Access to the Spleen Microenvironment Through Lymph

Access to the Spleen Microenvironment through Lymph Shows Local Cytokine Production, Increased Cell Flux, and Altered Signaling of Immune Cells during This information is current as Lipopolysaccharide-Induced Acute of October 6, 2021. Inflammation Elvira Semaeva, Olav Tenstad, Jørn Skavland, Marianne Enger, Per Ole Iversen, Bjørn Tore Gjertsen and Helge Wiig

J Immunol 2010; 184:4547-4556; Prepublished online 17 Downloaded from March 2010; doi: 10.4049/jimmunol.0902049 http://www.jimmunol.org/content/184/8/4547 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Access to the Spleen Microenvironment through Lymph Shows Local Cytokine Production, Increased Cell Flux, and Altered Signaling of Immune Cells during Lipopolysaccharide-Induced Acute Inﬂammation

Elvira Semaeva,*,† Olav Tenstad,* Jørn Skavland,†,‡ Marianne Enger,†,‡ Per Ole Iversen,x,{ Bjørn Tore Gjertsen,†,‡ and Helge Wiig*

The spleen is involved in fluid volume regulation, immune responses, and hematopoiesis. Yet,the composition of the fluid phase within the spleen microenviroment, the migratory routes of lymphocytes as well as the splenic response to bacterial endotoxin is incomplete. To address these issues, we isolated postnodal lymph in rats by cannulating an efferent lymphatic draining the spleen, and assessed the secretion of signaling substances during a septic response induced by LPS. Spleen lymph flow increased 8-fold after LPS exposure. The Downloaded from spleen exhibited a permeable microvasculature with low sieving of macromolecules that was absent after exposure to LPS. Further- more, after LPS exposure the spleen contributed significantly to the production of pro- and anti-inflammatory cytokines, and experiments in splenectomized rats suggested it may induce a protracted inflammation because of a dominant role in IL-6 production. A significant amount of lymphocytes exited via lymphatics draining the spleen in control rats. LPS-induced inflammation resulted in increased T cell and reduced B cell subset fractions, and gave a significant increase in CD4+ and CD8+ subset T cell efflux and a reduced B cell efflux in spleen lymph. Exposure of leukocytes to the spleen microenvironment affected their signaling status, and http://www.jimmunol.org/ by phosphorylation specific flow cytometry we could identify STAT3 and CREB as important mediators in the cellular signaling occurring during endotoxemia. We conclude that analysis of spleen lymph may unravel immune cell migration patterns and local signaling, and immune cells exit via lymph having acquired specific activation signatures after exposure to the spleen microenvironment. The Journal of Immunology, 2010, 184: 4547–4556.

