2 and MMP-9) are involved in the development of acute lung injury both experimentally and in clinical ARDS (31).

Oxidative Stress

Additional proteins that appeared to correlate with the pro-inflammatory activity of mesenteric lymph are associated with oxidative stress. These include glutathione S-transferase P/Mu4/omega1, peroxiredoxin-2 and -6, catalase, Cu/Zn and Mn superoxide dismutase, extracellular superoxide dismutase and protein DJ-1. Animal models have indicated that the GSTs function as an early and sensitive indicator of hepatocyte damage caused by various adverse conditions, including hemorrhagic shock (32). Peroxiredoxin-6 (PRDX6) is another protein that correlates with bioactivity from human mesenteric lymph samples and has a major role in lung phospholipid metabolism. Manevich and Fisher found that PRDX6 may offer cellular protection from oxidative stress (33). The DJ-1 protein, an oxidative stress response protein, correlates positively with inflammatory activity of mesenteric lymph. DJ-1 affects cell survival, in part, by modulating cellular signaling cascades such as PTEN/phosphatidylinositol 3-kinase/Akt (34) and altering p53 activity. Additionally, a number of studies have shown that DJ-1 can have protective functions against oxidative stress-induced injury. It was found that DJ-1 is required for the activity of Nrf2 (nuclear factor erythroid 2-related factor), a master regulator of response to oxidative stress (35).

Other Proteins

Lipopolysaccharide (LPS)-binding protein (LBP) had negative correlation with pro-inflammatory activity of human mesenteric lymph. It is a type I acute phase protein which markedly enhances cellular responses to LPS. LBP binds LPS and then interacts with membrane CD14 receptor and TLR4 to initiate a downstream signaling cascade, culminating in the activation of transcription factors such as NF-κB and the IFN regulatory factors (36). Elevated levels of LBP have been reported in infections and systemic inflammatory response syndrome (37). Macrophage migration inhibitory factor (MIF) was observed in lymph samples with high pro-inflammatory activity. MIF is a conserved 12.5 kDa pro-inflammatory cytokine secreted by immune and endocrine cells, and also by the epithelial lining of tissues in direct contact with the external environment upon inflammatory and stress stimulation. MIF participates in the pathogenesis of acute and chronic inflammatory and autoimmune disorders, such as sepsis, asthma, rheumatoid arthritis, and inflammatory bowel disease. It has been demonstrated that MIF can mediate inflammatory lung injury in acute respiratory distress syndrome (ARDS). The role of MIF as a promoter of intrapulmonary inflammation in ARDS was suggested by the observations that MIF augmented TNF-α and IL- 8 secretion from alveolar cells obtained from ARDS patients (38).

DISCUSSION

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 (1115) 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.

Supplementary Material

Supp1

Acknowledgments

To Debi Talamonti, Dr. Igal Kam, Dr. Michael Zimmerman, the University of Colorado Division of Transplant Surgery, Dr. Philip Stahel, Dr. Janeen Jordan, Dr. John Eun, Dr. David Mauchley, Lorrie Linquist, Steve Kelley, and the Donor Alliance organ procurement organization for their valuable assistance. This work was supported, in part, by National Institutes of Health Grants P30 CA046934-17 through the University of Colorado Comprehensive Cancer Center Core Support and S10RR023015 from the National Center for Research Resources. In addition, this work was supported, in part, by National Institutes of Health grants P50GM049222 and T32GM008315.

