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Proteomic Evaluation of Inflammatory Proteins in Rat Spleen Interstitial Fluid and Lymph during LPS-Induced Systemic Inflammation Reveals Increased Levels of ADAMST1 Eystein Oveland,* Tine V. Karlsen, Hanne Haslene-Hox, Elvira Semaeva, Bartlomiej Janaczyk, Olav Tenstad, and Helge Wiig Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway S Supporting Information *

ABSTRACT: The spleen is a part of the immune system and is involved in the response to a systemic inflammation induced by blood borne pathogens that may induce sepsis. Knowledge about the protein composition of the spleen microenvironment in a control situation and during systemic inflammation may contribute to our understanding of the pathophysiology of sepsis. To our knowledge, the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In order to access the proximal fluid surrounding the splenic cells, we collected postnodal efferent spleen lymph from rats by cannulation, and spleen interstitial fluid (IF) by centrifugation. The origin of the isolated spleen IF was assessed by the extracellular tracer 51 Cr-EDTA and the plasma tracer 125I-HSA. Spleen lymph, IF, and plasma samples were collected during lipopolysaccharide (LPS) induced systemic inflammation and analyzed using a cytokine multiplex assay and, for the first time, using label-free mass spectrometry based proteomics. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 increased severalfold in all fluids after LPS exposure. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were detected after LPS only. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was detected by proteomics (the pro- region) in lymph only after LPS. ADAMTS1 was assessed by ELISA (the metalloproteinase domain), and the concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. A dramatic increase in ADAMTS1 was detected in lymph, IF, and plasma after LPS exposure. In conclusion, the procedures we used to isolate IF and lymph from the spleen during LPS enabled detection of locally produced proteins. Furthermore, we have demonstrated that the inflammatory proteome is different in the spleen microenvironment when compared to that in plasma. KEYWORDS: inflammation, sepsis, spleen, lymph, interstitial fluid, proteomics

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INTRODUCTION Sepsis is a result of bacterial infection in the circulation and is associated with organ dysfunction, hypoperfusion, hypotension, and activation of the immune system.1,2 Severe sepsis is a serious condition, and the survival rate is only 50−75%.3 Experimentally, lipopolysaccharide (LPS) induced inflammation is frequently used as a model of sepsis. The spleen is important in innate and adaptive immunity, and it plays a central role in mounting a response against a systemic inflammation, e.g. induced by LPS.4 This organ plays a central role in B-cell differentiation and dendritic cell maturation occurring in the white pulp5,6 and is also involved in removal of damaged erythrocytes, blood filtration, iron recycling, and storage of plasma cells that takes place in the red pulp.4,7 The rat spleen is a representative model for the human spleen even though rodents do not have an inner and an outer marginal zone as do humans.4 Systemic inflammation and septicemia can be induced in rats by infusion of the LPS © 2012 American Chemical Society

endotoxin, and we have previously shown that the interstitial fluid (IF) surrounding the cells and vascular structures in the spleen are highly affected by such systemic inflammation.8 Furthermore, the spleen is the single most abundant source of the inflammatory cytokine TNF-α.9 Therefore, investigation of proteins specific to the spleen microenvironment during systemic inflammation might increase our understanding of the pathophysiology of sepsis. We recently collected spleen lymph in rats with LPS-induced septicemia and found that the spleen lymph had a protein concentration slightly lower than that of plasma,8 supporting the common notion that splenic blood vessels are discontinuous and unselective to macromolecules.4 In the same study, we demonstrated a local production and secretion of proinflammatory (TNF-α, IL-1β, and IL-6) and anti-inflammatory Received: June 22, 2012 Published: October 2, 2012 5338

dx.doi.org/10.1021/pr3005666 | J. Proteome Res. 2012, 11, 5338−5349

Journal of Proteome Research

Article

Induction of Septicemia by LPS Infusion

(IL-10) mediators during LPS-induced septicemia, an efflux of lymphocytes from the lymph into the general circulation, and that the spleen microenvironment affected leukocyte signaling.8 The isolated lymph, however, was postnodal and possibly modified in the lymph node, calling for additional methods to access and describe the spleen microenvironment. We have previously shown that IF from organs where such fluid is difficult to access can be isolated by exposing the tissue to an increased G-force using centrifugation.10−12 If applied to the spleen, the tissue centrifugation technique might enable isolation of fluid from the microenvironment directly surrounding the splenic cells, such that cytokines and proteins, increased in concentration during inflammation in this rat septicemia model, can be detected. To our knowledge the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In this study, we hypothesized that we could detect locally produced proteins important in the inflammatory process in rats in the spleen microenvironment during LPS-induced sepsis and activation of the immune system. Plasma served as a reference for global expression of proteins present in the systemic circulation. Using label-free proteomics, the plasma proteome was subtracted from the IF and lymph proteomes to detect proteins locally produced in the spleen. Several inflammation-associated cytokines (IL1-β, IL6, TNF-α, and IL10) and proteins (pancreatic α-amylase, peptidyl-prolyl cis− trans isomerase A, and galectin-1) were increased after LPS both in lymph and in IF. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was only detected in splenic lymph after LPS-administration using mass spectrometry (the pro-region). The ADAMTS1 concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. Our approach to address the spleen microenvironment by isolation of proximal fluids should be valuable to identify proteins involved in the pathogenesis of systemic inflammation.

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Septicemia and systemic inflammation was induced in rats by infusion of LPS from Escherichia coli (serotype 0127:B8, Sigma). The LPS was diluted in PBS containing 0.1% BSA to 0.25 mg/mL. The rats received a dose of 3.0 mg LPS/kg by infusion of stock solution intravenously and were observed for up to 3 h. Since using this approach the maximal response on cytokine levels has been found at 90 min,8 this period was used unless otherwise specified. Lymphatic Vessel Cannulation and Plasma Sampling