he mammalian spleen plays important roles in fluid volume tomized is asplenic overwhelming sepsis, most frequently because regulation as well as in modulation of immune responses of encapsulated bacteria (5). In addition to the immune function, and hematopoiesis. It has been shown that the spleen has T hematopoietic stem cells may initiate extramedullar hematopoiesis by guest on October 6, 2021 a very high blood flow, that it filters off cell-free fluid from the blood in severe bone marrow failure (6) and thus act as a specialized into the lymphatic system and that the rate of filtration is modulated hematopoietic stem cell-supportive microenvironment. by neuronal and humoral factors for example (1–3). Usually the microenvironment is considered as the insoluble With regard to immune function, the spleen combines the role of elements of the extracellular matrix and stromal cells surrounding the innate and adaptive immune system. Its distinctive location in the the parenchymal (or tumor) cells in a tissue. The interstitial fluid, circulatory system as well as its special structure makes the spleen however, contains signaling substances and growth factors that a unique lymphoid organ and a crucial site for early exposure to constitute the internal milieu of the tissue and is also an essential encapsulated bacteria (4). At the same time, splenectomy is a fre- part of the microenvironment. Identification of such locally se- quently used procedure in trauma surgery and in chronic diseases of creted factors like cytokines and chemokines, may significantly cell sequestration in the spleen, and the main risk for the splenec- enhance our understanding of the role of the spleen in immune system activation, hematopoiesis, as well as in fluid volume reg- *Department of Biomedicine and ‡Institute of Medicine, University of Bergen; ulation. There is, however, to our knowledge, no technique †Department of Internal Medicine, Haukeland University Hospital, Bergen; xDepartment of Nutrition, University of Oslo; and {Department of Hematology, Oslo available whereby native spleen interstitial fluid can be isolated. University Hospital, Ullevaal, Oslo, Norway Generally, prenodal lymph is representative for true interstitial Received for publication June 26, 2009. Accepted for publication February 16, 2010. fluid (7). Spleen lymph from rats has been shown to have a protein This work was supported by the Western Norway Regional Health Authority, the concentration slightly lower than plasma (2), supporting the com- Norwegian Cancer Society, and the Research Council of Norway. E.S. was a recipient mon notion that splenic blood vessels are discontinuous and un- of a Ph.D. scholarship from the Western Norway Regional Health Authority, and J.S., M.E., and B.T.G. received grants from the Research Council of Norway’s National selective to macromolecules (4). Data on lymph in the context of Program for Research in Functional Genomics. the spleen microenvironment are, however, scarce, calling for ad- Address correspondence and reprint requests to Prof. Helge Wiig, Department of ditional experiments to reveal the inherent regulatory mechanisms. Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. E-mail address: [email protected] We hypothesized that it would be possible to unravel extracellular signaling mechanisms occurring in the fluid surrounding the blood The online version of this article contains supplemental material. and lymphoid cells by studying the secretion of cytokines and Abbreviations used in this paper: aM, a2-macroglobulin; B, blood; C, complement; COP, colloid osmotic pressure; Fib, fibrinogen; Hapt, haptoglobin; L, lymph; LC, chemokines to the spleen lymph fluid phase. Moreover, analysis of liquid chromatography; MUG, muroglobulin; MS, mass spectrometry; RSA, rat se- the lymph cell fractions during development of inflammation could rum albumin. give important new information on cell modifications occurring in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 the splenic lymphatic bed in these conditions. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902049 4548 SPLEEN MICROENVIRONMENTAL RESPONSE TO LPS