Footnotes

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REFERENCES

1. Sauaia A, Moore FA, Moore EE, Norris JM, Lezotte DC, Hamman RF. Multiple organ failure can be predicted as early as 12 hours after injury. J Trauma. 1998;45(2):291–301. discussion 301–303. [PubMed]
2. Moore EE, Moore FA, Franciose RJ, Kim FJ, Biffl WL, Banerjee A. The postischemic gut serves as a priming bed for circulating neutrophils that provoke multiple organ failure. J Trauma. 1994;37(6):881–887. [PubMed]
3. Botha AJ, Moore FA, Moore EE, Kim FJ, Banerjee A, Peterson VM. Postinjury neutrophil priming and activation: an early vulnerable window. Surgery. 1995;118(2):358–364. discussion 364–365. [PubMed]
4. Lord RS. The white veins: conceptual difficulties in the history of the lymphatics. Med Hist. 1968 Apr;12(2):174–184. [PMC free article] [PubMed]
5. Leak LV, Liotta LA, Krutzsch H, Jones M, Fusaroa VA, Ross SJ, Zhao Y., III EFP. Proteomic analysis of lymph. Proteomics. 2004;4(3):753–765. [PubMed]
6. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg. 1998;228(4):518–527. [PubMed]
7. Deitch EA, Adams C, Lu Q, Xu DZ. A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability. Surgery. 2001;129(1):39–47. [PubMed]
8. Gonzalez RJ, Moore EE, Ciesla DJ, Biffl WL, Johnson JL, Silliman CC. Mesenteric lymph is responsible for post-hemorrhagic shock systemic neutrophil priming. J Trauma. 2001;51(6):1069–1072. [PubMed]
9. Jordan JR, Moore EE, Sarin EL, Damle SS, Kashuk SB, Silliman CC, Banerjee A. Arachidonic acid in post shock mesenteric lymph induces pulmonary synthesis of leukotriene B4. J Appl Physiol. 2008;104(4):1161–1166. [PubMed]
10. Kaiser VL, Sifri ZC, Senthil M, Dikdan GS, Lu Q, Xu D, Deitch EA. Albumin peptide: a molecular marker for trauma/hemorrhagic-shock in rat mesenteric lymph. Peptides. 2005;26(12):2491–2499. [PubMed]
11. Peltz ED, Moore EE, Zurawel AA, Jordan JR, Damle SS, Redzic JS, Masuno T, Eun J, Hansen KC, Banerjee A. Proteome and system ontology of hemorrhagic shock: exploring early constitutive changes in post shock mesenteric lymph. Surgery. 2009;146(2):347–357. [PubMed]
12. Mittal A, Middleditch M, Ruggiero K, Loveday B, Delahunt B, Jüllig M, Cooper GJS, Windsor JA, Phillips ARJ. Changes in the mesenteric lymph proteome induced by haemorrhagic shock. Shock. 2010;34(2):140–149. [PubMed]
13. Mittal A, Middleditch M, Ruggiero K, Buchanan CM, Jullig M, Loveday B, Cooper GJS, Windsor JA, Phillips ARJ. The proteome of rodent mesenteric lymph. Am J Physiol Gastrointest Liver Physiol. 2008;295(5):G895–G903. [PubMed]
14. Mittal A, Phillips ARJ, Middleditch M, Ruggiero K, Loveday B, Delahunt B, Cooper GJS, Windsor JA. The proteome of mesenteric lymph during acute pancreatitis and implications for treatment. JOP. 2009;10(2):130–142. [PubMed]
15. Fang JF, Shih LY, Yuan KC, Fang KY, Hwang TL, Hsieh SY. Proteomic analysis of post-hemorrhagic shock mesenteric lymph. Shock. 2010;34(3):291–298. [PubMed]
16. Caraux G, Pinloche S. PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics. 2005;21(7):1280–1281. [PubMed]
17. Kakhniashvili DG, Bulla LA, Goodman SR. The Human Erythrocyte Proteome. Mol Cel Proteomics. 2004;3(5):501–509. [PubMed]
18. Silliman CC, Curtis BR, Kopko PM, Khan SY, Kelher MR, Schuller RM, Sannoh B, Ambruso DR. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model. Blood. 2007;109(4):1752–1755. [PubMed]
19. Kurdowska A, Miller EJ, Noble JM, Baughman RP, Matthay MA, Brelsford WG, Cohen AB. Anti-IL-8 autoantibodies in alveolar fluid from patients with the adult respiratory distress syndrome. J Immunol. 1996;157(6):2699–2706. [PubMed]
20. Puel A, Picard C, Lorrot M, Pons C, Chrabieh M, Lorenzo L, Mamani-Matsuda M, Jouanguy E, Gendrel D, Casanova J-L. Recurrent Staphylococcal Cellulitis and Subcutaneous Abscesses in a Child with Autoantibodies against IL-6. J Immunol. 2008;180(1):647–654. [PubMed]