Efferent lymph vessel cannulation was performed on rats before and 3 h after LPS infusion as previously described.8 Briefly, after anesthesia, the rat was laparotomized and the spleen carefully cleared from its attachments to the stomach. The intestine was deflected out of the abdominal cavity and wrapped in parafilm. The spleen was carefully laid against the stomach, exposing its dorsal side and vessels. The spleen lymph nodes were then identified, and a major lymphatic efferent vessel could be seen as a transparent conduit close to the splenic artery. After ligation with a microvascular clamp, the lymph vessel swelled, and after incision, a polypropylene tubing, pulled out to an outer diameter of about 0.2 mm and filled with undiluted heparin, could be inserted into the vessel and secured. Lymph was collected into heparinized microhematocrit tubes, allowing quantification of lymph flow. To prevent proteolysis, the tubes were pretreated with aprotinin and EDTA and were kept on ice during sampling. Blood was collected in heparinized vials from PE-catheters placed in the femoral artery, before and after LPS. The samples were centrifuged at 14 000g for 10 min to allow isolation of plasma. All samples were stored at −80 °C until processed for analysis. Isolation of IF from the Spleen by Tissue Centrifugation

Rats were infused with LPS (as described above) or with an equal volume of 0.9% NaCl. Ninety minutes later, the rats were euthanized and immediately transferred to an incubator with 100% relative humidity to prevent evaporation during handling of the spleen. The spleen was carefully removed and folded with its visceral side inward and the diaphragmatic side facing a nylon mesh basket (pore size ∼15 × 20 μm) and put into a 2 mL microcentrifuge tube.11,15 The preweighed tube with the nylon basket was immediately capped and reweighed after adding the spleen. The tube was centrifuged at 239g (1500 rpm) for 10 min at 4 °C in an Eppendorf 5417R centrifuge. The spleen IF was collected from the centrifugation tubes inside the humidity chamber. The volumes of extracted fluid from the spleens ranged from 3 to 10 μL with no significant difference between control and LPS-treated animals.

MATERIAL AND METHODS

Experimental Animals

The experiments were performed in Long-Evans rats of both sexes (range 350−20 g, median 436 g) that were fed a standard laboratory diet. Both sexes were represented to equalize effects related to the sex of the rats, since male and female rats might express differences in the acute phase response.13,14 All rats were exposed to light on a 12:12-h cycle in a humidity- and temperature-controlled environment. Before the experiments the rats were fasted overnight and had free access to water only. Anesthesia was induced with sodium pentobarbital, 50 mg/ kg body weight, given intraperitoneally. While anesthetized, the body temperature was maintained at 37 ± 1 °C using a heating pad and a lamp. Polyethylene (PE-50) catheters were placed in the femoral vein for injection of tracers and substances (see below) and in the femoral artery for blood sampling and monitoring of blood pressure. Upon termination of the experiment, the rats, while anesthetized, were euthanized by cardiac arrest caused by an intravenous injection of saturated potassium chloride. All animal experiments were conducted in accordance with the regulations of the Norwegian State Commission and with preapproval from the Local Ethical Committee at the University of Bergen.

Validation of the Fluid Isolated by Centrifugation of the Spleen

After anesthesia and catheter placement, the rats were bilaterally nephrectomized through flank incision to prevent excretion of the extracellular tracer 51C-EDTA. Rats (n = 7) were injected intravenously with 0.1 MBq 51Cr-EDTA (Institute for Energy Technology, Kjeller, Norway), that was allowed an equilibration phase of 120 min. Before termination of the experiment, 0.02 MBq of 125I-albumin (Institute for Energy Technology) was injected intravenously and allowed a circulation time of 5 min as previously described.11 After the 120 min equilibration phase, a blood sample was collected from the arterial catheter, before the rats were euthanized with an 5339



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into 17 mRP-fractions and the IF samples from the centrifugation experiment into 7 fractions. SEC. SEC HPLC of the immunodepleted protein samples was performed on an Ettan LC System (GE Healthcare) using two 4.6 mm i.d. × 30.0 cm TSKgel Super SW3000 columns (Tosoh Biosciences, Stuttgart, Germany) coupled in series (optimal separation at 10−500 kDa). The buffer/mobile phase was 0.1 M Na2SO4 in 0.1 M phosphate buffer pH 6.7−7.0. The protein concentration in the elution fluid was measured by UV detection at 220 nm. Strong Cation Exchange of Peptides (SCX). The mRP fractions were trypsinized (described below) and further separated using HPLC with an SCX column (5065−9942, ZORBAX BioSCX Series II, Agilent Technologies) with settings as recommended by the manufacturer. A flow rate of 100 μL/min was applied, and 23 fractions of 0.6 min each were collected, resulting in 60 μL per fraction. Each of the fractions that contained peptides (gave a chromatographic UV-signal) were evaporated and prepared for LC-MS (≤10 fractions each mRP fraction).

intravenous injection of saturated potassium chloride. The spleen was surgically removed, IF was isolated by centrifugation as described above, and the levels of tracers in IF and serum were measured in an LKB gamma counter as described previously.10 Label-Free Proteomics for Relative Protein Quantification

Lymph, spleen IF, and plasma samples, respectively, from several animals were pooled in order to get a representative selection and sufficient amount of protein for fractionation prior to high performance liquid chromatography mass spectrometry (LC-MS). Using an equal fluid volume from each sample, two sample pools were generated from lymph (L) and two from plasma (P) collected from experimental rats (n = 10), before the intervention (L-CTR, P-CTR), as well as 90 min after the LPS administration (L-LPS, P-LPS). Using equal amounts of protein from each sample (measured by size exclusion chromatography, as described below), one sample pool was generated from IF collected by centrifugation from control rats (IF-CTR, n = 5) and one from rats infused with LPS (IF-LPS, n = 5) using 3−6 μL from each individual to a total volume of 30 μL. The IF samples had variable amounts of hemoglobin due to slight hemolysis that occurred during sample preparation, and hemoglobin was therefore subtracted from the calculation of the protein concentration based on its peak distinguished in the size exclusion (SEC) chromatogram. The L-CTR and L-LPS pools were immunodepleted and fractioned into 17 fractions by macroporous reversed-phase HPLC (mRP) as described for fractionation below. Each of the 17 mRP fractionations were trypsinized and further separated into 10 strong cation exchange (SCX) fractions. The 187 samples (17 + 17 × 10) were analyzed by LC-MS. With respect to the L-CTR, a second mRP fractionation was run and the 17 protein fractions were further separated into 9 fractions by SEC-HPLC followed by trypsination. The additional 170 samples (17 + 17 × 9) were analyzed by LC-MS and the results included in the L-CTR in order to ensure that the proteins associated with inflammation were not discovered in the L-LPS proteome by chance only. The P-CTR and P-LPS pools were immunodepleted and fractioned into 17 fractions by mRP, trypsinized, and analyzed by LC-MS. The mRP fractionation was repeated for both plasma pools, and each of the 17 mRP fractionations were trypsinized and further separated into 6 SCX fractions. All the samples (17 + 17 + 17 × 6 = 136) were analyzed by LC-MS. The IF-CTR and IF-LPS pools were immunodepleted and fractioned into 7 fractions by mRP. Each of the 7 mRP fractions were trypsinized and analyzed by LC-MS in triplicate.