By isolation of spleen lymph, we show that there is a significant HPLC of plasma and lymph efflux of lymphocytes and that the spleen produces significant The distribution of macromolecules in spleen lymph and serum was de- amounts of pro- and anti-inflammatory agents that are secreted into termined by HPLC. We used two 4.6 mm ID 3 30.0 cm TSKgel Super the general circulation during an LPS-induced inflammation, thus SW3000 columns coupled in series (Tosoh Biosciences, Stuttgart, Ger- demonstrating in a quantitative manner the importance of the many) with an optimal separation range for globular proteins of 10–500 spleen in a systemic immune reaction. LPS stimulation resulted in kDa. The protein concentration in the elution fluid was measured by UV detection at 220 nm on a Ettan LC System (GE Healthcare Europe, Hill- a specific lymphatic secretion of IL-6 that was reflected in systemic erød, Denmark) and the buffer/mobile phase was 0.1 M Na2SO4 in 0.1 M serum levels, whereas the rise in serum IL-6 was strongly atten- phosphate buffer pH 6.7–7.0. uated in the splenectomized animals. Furthermore, IL-6 induced Identification of plasma proteins by HPLC-mass spectrometry STAT3 and CREB phosphorylation in lymphatic vessel lymphocytes in vivo, demonstrating an integration of extracellular and After HPLC separation as described previously, macromolecular fractions intracellular signaling that may explain the complex cytokine from plasma and lymph were identified by mass spectrometry (MS) as described in detail elsewhere (10). Briefly, samples were exchanged into pattern observed in septicemia. 100 mM ammonium bicarbonate and concentrated to a final volume of 25 ml and denatured by addition of 25 ml trifluoroethanol (T-8132; Sigma- Materials and Methods Aldrich, St. Louis, MO), 2.5 ml DTT (D-5545; Sigma-Aldrich) at 60˚C Experimental animals and housing for 45 min, and digested at 37˚C over night by trypsin (T6763; Sigma- Aldrich). Digested proteins from plasma and lymph were analyzed using The experiments were performed in Long-Evans rats of either sex (range an Agilent 1100 LC/MSD Trap XCT Plus system consisting of a nanoflow 350–520 g, median 436 g) that were fed a standard laboratory diet. The pump, well-plate sampler, capillary pump, HPLC-Chip/MS interface, and numbers of rats used in the various experiments are specified in Results. All a Trap XCT Plus mass spectrometer. Processing of the MS/MS data were rats were exposed to light on a 12:12 h cycle in humidity- and temperature- performed with Spectrum Mill MS proteomics workbench software (Rev Downloaded from controlled environment. Before the experiment the rats were fasted over- A.03.02.060) using identity mode and default settings. night and had free access to water only. Colloid osmotic pressure Anesthesia and surgery Colloid osmotic pressure (COP) was measured in a colloid osmometer Anesthesia was induced with pentobarbital sodium, 50 mg/kg body weight, designed for submicroliter samples (11) using membranes with a cutoff size given i.p. When anesthetized, the body temperature was maintained at 37˚C of 30 kDa. Usually, samples of 0.5–1 ml were applied to the osmometer 6 1˚C using a heating pad and lamp. Polyethylene (PE-50) catheters were membrane. http://www.jimmunol.org/ placed in one femoral vein for injection of tracers and substances (see below) and in one femoral artery for blood sampling and monitoring blood Analysis of inflammatory mediators in serum and lymph pressure. On termination of the experiment, the rat was killed by cardiac The proinflammatory cytokines IL-1b, IL-6, TNF-a, and the anti-in- arrest induced under anesthesia with an i.v. injection of saturated potas- flammatory cytokine IL-10 were measured simultaneously in serum and sium chloride. All animal experiments were conducted in accordance to lymph samples (n = 10) using Lincoplex kit (Linco research, St. Charles, the regulation of the Norwegian State Commission and with approval from MO) according to the manufacturer’s instructions and as described in the Local Ethical Committee at University of Bergen. detail elsewhere (10). A broad range of standards (4.8–20000 pg/ml) was Lymphatic vessel cannulation provided in the multiplex kit and the multiplexed assay was analyzed on a flow cytometer (Luminex100; Luminex, Austin, TX) with STarStation To sample lymph from the spleen, we used the approach described by 2.0 software (Applied Cytometry System, Sheffield, U.K.). The minimum by guest on October 6, 2021 Kaufman et al. (2). After anesthesia, the rat was laparotomized, and the detection level ranged from 2.3 (IL-1b) to 5.4 (IL-10) pg/ml. spleen was carefully cleared from its attachments to the stomach. The intestine was deflected out of the abdominal cavity and wrapped in par- Flow cytometric analysis afilm. The spleen was carefully laid against the stomach, exposing its Spleen lymph and blood were stained with the following mAbs: PE Cy5 dorsal side and vessels. We could then observe multiple small efferent conjugated mouse anti-rat CD 4, allophycocyanin-conjugated mouse anti- lymphatic tributaries originating in the spleen traversing the spleen rat CD3, FITC-conjugated mouse anti-rat CD 8a, and rat T ⁄ B ⁄ NK cell omentum. Cannulation of such efferent tributaries (afferents to the splenic mixture (BD Biosciences, San Jose, CA). nodes) was deemed impracticable. In some cases, it was difficult to Analyses were performed on a FACSAria flow cytometer (BD Bio- identify one main lymph vessel that with certainty was of splenic origin, sciences), equipped with a 488 nm 100 mW, 532 nm 150 