21. Höflich C, Sabat R, Rosseau S, Temmesfeld B, Slevogt H, Döcke WD, Grütz G, Meisel C, Halle E, Göbel UB, Volk HD, Suttorp N. Naturally occurring anti-IFN-gamma autoantibody and severe infections with Mycobacterium cheloneae and Burkholderia cocovenenans. Blood. 2004;103(2):673–675. [PubMed]
22. Lee JS, Gladwin MT. Bad Blood: The risks of red cell storage. Nat Med. 2010;16(4):381–382. [PubMed]
23. Oh SK, Pavlotsky N, Tauber AI. Specific binding of haptoglobin to human neutrophils and its functional consequences. J Leukoc Biol. 1990;47(2):142–148. [PubMed]
24. Tashian RE. The carbonic anhydrases: Widening perspectives on their evolution, expression and function. Bioessays. 1989;10(6):186–192. [PubMed]
25. Pelsers MMAL. Fatty acid-binding protein as marker for renal injury. Scand J Clin Lab Invest Suppl. 2008;241:73–77. [PubMed]
26. Ljungqvist O, Khan A, Ware J. Evidence of increased gluconeogenesis during hemorrhage in fed and 24-hour food-deprived rats. J Trauma. 1989;29(1):87–90. [PubMed]
27. Suurna MV, Ashworth SL, Hosford M, Sandoval RM, Wean SE, Shah BM, Bamburg JR, Molitoris BA. Cofilin mediates ATP depletion-induced endothelial cell actin alterations. Am J Physiol Renal Physiol. 2006;290(6):F1398–F1407. [PubMed]
28. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 7285;464:104–107. [PMC free article] [PubMed]
29. Bogenhagen DF, Wang Y, Shen EL, Kobayashi R. Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes. Mol Cell Proteomics. 2003;2(11):1205–1216. [PubMed]
30. Torii K, Iida K, Miyazaki Y, Saga S, Kondoh Y, Taniguchi H, Taki F, Takagi K, Matsuyama M, Suzuki R. Higher concentrations of matrix metalloproteinases in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Am J Respir Crit Care Med. 1997;155(1):43–46. [PubMed]
31. Redl H, Schlag G, Paul E, Davies J. Plasma glutathione S-transferase as an early marker of posttraumatic hepatic injury in non-human primates. Shock. 1995;3(6):395–397. [PubMed]
32. Manevich Y, Fisher AB. Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism. Free Radic Biol Med. 2005;38(11):1422–1432. [PubMed]
33. Kim RH, Peters M, Jang Y, Shi W, Pintilie M, Fletcher GC, DeLuca C, Liepa J, Zhou L, Snow B, Binari RC, Manoukian AS, Bray MR, Liu F, Tsao M, Mak TW. DJ-1, a novel regulator of the tumor suppressor PTEN. Cancer Cell. 2005;7(3):263–273. [PubMed]
34. Clements CM, McNally RS, Conti BJ, Mak TW, Ting JP. DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci U S A. 2006;103(41):15091–15096. [PubMed]
35. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990;249(4975):1431–1433. [PubMed]
36. Myc A, Buck J, Gonin J, Reynolds B, Hammerling U, Emanuel D. The level of lipopolysaccharide-binding protein is significantly increased in plasma in patients with the systemic inflammatory response syndrome. Clin Diagn Lab Immunol. 1997;4(2):113–116. [PMC free article] [PubMed]
37. Lai KN, Leung JCK, Metz CN, Lai FM, Bucala R, Lan HY. Role for macrophage migration inhibitory factor in acute respiratory distress syndrome. J Pathol. 2003;199(4):496–508. [PubMed]
38. Omenn GS, States D, Adamski M, Blackwell T, Menon R, Hermjakob H, Apweiler R, Haab B, Simpson R, Eddes J, Kapp E, Moritz R, Chan D, Rai A, Admon A, Aebersold R, Eng J, Hancock W, Hefta S, Meyer H, Paik Y, Yoo J, Ping P, Pounds J, Adkins J, Qian X, Wang R, Wasinger V, Wu CY, Zhao X, Zeng R, Archakov A, Tsugita A, Beer I, Pandey A, Pisano M, Andrews P, Tammen H, Speicher D, Hanash S. Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics. 2005;5(13):3226–3245. [PubMed]
39. Swartz MA. The physiology of the lymphatic system. Adv Drug Deliv Rev. 2001;50(1–2):3–20. [PubMed]