Digestion of Protein Samples with Trypsin

The fractionated protein samples were exchanged into 100 mM ammonium bicarbonate and concentrated using Amicon Ultra4 Centrifugal Filter Units with 3 kDa cutoff (Millipore, Milford, MA). The protein concentration, if not established by HPLC, was determined with a Qubit Quant-IT Protein Assay Kit (Invitrogen), and tryptic peptide samples were prepared as previously described.12 Briefly, protein samples (30 μg) were denatured, reduced/alkylated, and trypsinized (enzyme/protein ratio of 1:30). The tryptic peptide samples were purified using PepClean C-18 spin columns (Thermo Scientific), evaporated in a speed-vac concentrator (Eppendorf) and reconstituted in 100 μL of 0.1% formic acid. Protein Identification by Mass Spectrometry

The peptide samples (1 μL) were analyzed using LC-MS. The instrument used was an 1100 cap/nano HPLC with a microwell-plate sampler coupled to a chip-cube-LC/MSD XCT Plus ion trap mass spectrometer (Agilent Technologies), and the Spectrum Mill workbench (Rev B.04.00, Agilent Technologies) was used for data extraction, searching, and validation as previously described.12,16 The Spectrum Mill settings for the extraction of the raw data were as follows: carbamidomethylation (C) as fixed modification, fragment mass tolerance MH+ interval 600−4000, merge Spectral Similarity and RT and m/z, find precursor C12 with charge and maximum +7 and the S/N minimum 25. The database search settings were as follows: precursor mass tolerance of 2.5 Da and product mass tolerance of 0.7 Da, allow two trypsin miss-cleavages, fixed carbamidomethylation, and variable oxidation of methionine. The database used was the Rattus norwegicus SwissProt version October 2011. Reverse database scores were calculated to allow reversed database false discovery rate (FDR) calculation. The default fixed autovalidation settings were applied for both (1) protein and (2) peptide level, including “calculate FDR using reverse hits”. Proteins were accepted without filtering at precursor charge +2 if the score >6 and %SPI >60 and at charge +3 if the score >8 and %SPI >70. Proteins with a total score >20 were accepted. Peptides were accepted without filtering at precursor charge +2 if the score >11 and %SPI >60 and at charge +3 if the score >13 and %SPI >70.

Fractionation of Protein Samples

Different HPLC-fractionation strategies were used to prepare the pooled IF and lymph samples for LC-MS analyses: Immunodepletion. Samples were depleted of the 7 most abundant plasma proteins (albumin, IgG, α1-antitrypsin, IgM, transferrin, haptoglobin, and fibrinogen) using the Pre-Packed Seppro IgY-R7 LC2 column (Genway Biotech, San Diego, CA, USA) with an Ettan 900 LC System (GE Healthcare, formerly Amersham Biosciences,) according to the manufacturer’s recommendation. mRP. Immunodepleted samples (flow-through) were denatured by adding 0.48 g/mL urea and 13 μL/ml neat acetic acid, injected into an mRP C18 column (Agilent Technologies, 5188−5231) and fractionated as previously described.12 The lymph and plasma samples were separated 5340



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The data generated in the multiplex assay were analyzed by ANOVA followed by Bonferroni posthoc tests. P < 0.05 was considered as statistically significant. Values are given as mean ± SD. SigmaPlot (Version 12.0 Systat Software Inc.) was used for statistical analysis and graphical presentation. The ELISA data were compared using Mann−Whitney test using GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, USA). Values are given as mean ±1 SD, and p < 0.05 was considered statistically significant.

The FDR was less than 1%. Details about the extraction, searching, and validation are presented in Table 1 of the Supporting Information together with the proteomics results. Proteins with ≥1 unique peptide assigned were accepted when ≥2 spectra were identified. Detection of Cytokines Using Multiplex Assays

The pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and the anti-inflammatory cytokine IL-10 were measured simultaneously in serum and lymph samples (n = 10) and in IF in control (n = 5) and LPS-treated rats (n = 5) using a Lincoplex kit (Linco research, St. Charles, MO, now available from Millipore as RCYTMAG-65) according to the manufacturer’s instructions, as previously described.8,10 The multiplexed assay was analyzed on a flow cytometer (Luminex100; Luminex, Austin, TX) with STarStation 2.0 software (Applied Cytometry System, Sheffield, U.K.). The minimum detection level ranged from 2.3 (IL-1β) to 5.4 (IL-10) pg/mL.

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RESULTS

Experimental Design

The levels of inflammation-associated cytokines in plasma and spleen extracellular fluids were analyzed using a multiplex assay (n = 5). In the first proteomics experiment, spleen lymph and plasma samples were collected from the same rats before and 1.5 h after LPS stimulation (n = 10). In the next proteomics experiment, extracellular fluid was isolated by centrifugation of the spleen from control rats (n = 6) and the spleen from LPSexposed rats (n = 6). The protein complements from the two experiments were compared, LPS-associated proteins detected, and candidates assessed with ELISA.

Validation of Protein Candidates Using ELISA Assays

ELISA assay kits (purchased from Alfa Lab AS, Oslo, Norway) were used according to the manufacturer’s instructions to detect and quantify the following proteins in lymph, IF, plasma: Cyclophilin A (E90979Ra, USCN Life Science Inc., Wuhan, China) and ADAMTS1 (E91973Ra, USCN Life Science Inc., Wuhan, China). A sample volume of 1 to 20 μL was diluted in 20 mg/mL BSA in PBS to the recommended volume (100 μL). The optical density was measured using a microplate reader (Spectramax, Molecular Devices, USA) set to 450 nm with wavelength correction and data analyzed and corrected for dilutions using the SOFT max PRO software.