mW, 638 nm but usually (in 70–80% of the rats) a large common lymphatic efferent 30 mW, and 405 nm 50 mW laser. Labeled cells were acquired and vessel draining one or more splenic lymph nodes could be identified and immunophenotyping performed using BD FACSDiva (BD Biosciences) cannulated along the splenic artery. To reduce the trauma involved during software version 6.0. T cells, B cells, NK cells, and CD4/CD8 subsets of sampling, we did not perform extensive surgery to ligate vessels in the CD3 positive T cells were analyzed. vicinity of the spleen to avoid contamination by extrasplenal lymph as In one series of experiments (n = 10), mAbs were put into 5 ml flow described before, during spleen lymph sampling in dogs (8). After ligation cytometry tubes and 50 ml lymph was added. Tubes were incubated for 30 using an ethicon 7-0 chirurgical nonabsorbable suture, the lymph vessel min at room temperature. Then 1 ml BD Pharm Lyse Lysing Buffer was swelled, and after incision, a polypropylene tubing pulled out to an outer ∼ added and samples were incubated for another 8 min. Washing was per- diameter of 0.2 mm and filled with undiluted heparin could be inserted formed three times with 2 ml PBS containing with 0.5% BSA. Samples into the vessel and secured. The cannulation procedure was technically were centrifugated at 350g for 5 min between each washing. Then 250 ml demanding due to a difficult access to the lymph vessels below the left 2% paraformadehyde was added into each tube before analysis. dome of the diaphragm and respiratory movements. After optimization of ∼ To determine absolute counts of leukocytes in spleen lymph in control the technique, cannulation was successful in 75% of the rats. After animals and after LPS treatment, mAbs and 50 ml lymph were added to successful lymph vessel cannulation, the vessel could usually be kept open bead-containing tubes (BD TruCount tubes, BD Biosciences, Becton for the 3–4 h duration of the experimental period (see below). Postnodal Dickinson, San Jose, CA). Tubes were incubated for 15 min at room lymph was thus sampled from a major efferent lymphatic vessel collecting m m temperature before 450 l BD Pharm Lyse Lysing Buffer was added. from several prenodal lymphatics (diameter 40–80 m) that could be Samples were analyzed the same day and the absolute count using Tru- followed from their origin and deriving exclusively from the spleen. That Count tubes was calculated from the appropriate dot plot values entered the lymph originated from the spleen was verified by visual examination in into a spreadsheet that was formatted to use the formula [(no. of events in every experiment. Furthermore, to verify the origin of the sampled lymph, quadrant containing cell population)/(no. of events in absolute-count bead we injected Evans blue bound to human serum albumin in the spleen at the region)] 3 [(total no. of absolute-count beads)/(test volume [50 ml])]. end of the experiment and found that the blue dye appeared in sampled lymph 4–6 min after injection, in agreement with observations in spleen of Determination of single-cell phosphosignaling mice (9). Dye injected in the stomach wall did not appear in the lymph. Lymph was collected into heparinized microhematocrit tubes allowing Single-cell phosphoflow cytometry of blood and spleen lymph cells was quantification of lymph flow. Sampled lymph was either processed for determined as described previously (12). Briefly explained, 150 ml spleen analysis directly or stored immediately at 280˚C for later analysis. lymph was collected and immediately fixed in 850 ml 2% paraformadehyde The Journal of Immunology 4549 for 5 min at room temperature before centrifuged at 750g for 5 min. Collected blood cells were immediately added to BD-Lysis/fix buffer for 10 min at 37˚C. For in vitro experiments, collected blood was split in aliquots and stimulated with IL-6 (100 ng/ml) or LPS (1 mg/ml) at 37˚C for indicated periods (5–120 min). Stimulation was immediately stopped with BD-Lysis/fix buffer at indicated time points. One milliliter 100% ice-cold methanol was added to the prevertexed pellet before samples were stored at 280˚C for later analysis. Paraformadehyde-fixed, methanol-permeabilized cells were rehydrated by adding 4 ml PBS containing0.5%BSA and washed(centrifugationat 750g for 5 min) twice. Directly conjugated Abs were added at optimized concentrations and incubated protected against light and at room temperature for 30 min. Cells from the spleen lymph were stained with the following Abs: p-p38 Alexa 488 (BD Biosciences, Cat. 612595, 6 ml per sample), p-STAT3 Alexa 488 (BD Biosciences, Cat. 557418, 10 ml per sample), and p-CREB PE (BD Biosciences, Cat. 558436, 5 ml per sample). Data were collected using BD FACSDiva software and analyzed using FlowJo (TreeStar, Ashland, OR) and Cytobank (www.cytobank.org), an open- source flow cytometry storage and analysis application. For all samples, a tight gate was drawn around the lymphocyte populations that were gated using forward light scatter versus side light scatter (SSC) and were deemed viable and used for analysis. The normalized p-STAT3 (or p-p38 or p-CREB) response was calculated by transforming the raw data using the inverse hyperbolic sine part of the Downloaded from biexponential class of functions used for digital flow cytometry data (13), and calculating a Log10 change in the 95th percentile of p-STAT3 response between a LPS-stimulated sample and its unstimulated/basal state. Experimental interventions LPS-induced acute inflammation. We mimicked early sepsis by infusion of

LPS from Escherichia coli (serotype 0127:B8, Sigma-Aldrich). The LPS http://www.jimmunol.org/ was diluted in PBS containing 0.1% BSA, and the stock solution had a concentration 0.25 mg/ml. The rats received a dose of 3.0 mg/kg LPS by infusion of stock solution i.v. and were observed for 3 h. In these experiments, the femoral artery was catheterized for monitoring arterial pressure, after the injection of LPS. One subgroup of these rats was used for lymph sampling by lymph vessel cannulation, another subgroup treated identically was used for sampling of blood for ﬂow cytometric analysis and yet another subgroup was subjected to splenectomy (see below). Samples from these were analyzed the same day or stored at 280˚C for later analysis.

Splenectomy by guest on October 6, 2021 To assess the quantitative role of the spleen in the cytokine response after an acute LPS-induced inflammation we added a series of experiments using FIGURE 1. High resolution size exclusion chromatography of plasma splenectomized rats. After anesthesia, laparotomy was made; the spleen was and spleen lymph. Direct comparison of high resolution size exclusion carefully cleared from its attachments to the stomach. The spleen was pherograms from plasma and spleen lymph samples using two 4.6 mm 3 isolated, appropriate blood vessels ligated and the spleen removed. Control 30 cm SuperSW3000 columns coupled in series. The dominant proteins in animals were treated identically, except the spleen was not ligated or re- the peaks were identified by nano LC-/MS/MS, and the curves have been moved. After a postoperative stabilization period of 30 min, LPS was normalized with respect to RSA. Representative patterns for plasma (red injected as described previously. Rats were observed for 180 min either in control or after LPS treatment. Serum was stored immediately at 280˚C for line) and spleen lymph (blue line) in control situation (upper panel) and later analysis. 180 min after exposure to LPS (lower panel) are shown. Elution time at 0.2 ml/min, UV signal at 220 nm in arbitrary units. aM, a2-macroglobulin; C, Statistical methods complement; Fib, fibrinogen; Hapt, haptoglobin; MUG, muroglobulin. All values are means 6 SEM unless otherwise stated. Differences were tested with two-tailed t test using paired comparison when appropriate or ANOVA, followed by Dunn’s or Bonferroni’s test. p , 0.05 was consid- together with that of plasma to compare the relative distribution of ered statistically significant. differently sized macromolecules. As evident from Fig. 1, upper panel, the protein distribution patterns for the two fluids were Results almost similar. Plasma and lymph from seven rats were also an- Lymph was clear without any macroscopic traces of erythrocytes. alyzed by online reversed-phase nano liquid chromatography The flow rate during control conditions ranged from 1.8–3.8 ml/ coupled with ion trap MS to establish the identity of the most min, averaging 2.3 6 0.5 ml/min when assessed in n = 10 rats. abundant proteins in lymph. This analysis showed that the 20–30 of the most abundant plasma proteins were also found in lymph. In Protein distribution pattern and COP in control situation Table I, we have summarized the relative fraction of some abun- The spleen exchange vessels are assumed to be highly permeable to dant plasma proteins identified by MS. All concentrations are plasma proteins. To address the sieving properties of the spleen based on peak area relative to that of rat serum albumin (RSA). microvessels and some of the factors determining transcapillary We observe that there was a lower relative concentration of some fluid flux in this organ, we assessed the protein distribution pattern of the high m.w. plasma proteins in lymph than in plasma, most of spleen lymph. These pherograms were used for a quantification notable for IgM and a2-macroglobulin. Taken together, a similar of the amount of proteins in the various fractions as described protein distribution pattern with a lower mass of some plasma previously (10, 14). Furthermore, we normalized all pherograms proteins suggest a small but significant sieving of high m.w. with respect to albumin and plotted the elution curves for lymph plasma proteins in the spleen microvasculature. 4550 SPLEEN MICROENVIRONMENTAL RESPONSE TO LPS