Validation of Whether Collected Fluids Are Representative for the Spleen Microenvironment

In order to analyze the rat spleen microenvironment during LPS-induced septicemia, two different approaches were used: spleen lymph vessel cannulation and spleen centrifugation. Spleen lymph has previously been validated by our group,8 and the sampled lymph was clear and apparently colorless, and had a size distribution of the most abundant proteins according to molecular weight based on SEC similar to that of plasma as previously shown.8 The individual spleen IF samples collected by tissue centrifugation ranged from 3 to 10 μL, averaging 6.3 μL ± 3.5, and they were clear but occasionally had some hemolysis. The distribution of the most abundant proteins in the IF was analyzed using SEC, and the chromatographic profile resembled that of plasma (results not shown). In order to assess the origin of the fluid isolated by centrifugation, we used an approach similar to that introduced upon development of the centrifugation method;11 dilution of an equilibrated extracellular tracer (i.e., 51Cr-EDTA) by intracellular fluid not containing tracer would result in a tracer IF/plasma ratio < 1.0. To address this effect, we measured recovered 51Cr-EDTA in centrifugates and related these concentrations to those of the corresponding plasmas. This ratio averaged 0.7 ± 0.1 (n = 7) and was significantly different from 1.0, showing that fluid not containing tracer, most likely intracellular fluid, had been added to the centrifugate. We also determined the amount that derived from plasma by the recovery in the centrifugate of the intravascular tracer 125I-HSA, that averaged 24.5% ± 11.3% (n = 7). This shows that a significant part of the isolated fluid derived from plasma, reflecting the high degree of vascularity of the spleen and possibly also the discontinuous nature of spleen vessels. Even with these reservations, the fluid isolated by centrifugation of the spleen will be referred to as IF.

Analysis of Data from Proteomics, Multiplex, and ELISA Assays

ProteinCenter (Software Version 3.7, Thermo Fischer Scientific) was used to organize and compare the identified proteins. We intended to investigate the proteins up-regulated after LPS, and therefore, the control proteomes were covered by an additional set of fractionation and LC-MS runs as described in the design of the experiments above. Thus, when a protein was identified in IF or lymph only after LPS, it was considered likely to be increased due to the infection. The proteins identified in the ionTrap MS analysis were measured based on spectral counting, which gives semiquantitative information about protein abundance. The spectral count for a protein was normalized by dividing by the total number of spectra for the respective LC-MS runs, and it was given as log10. Since the distribution of ratios was expected to be non-Gaussian, i.e. containing up- and down-regulated proteins, upper and lower SD values using percentiles were calculated. The z-statistics for quantitative proteomics has recently been described for iTRAQ data17 and is based on the algorithms implemented in the label-free software MaxQuant.18 Briefly, z-scores were calculated for each protein using the upper SD for protein ratios above the median and the lower SD for ratios below the median. Based on the z-scores, the probability of regulation for the upper and lower population, respectively, was calculated using the Gaussian probability function, with the z-table values ensuring a probability of 5%. The p-values obtained were then corrected for multiple hypotheses testing by the Benjamini−Hochberg method as described previously.17 Statistically significant regulation of a protein was reported with more than 5% probability (p < 0.05) both with and without the Benjamini−Hochberg correction.

Elevated Cytokine Levels in the Spleen IF during LPS-Induced Sepsis

We have previously shown that pro-inflammatory (TNF-α, IL1β, IL-6)- and anti-inflammatory (IL-10) cytokines are locally produced in the spleen during septicemia, since they were 5341



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Figure 1. Concentration of cytokines in spleen lymph and spleen IF during LPS infusion. Concentration of the pro-inflammatory cytokines IL-1β (A), IL-6 (B), and TNF-α (C) and the anti-inflammatory cytokine IL-10 (D) in spleen lymph isolated by cannulation and spleen IF isolated by centrifugation in a control situation and 90 min after induction of sepsis by i.v. injection of LPS (3.0 mg/kg) (n = 5). Abbreviations: §, P < 0.05 when compared to the corresponding value in lymph; *, p < 0.05 when compared to the corresponding control value. Concentrations were compared using ANOVA followed by a Bonferroni post hoc test. The corresponding concentrations of these cytokines in plasma have previously been published by our group.8.

significantly higher in lymph than in plasma after LPS.8 Since this analysis was performed on postnodal lymph that may have been modified in the lymph node, we measured the level of these cytokines in IF isolated by centrifugation of spleens from control rats and from rats 90 min after LPS infusion. The concentrations of the pro-inflammatory IL-6, TNF-α, and IL-1β after LPS were 3, 4, and 45 times higher in IF than in lymph, respectively (Figure 1A−C), and that of the anti-inflammatory IL-10 after LPS was 4 times higher in IF than in lymph (Figure 1D). Clearly, LPS administration to the systemic circulation activated the immune response in the spleen, and pro- and antiinflammatory cytokines were secreted to the IF phase. For all the cytokines determined, the concentration gradient from plasma was higher for IF than for lymph.

differentially expressed proteins. Thus, six sample pools were generated and the proteins fractionated, trypsinized, further subfractionated, and analyzed on an LC-ion-trap mass spectrometer. The LC-MS data files were extracted, searched against the Uniprot database (Rattus norwegicus), and validated using Spectrum Mill. The identified proteins in the six groups (averaging 182 proteins each pool) were clustered at 99% homology to reduce ambiguity between protein entries, and the proteomes were compared using ProteinCenter (see Supporting Information Table 1 for complete protein lists). The proteins identified in P-CTR (187 proteins) and P-LPS (164 proteins) were compared, and 49 proteins were detected after LPS only (Figure 2A). These proteins were typically involved in complement activation and acute phase reaction, confirming the onset of a systemic inflammation. The P-CTR and P-LPS proteomes were merged to generate a proteome representative for the systemic circulation (236 plasma proteins) in the two situations. Several proteins were only detected in lymph and IF in response to LPS (not in lymph and IF control) when the systemic plasma proteome was subtracted

Proteins with Increased Levels in Spleen Lymph, Spleen IF, and Plasma after LPS

Spleen lymph and plasma were collected from the same rats before (L-CTR, P-CTR) and 90 min after LPS exposure (LLPS, P-LPS), and spleen IF was isolated from control (IFCTR) and LPS-exposed rats (IF-LPS) for proteomic analysis of 5342