Table I. Concentration of plasma proteins relative to albumin in control situation and after exposure to LPS

IgM aM Fib MUG Hapt IgG C Control Plasma 0.006 6 0.002 0.150 6 0.036 0.015 6 0.003 0.271 6 0.062 0.048 6 0.008 0.104 6 0.017 0.110 6 0.026 Lymph 0.004 6 0.001* 0.103 6 0.017* 0.016 6 0.004 0.234 6 0.035 0.053 6 0.007 0.109 6 0.014 0.120 6 0.018 LPS 30–60 min Plasma 0.006 6 0.002 0.144 6 0.039 0.016 6 0.004 0.251 6 0.064 0.043 6 0.008 0.095 6 0.017 0.104 6 0.026 Lymph 0.005 6 0.001 0.115 6 0.026 0.015 6 0.002 0.225 6 0.043 0.046 6 0.007 0.099 6 0.012 0.110 6 0.023 LPS 60–90 min Plasma 0.007 6 0.003 0.117 6 0.037 0.015 6 0.005 0.210 6 0.072 0.038 6 0.010 0.079 6 0.021 0.088 6 0.027 Lymph 0.005 6 0.002 0.088 6 0.024 0.012 6 0.003 0.185 6 0.058 0.035 6 0.008 0.074 6 0.017 0.091 6 0.029 Concentrations expressed as plasma protein peak area relative to that of RSA, set to 1.0, having an average concentration of 35 mg/ml in plasma. pp , 0.05 (paired t test) when compared with corresponding protein in plasma. Values are mean 6 SEM. n =7. aM, a2- macroglobulin; C, complement; Fib, ﬁbrinogen; Hapt, haptoglobin; MUG, muroglobulin.

Effect on the splenic lymphatic bed by LPS very low levels of the proinflammatory cytokines IL-1b, IL-6, and a Based on the central role of the spleen in a systemic inflammatory TNF- in samples from serum and spleen lymph, whereas the response, we asked if and how a systemic inflammation induced by anti-inflammatory cytokine IL-10 was not detectable (Fig. 3). Induction of a systemic inflammatory response produced sig-

LPS affected lymph flow and vascular sieving properties. In this Downloaded from series, collecting lymph into heparinized microhematocrit tubes nificant changes in all measured cytokines. In LPS-treated animals b a also allowed quantification of lymph flow. Lymph was sampled the levels of IL-1 , IL-6, IL-10, and TNF- in serum and spleen during a control period of 30 min and then for 3 h after i.v. injection of LPS (n = 6). As evident from Fig. 2A, LPS injection had a significant effect on spleen lymph flow. Flow increased gradually from 2.9 6 0.3 ml/min during the first hour after injection to 16 6 http://www.jimmunol.org/ 1.6 ml/min 3 h after injection. In control rats, there was a slight gradual increase in lymph flow during the experiment, from 1.8 6 0.2 during the first hour to 3.8 6 0.5 ml/min at 180 min (Fig. 2A). These experiments suggest that there is a substantial increase in transcapillary fluid filtration during LPS exposure. Protein distribution pattern and COP during LPS LPS is known to affect the microvascular barrier, for example (15).

The control experiments suggested that some sieving took place in by guest on October 6, 2021 spleen microvessels, and we asked whether LPS affected the spleen vascular barrier by assessing protein distribution pattern, protein quantity in the various m.w. fractions and COP. The total protein concentration in lymph fell as an effect of LPS injection, whereas the relative distribution of proteins in the peaks was similar to that in control situation. There was, however, a relative increase in large proteins, in particular a notable increase in the IgM fraction, as exemplified in Fig. 1, lower panel. The COP in spleen lymph in control situation (n = 5) and after LPS treatment (n = 5) are shown in Fig. 2B. Pressure in control lymph was 17.0 6 0.4 mm Hg in lymph sampled during the first 30 min after cannulation and was stable throughout the duration of the experiment. The corresponding control pressure in plasma was 19.7 6 0.4 mm Hg (Fig. 2B). Endotoxemia induced by infusion of LPS resulted in reduction in plasma and spleen lymph COP, from 19.0 6 0.8 mm Hg to 17.0 6 0.4 mm Hg in plasma and from 17.0 6 0.4 mm Hg to 15.4 6 0.2 mm Hg in lymph. These experiments suggest that although small, some sieving properties are maintained in the spleen microvasculature during an inflammation induced by LPS. FIGURE 2. Spleen lymph flow and COP in experimental endotoxemia. A, Spleen lymph flow after experimental endotoxemia induced by i.v. in- Cytokine concentrations in serum and spleen lymph jection of LPS (3.0 mg/kg) (o). (n = 6). Experiment ended 180 min after d We next asked whether a systemic inflammation produces de- LPS administration. Controls (n =5)( ) received vehicle alone and were 6 p , tectable changes in inflammatory mediators in spleen lymph and kept for 180 min. Values are means SEM. ANOVA, p 0.05 compared with beginning of experiment; xp , 0.05 when compared with respective whether there was a local production of cytokines in spleen. To be values in control animals. B, COP in plasma (circles) and spleen lymph able to monitor whether a systemic inflammatory process was (triangles) in control situation (n = 5) (filled symbols) and in experimental reflected in the splenic lymphatic bed, the proinflammatory endotoxemia induced by i.v. injection of LPS (3.0 mg/kg) (open symbols) cytokines IL-1b, IL-6, TNF-a, and the anti-inflammatory cytokine (n = 5). Values are means 6 SEM. ANOVA, pp , 0.05 compared with IL-10 were measured simultaneously in serum and lymph samples corresponding plasma COP; xp , 0.05 when compared with lymph control (n = 10) using a multiplex technique. In control animals, we found value; #p , 0.05 when compared with plasma control value. The Journal of Immunology 4551

FIGURE 3. Concentration of cytokines in serum and lymph during endotoxemia. Concentration of the proinﬂammatory cytokines IL-1b (A), IL-6 (B), and TNF-a (C) and the anti-inﬂammatory cytokine IL-10 (D) in serum (d) and spleen lymph (o) samples in experimental endotoxemia induced by i.v. injection of LPS (3.0 mg/kg) (n = 5). The experiment ended 180 min after LPS administration. Controls (n =5) received vehicle alone and were kept for 180 min. Serum and spleen lymph samples from control animals exhibited low or undetectable levels of cytokines and data are not shown. ANOVA, pp , 0.05 compared with experimental serum. Values are mean 6 SEM. Downloaded from http://www.jimmunol.org/

lymph were increased compared with control values (Fig. 3). In- whereas at 180 min the level of TNF-a in serum in control ani- terestingly, we found that the average concentration of IL-6 was mals was less (299 pg/ml) than that in splenectomized animals significantly higher in spleen lymph compared with that in serum (333 pg/ml) (Fig. 4B). The levels of anti-inflammatory cytokine during the last part of the experiment (Fig. 3B). LPS also gave an IL-10 were slightly but not significantly higher in the control increase in TNF-a concentration from 60 min duration of LPS group (3765 pg/ml, 3433 pg/ml, 1913 pg/ml at 60 min, 120 min,