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Figure 2. Detection of up-regulated proteins in spleen lymph and spleen IF during LPS infusion. Spleen lymph and plasma were collected from the same rats before (L-CTR, P-CTR) and after LPS (L-LPS, P-LPS), and spleen IF was isolated from control rats (IF-CTR) and LPS-exposed rats (IFLPS). Six sample pools were generated (L-CTR, L-LPS, P-CTR, P-LPS, IF-CTR, and IF-LPS) and the proteins fractionated, trypsinized, fractionated further, and analyzed on an LC-ion-trap mass spectrometer. The LC-MS data files were extracted, searched against the Uniprot database (Rattus norwegicus), and validated using Spectrum Mill. The identified proteins were clustered at 99% homology and compared using ProteinCenter. (A) Venn diagrams indicating the differences between the plasma proteome before and after LPS. The plasma protein entries were merged to generate a plasma reference proteome. (B) The plasma reference proteome was subtracted, and proteins only identified in L-LPS and IF-LPS used for validation with ELISA are highlighted. (C) Proteins identified in L-CTR plus L-LPS (10) and in IF-CTR plus IF-LPS (76) were analyzed further using z-statistics. Proteins present with significantly higher levels after LPS are indicated at p < 0.05, and the p-value correction method used was that of Benjamini−Hochberg.17.

(Figure 2B). The proteins only identified in L-LPS (68) and IFLPS (10) were investigated and those of extracellular/

membrane origin presented (Tables 1A and 2A). Among these proteins were the inflammation associated galectin-1, 5343



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Table 1. Extracellular/Membrane Proteins Identified in Spleen Lymph after LPS Administration (A) no.

protein name

1 2

Pancreatic α-amylase (PA)a A disintegrin and metalloproteinase with thrombospondin motifs 1 Peptidyl-prolyl cis−trans isomerase A (PPlase A, Cyclophilin A)a

4 5 6

3

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

uniprot

gene

pep

spec

score

cov

aa

P00689 Q9WUQ1

Amy2 Adamts1

9 5

13 9

112 73

23 9

508 967

tm

ss

P10111

Ppia

5

8

63

28

164

Ab2-162; Fam65b Galectin-1a

Q7TP54 P11762

Fam65b Lgals1

5 4

6 6

69 13

4 11

1310 135

Phosphatidylethanolamine binding protein 1 Lymphatic vessel endothelial hyaluronic acid receptor 1 Lymphocyte specific 1, isoform CRA_a Vascular cell adhesion protein 1 Chemokine (C-X-C motif) ligand 1 WAP four-disulfide core domain protein 2 RT1 class I histocompatibility antigen, AA alpha chain Protein NOV homologue; nephroblastoma overexpressed gene Short isoform growth hormone receptor Protein C, isoform CRA_b Mimecan; Osteoglycin

P31044

Pebp1

4

4

53

33

208

D3ZD19

Lyve1

4

4

51

12

343

Q4QQV6

Lsp1

3

5

37

12

331

P29534

Vcam1

2

4

30

3

739

P14095

Cxcl1

2

4

28

27

96

Y

Q8CHN3−1

Wfdc2

2

3

24

14

168

Y

P16391

RT1-EC2

2

3

20

6

371

Q9QZQ5

Nov

2

3

22

8

Q5RJL5

Ghr

2

2

26

Q68FY8 D3ZVB7

Proc Ogn

2 2

2 2

Metalloproteinase inhibitor 2 Insulin-like growth factorbinding protein 2 Hepatoma-derived growth factor GM2 activator protein Chitinase 3-like 1, isoform CRA_a Cathepsin L1 preproprotein

P30121 P12843

Timp2 Igfbp2

2 2

Q8VHK7

Hdgf

Q8CJH4 Q9WTV1

Neutrophil antibiotic peptide NP-4

Y Y

Extracellular form can directly induce leucocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases.42 May be involved in cytoskeletal rearrangement. Glycan-binding, expressed in inflammatory and cancerous tissues, immunoregulation.43 Lipid binding, involved in the extracellular Raf/ Mek/Erk pathway. Mainly expressed in endothelial cells lining lymphatic vessels. Involved in neutrophil activation and chemotaxis. May be involved in immune responses and leucocyte chemotaxis. Neutrophil chemoattractant; may be involved in acute phase inflammatory responses. Serine protease inhibitor, secretory.

Y

Y

Y

Y

Y

Y

protein characteristics Endohydrolysis of oligosaccharides. Involved in inflammation and aggrecan cleavage.

Y

Involved in the presentation of foreign antigens to the immune system.

351

Y

May act as a growth factor.

11

279

Y

28 25

4 8

461 298

2 2

26 23

9 8

220 304

2

2

24

8

237

Gm2a Chi3l1

2 2

2 2

26 29

15 6

199 381

Y Y

P07154

Ctsl1

2

2

21

8

334

Y

Q62714

Np4

1

2

17

10

93

Y

Activates the JAK2/STAT5 pathway. Promotes lymphangiogenesis.44 Catalyzes the inactivation of blood coagulation. May induce bone formation in conjunction with TGF-beta-1 or TGF-beta-2. Irreversibly inactivates metalloproteinases. May inhibit IGF-mediated growth by binding to IGF2 and IGF1. Induces DNA replication in vascular smooth muscle cells. May be involved in sphingolipid metabolism. May be involved in defense against pathogens and tissue remodeling. Important for the overall degradation of proteins in lysosomes. Active in vitro against S. aureus, fungi, Grampositive and Gram-negative bacteria.

Y

Y Y Y Y

(B) no.

protein name

uniprot

gene

spec/pep LPS

spec/pep CTR

norm LPS/ CTR

p

Ben. Hoch.

1 2

Ab2-001 RCG49849

Q7TMC0 D3ZAE6

Mbl2 Vasn

8/3 5/3

2/2 2/2

7.7 4.8

n.s n.s

n.s n.s

3

Cofilin-1

P45592

Cfl1

12/5

5/2

4.6

n.s

n.s

protein characteristics Positive regulation of complement activation. Similar to the human membrane protein vasorin, which may inhibit TGF-β signaling. Regulates actin cytoskeleton dynamics.

(C) no. 1 2 3 4

protein name Nucleobindin-1 Insulin-like growth factor-binding protein 1 Acyl-CoA-binding protein Urinary protein 2/3b

uniprot

gene

pep

spec

score

cov

aa

Q63083 P21743

Nucb1 Igfbp1

7 4

7 10

77 45

18 25

459 272

P11030

LOC 100365425 LOC 680367

3

7

43

33

87

27/17

30/13

P81828/ P83121

2/1

12/2

101

tm

ss

protein characteristics

Y Y

Major calcium-binding protein of the Golgi. Prolongs the half-life of the IGFs and promotes cell migration (in humans).

Y

Intracellular carrier of acyl-CoA esters, possibly also functioning as a neuropeptide. Secreted protein; function not well established.

(A) Proteins only identified in lymph after the rats were exposed to LPS (L-LPS) and not in lymph of control rats (L-CTR) or in plasma neither before (P-CTR) nor after LPS infusion (P-LPS). (B) Proteins increased in level in L-LPS compared to L-CTR and not identified in plasma. Abbreviations: Ben.Hoch., Benjamini-Hochberg multiple hypothesis test corrected p-values; n.s, not significant. (C) Proteins identified in L-LPS and not L-CTR but also identified in P-LPS, exclusively, or increased in P-LPS more than 3 times compared to P-CTR. Abbreviations: pep, number of 5344



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Table 1. continued unique peptides identified; spec, spectra identified; score, protein score from Spectrum Mill; cov, percent protein sequence coverage; aa, amino acids in full-length protein; tm, transmembrane motif; ss, signal sequence (Y = yes); Norm LPS/CTR, normalized number of spectra in L-LPS divided by spectra in L-CTR; p, p-value (calculated using z-statistics; the protein characteristics were retrieved from ProteinCenter and the protein databases Uniprot, NCBInr and RGD). aUp-regulated both in lymph and IF after LPS. bUrinary protein 3 is 96% similar to urinary protein 2, which is identified in plasma, but 2-fold higher in amount after LPS.

Table 2. Extracellular/membrane proteins identified in spleen-IF after LPS administration (A) no.

uniprot

gene

pep

spec

score

cov

aa

Protein S100-A8/A9 heterotetramer Galectin-1

P50115/ P50116 P11762

S100a8/ S100a9 Lgals1

4

17

14

26

202

4

6

33

37

135

3

Leukemia inhibitory factor receptor

O70535

Lifr

3

6

34

3

1093

4

Insulin-like growth factor-binding protein complex acid labile subunit Carboxypeptidase A2

P35859

Igfals

2

6

25

5

603

P19222

Cpa2

2

3

23

5

417

1 2

5

protein name

tm

ss


Y

Y

Y

Y

May be involved in development of endotoxic shock in response to LPS. Glycan-binding, expressed in inflammatoryand cancerous tissues, immunoregulation.43 Type I cytokine receptor family. Stimulation of acute-phase protein synthesis in hepatocytes. Regulating the access of circulating IGFs to the tissues.

Y

Expressed in mesenteric lymph during acute pancreatitis.30

(B) spec/ pep CTR

norm LPS/ CTR

p

Ben. Hoc.

uniprot

gene

spec/pep LPS

P19223 P10111

Cpb1 Ppia

15/5 19/8

2/2 6/4

7.6 3.2

0.00 0.02

0.000 0.175

3

Carboxypeptidase B Peptidyl-prolyl cis−trans isomerase A (PPlase A, Cyclophilin A) Pancreatic α-amylase (PA)

P00689

Amy2

37/15

13/9

2.9

0.03

0.219

4

α-1B-glycoprotein

Q9EPH1

A1bg

119/18

47/15

2.6 (C)

0.04

0.229

no.

protein name

1 2

no.

protein name

1 2 3

Zinc-α-2-glycoprotein Complement factor D Histidine-rich glycoprotein Insulin-like growth factor 1 Complement factor I

4 5

uniprot

tm

ss

protein characteristics Coagulation and anti-inflammatory properties.45 Extracellular form can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases.42 Endohydrolysis of oligosaccharides, increased during pancreatitis. Acute phase protein upregulated during LPS.46

gene

pep

spec

score

cov

aa

Q63678 P32038 Q99PS8

Azgp1 Cfd Hrg

4 3 3

7 7 5

35 48 33

17 21 6

296 263 525

Y Y Y

P08025−1

Igf1

2

4

29

18

153

Y

Involved in lipolysis. MHC class family. Alternative (innate) complement pathway. Regulation of macrophage Fc receptor expression, phagocytosis, thrombosis. Extracellular cytokine, involved in arthritis.


Q9WUW3

Cfi

2

2

22

4

604

Y

Inactivates complement subcomponents.

(A) Proteins only identified in spleen IF after the rats were exposed to LPS (IF-LPS) and not in IF of control rats (IF-CTR) or in plasma before (PCTR) or after LPS (P-LPS). (B) Proteins increased in level in IF-LPS compared to IF-CTR. (C) Proteins identified in IF-LPS and not IF-CTR but also identified in P-LPS and P-CTR. See Table 1 for abbreviations.

“extracellular” were 68% for the plasma reference proteome (P-LPS + P-CTR), 56% for lymph (L-LPS + L-CTR), and 41% for IF (IF-LPS + IF-CTR).

peptidyl-prolyl cis−trans isomerase A (PPlase A, also named Cyclophilin A), pancreatic amylase (PA, also named alphaamylase), and ADAMTS1 (Figure 2B). The two proteins ADAMTS1 and PPlase A were selected for validation using ELISA (see below). Proteins identified in both L-LPS and L-CTR (10) and in both IF-LPS and IF-CTR (76) were analyzed using z-statistics. The probabilities were calculated to determine whether the proteins were significantly elevated in concentration after LPS exposure (Figure 2C and Tables 1B and 2B). Some proteins were identified in lymph or IF after LPS only, and they were also present in plasma, although with more than 3-fold higher levels in P-LPS than in P-CTR (Tables 1C and 2C). Many of the proteins that were found to be elevated in lymph and IF after LPS injection have been associated with acute inflammation. The characteristics and relevance to the spleen microenvironment and sepsis for the candidate proteins are summarized in Tables 1 and 2. The percentages of proteins assigned by ProteinCenter to the gene ontology (GO)

Validation of Proteins Only Detected in IF and Lymph after LPS Administration

From the proteins that were elevated in lymph after LPS in the proteomics experiment (only detected in L-LPS), the two most abundant secretory proteins were chosen to be validated as potential disease markers for septicemia using ELISA: ADAMTS1 and PPlase A. The concentration of the ADAMST1 protein increased significantly after LPS administration in lymph (3 times), IF (4 times), and also plasma (51 times) (Figure 3). This verifies the proteomic results for the lymph samples and highlights ADAMTS1 as a protein involved in the systemic inflammation caused by LPS in rats. Increasing levels of PPlase A (Cyclophilin A) after LPS administration were observed in lymph (25 ng/mL in CTR to 38 in LPS) and plasma (73 ng/mL in CTR to 82 in LPS) but 5345



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detected after LPS only (Supporting Information Table 1). The most intriguing of these proteins was ADAMTS1, found in lymph after LPS only. ADAMTS1 was validated using ELISA and had a higher concentration in lymph and IF after LPS exposure, showing that a quantitative proteomic approach applied to the spleen subproteome was useful when attempting to identify potential candidates involved in the pathogenesis of systemic inflammation. IF and lymph Representing the Extracellular Microenvironment of the Spleen

Identification of proteins in IF and lymph after LPS but not in plasma indicates a local production in the spleen, secretion to the IF, and drainage of fluid containing the protein to systemic lymph. Proteins being transported across the microvasculature to the IF and lymph from plasma will be present in lower concentrations in the IF and lymph than in plasma.19 Therefore, most proteins and substances with higher concentrations in the spleen microenvironment than in plasma after LPS-infusion are locally produced from cells within the spleen (e.g., by activated immune cells). These proteins will originate from active secretion, shedding of cell membranes, cell lysis, and, to a lower extent, plasma. Using tracers, we observed that as much as ∼30% of the collected IF could originate from spleen cells. An explanation for this is mechanical cell lysis and ex-vivo protein production during the removal and centrifugation of the spleen. A higher degree of intracellular proteins in IF than in lymph and plasma samples was also demonstrated by the GO analysis. Such a contribution of intracellular fluid to IF is an explanation of our finding of several-fold higher concentrations of IL1β, IL-6, TNF-α, and IL-10 in the IF than in the lymph after LPS administration. The isotope experiments show that a significant portion of the proteins in IF derived from plasma, which will dilute the IF/plasma gradient in situations with high local production of, for example, inflammatory proteins. Accordingly, the centrifugation method for isolation of IF may not be as useful as in organs and tissues evaluated earlier, but it may be a useful supplement in situations where lymph is not available. For illustrations of the physiology of interstitial fluid and lymph, see previously published work by our group.20,21 Surgical interventions will, to some extent, affect the protein complement and induce stress reactions during fluid sampling

Figure 3. Detection of ADAMTS1 in spleen lymph, spleen IF, and plasma. (A) The concentration of A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was determined in spleen lymph, spleen IF, and plasma before and after LPS administration. ELISA was used as the method of detection. The p-values for the respective fluids before and after LPS were calculated using the twoway Mann−Whitney test (nonparametric).

not in spleen IF; however, the values did not reach statistical significance (results not shown).

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DISCUSSION We have assessed the microenvironment of the spleen, important for immunological function during systemic inflammation, through spleen lymph vessel cannulation and by spleen centrifugation during LPS-induced sepsis in rats. In agreement with our previous study, we observed that the proinflammatory cytokines TNF-α, IL-1β, and IL-6 and the antiinflammatory cytokine IL-10 increased significantly in the spleen IF after LPS administration, as previously shown in the spleen lymph,8 but that the absolute increase in the microenvironment is even more pronounced than indicated by lymph. To our knowledge, we are the first to present the subproteome of the spleen IF and the proteomic changes occurring in the interstitial microenvironment during acute septicemia. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were

Figure 4. Hypothesis for ADAMTS1 regulation during inflammation. ADAMTS1 proteolytic processing events result in several forms with different biochemical properties.27,29 Removal of the signal peptide generates the zymogen pro-ADAMTS1 (110 kDa). Pro-ADAMTS1 is cleaved by a Golgiassociated or an extracellular endopeptidase to the mature form of ADAMTS1 (87 kDa). Further processing of the C-terminal by autoproteolysis of the metalloproteinase domain generates other fragments of the protein (e.g., 65 kDa).27 The peptides identified with MS belong to the pro-region of ADAMTS1 (7% sequence coverage by five unique peptides and nine MS/MS spectra; Supporting Information Table 1). This region was only identified in lymph after LPS exposure and indicates that processing of pro-ADAMTS1 is performed in lymph by an extracellular endopeptidase. The conventional cleavage of the pro-ADAMTS1 occurs in Golgi by the furin endopeptidase; however, extracellular cleavage has been reported and enables targeting of ADAMTS1 to various tissues before activation.27−29 ADAMTS1 was detected by ELISA in IF, lymph, and plasma. The immunogen of the antibodies in the E91973Ra ELISA kit is E. coli-derived recombinant rat ADAMTS1 (amino acid residues 258−467) according to the developer (USCNK). This region is referred to as the peptidase M12B domain, and its sequence is unique for ADAMTS1. 5346



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also increased, probably as a counter-response to stabilize the dramatic increase in acute phase proteins after the systemic inflammation (e.g., Cpb1 and Cfi). Proteins involved in structural modeling of the extracellular matrix increased in IF and lymph after LPS. These proteins regulate cleavage of carbohydrates, proteoglycans, ribonucleic acids, lipids, and proteins (e.g., ADAMTS1 and PA). PA was elevated both in lymph and IF after LPS, and carboxypeptidase B was elevated in IF, which is in accordance with a previous study on mesenteric lymph from rats during inflammation (acute pancreatitis).30 Some of the proteins identified to be elevated after septicemia are likely to be involved in the same biological processes or the same pathways or be directly/indirectly interacting with each other. To study this, the proteins increased in lymph and IF after LPS (Tables 1 and 2) were investigated using STRING.31 A network of interactions between six of these proteins was found (Figure 5). These

from the research animals. In the spleen centrifugation experiment, to isolate IF, we had a surgical intervention control group with Ringer solution. In the lymphatic vessel cannulation experiment, to collect lymph, the L-CTR and L-LPS proteomes were generated from lymph collected from the same animals; however, the animals were exposed to anesthesia and surgery for 90 min longer due to LPS treatment to collect the LPS lymph. Discovery of ADAMTS1 by Mass Spectrometry and Validation Using ELISA

ADAMTS1 was identified in the spleen lymph after LPS infusion of the rats but not detected by MS in plasma or in the spleen IF, neither before nor after the LPS infusion. However, according to the ELISA assay, ADAMTS1 was in fact present in IF and in plasma and increased in concentration after LPS (Figure 3B). Antibody-based quantification of ADAMTS1 with ELISA relies on protein epitopes and the label-free semiquantitative mass-spectrometry approach on peptide spectra matches. ELISA is a target specific approach, which is more sensitive in detection of ADAMTS1 than label-free proteomics. The detection of ADAMTS1 and our hypothesis is explained in Figure 4. ADAMTS1 is a secreted extracellular matrix protease that is positively regulated by the pro-inflammatory IL-1β22 and is involved in tissue organization by cleaving proteoglycan substrates such as versican and aggrecan.23,24 ADAMTS1 is expressed in various tissues during development in mice, and the transcript has been detected in the spleen;25 however, protein expression in the spleen has not been reported. Accordingly, this is a novel observation suggesting that the protein has a role in the LPS-induced disease process. Interestingly, this role can only be demonstrated by considering the control situation, since it is camouflaged by the systemic activation of the immune system when the full-blown reaction to LPS is established. Only one splice-form of the rat ADAMTS1 has been reported, and this encodes a 967 residue protein (Ensembl.org gene annotation); however, several proteolytically processed forms have been published.26−29 The presence of multiple processed forms of ADAMTS1 involving the tryptic peptides or the epitope will give different results between MS and ELISA with respect to pro-ADAMTS1 and the cleaved forms of ADAMTS1. An alternative approach would be to use targeted mass spectrometry of ADAMTS1 forms using selected reaction monitoring (SRM). Cleavage of the propeptide is a prerequisite for activation of ADAMTS1.28 Therefore, it is likely that we identified only the propeptide part of ADAMTS1 in lymph using MS but that the ADAMTS1 protein (zymogen or activated) is increased in lymph, IF, and plasma after LPS. We postulate that ADAMTS1 containing the propeptide (the zymogen) is secreted into lymph and cleaved by an extracellular endopeptidase, possibly postnodal, after LPS infusion.

Figure 5. Interactions between proteins up-regulated in spleen lymph and/or spleen IF after LPS. The entries of the 46 up-regulated proteins in lymph and IF after LPS (Tables 1 and 2) were analyzed in the bioinformatics software STRING 9.031 to investigate whether these proteins have known interactions with each other. Such interactions can be molecular binding, molecular catalyzation, molecular reaction, or the presence in the same study by literature mining (Pubmed, NCBI, NIH, USA). The analysis showed that several of the upregulated proteins are players of the multifunctional growth hormone/ insulin like growth factor axis important for regulation of the immune system during inflammation.

proteins play a role in the multifunctional growth hormone/ insulin like growth factor axis, and the representative proteins have been associated with regulation of the immune system during inflammation.32 In general, systemic down-regulation of IGF-1 is associated with septic shock; however, there is evidence that local production or sequestration of IGF-1 during inflammation may contribute to tissue repair.32,33 An interaction between the proteins S100a8 and S100a9 and between histidine-rich glycoprotein and complement factor D was also postulated (Figure 5). Histidine-rich glycoprotein sequesters the complement factor D and might be involved in regulation of the complement activation,34 which is central in

Proteins Discovered by Proteomics

Abundant acute phase proteins dominated the proteins identified during LPS administration and were subtracted, since they also had high levels in plasma (Figure 2). The presence of proteins involved in activation of an immune response, such as lymphocyte activation and chemotaxis (e.g., Lsp1 and Cxc1) and activation of the complement (e.g., Mbl2), was observed in lymph and IF only after LPS (Tables 1 and 2). Interestingly, some proteins inhibiting these processes were 5347



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systemic inflammation. Interestingly, the inflammatory heterotetramer S100-a8/a9 was up-regulated in the spleen IF and not found in the spleen lymph or plasma, which could indicate a local function in the spleen in addition to a local production. To our knowledge, this is the first time the proteome of the spleen microenvironment has been investigated during systemic inflammation. However, the reference proteomes of spleen cells and lymph nodes in healthy mice have been described35,36 and phosphopeptides representing proteins involved in inflammation (e.g., Dido1, Dok1, GM12250, Hemgn, and Map3k5) were identified in a spleen lysate from mice exposed to Anthrax toxin.37 Furthermore, gene expression microarrays have been used to study alterations to the spleen transcriptome during different inflammatory burdens in mice and pigs.38,39 Proteins involved in hydrolysis of proteins, oligosaccharides, and ribonucleic acids were up-regulated in mesenteric lymph during inflammation in an acute pancreatitis rat model,30 and a few intracellular nitrosative stress proteins have been described in rat spleen during aniline exposure.40 The proteins gelsolin, hemopexin, and α-1-B glycoprotein have been identified in gastric lymph and were up-regulated in nematode-infected sheep.41 Summary and Perspectives

Our results may be considered in a translational perspective, and proteins involved in the inflammatory process caused by septicemia might be investigated further and evaluated as treatment targets. As an example of such previous successful translation, treatment with recombinant human activated protein C (rhAPC), an endogenous anticoagulant with antiinflammatory properties, has been shown to improve survival in severe sepsis, as shown in a recent clinical trial.3 The way to access the rat spleen microenvironment presented here via efferent lymph vessel cannulation and by centrifugation of the spleen may also be useful in other model systems involving activation of the immune system. Taken together, our results show the importance of proximal fluids in the investigation of the spleen subproteome when attempting to identify candidates involved in the pathogenesis of systemic inflammation induced by LPS.

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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Studies on Spleen Microenvironment and Lymph

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +47 55586767. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge Harald Barsnes and Frode S. Berven at The Proteomics Unit at University of Bergen (PROBE) for helpful advice in proteomics and bioinformatics, Odd Kolmannskog for excellent technical assistance, and Ian F. Pryme for proofreading the manuscript. 5348



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