exposure. Although the concentration in lymph tended to be and 180 min, respectively) than that in splenectomized animals by guest on October 6, 2021 higher than in serum at all times, this difference was significant at (3591 pg/ml, 2846 pg/ml, 1717 pg/ml at corresponding time 75 min only (Fig. 3C). points). Similarly, the average IL-1b concentrations were slightly Clearly, LPS treatment also resulted in secretion of the anti- but not significantly higher in the group with intact spleen (315 pg/ inflammatory cytokine IL-10 (Fig. 3D). Thus, at 180 min, lymph ml, 1495 pg/ml, 943 pg/ml at 60 min, 120 min, and 180 min, IL-10 averaged 6593 pg/ml, as compared to 308 pg/ml in the respectively) than in the splenectomy group (181 pg/ml, 811pg/ control situation. The level of IL-10 in spleen lymph samples was ml, 643 pg/ml at corresponding time points). These data show the markedly higher than the concentration of this cytokine found in quantitative role of spleen in cytokine production, especially IL-6. serum (Fig. 3D). Flow cytometric analysis of spleen lymph Taken together, these observations suggest that there was a local production of inflammatory as well as anti-inflammatory cytokines To characterize the cellular elements in spleen lymph and to study in spleen after LPS exposure, and that the spleen may provide the whether this pattern was changed during systemic inflammation, systemic circulation with cytokines that can modify the ensuing we performed flow cytometry analysis of spleen lymph and inflammatory response. compared these with corresponding fractions in blood. Gating of lymphocytes was based on flow cytometric forward light scatter Cytokine concentrations in serum in splenectomized rats and SSC and expression of the T lymphocyte markers CD3+, CD4+, treated with LPS CD8+, and the B lymphocyte marker CD45RA. The lymphocyte To investigate more quantitatively the role of spleen as a provider of population could be identified as a separate population in the FCS- cytokines to the general circulation in a systemic inflammation, SSC diagram at the identical place as human lymphocytes (Fig. we studied cytokine production in splenectomized rats (n = 10). As 5). Throughout our data presentation, we describe the results for for animals with intact spleen, induction of systemic inflammatory both blood and lymph samples in control (n = 5) and LPS study response resulted in changes in all of cytokines that were mea- (n = 6). A total of 30,000 events were acquired on average. The sured (Fig. 4). In the group with intact spleens, the mean con- absolute number of T and B cells (determined using Trucount centration of the proinflammatory cytokine IL-6 in control rats tubes) were 7.7 6 2.3 and 1.7 6 1.0 3 106 cells/ml in the control was markedly higher (70,764 pg/ml, 183,580 pg/ml, 201,046 pg/ situation (n = 6). ml at 60 min, 120 min, and 180 min, respectively) than the re- We found that expression of all T cell subsets monitored was spective concentration in splenectomized rats (1060 pg/ml, 22,980 significantly higher in spleen lymph than in blood in control as well pg/ml, 46,232 pg/ml, respectively) (Fig. 4A). Although the dif- as LPS-treated animals (data not shown). Moreover, LPS admin- ference was less pronounced, the average TNF-a levels at 60 min istration resulted in an increase in the percentage of CD3+T cells in and 120 min (1236 pg/ml and 944 pg/ml, respectively) were spleen lymph, from 70.1 6 4.6% in control situation to 82.8 6 higher than that in splenectomized rats (669 pg/ml and 692 pg/ml), 4.5% after LPS (Fig. 6A). Corresponding numbers for blood were 4552 SPLEEN MICROENVIRONMENTAL RESPONSE TO LPS Downloaded from

FIGURE 5. Flow cytometric analysis of spleen lymph. Original ﬂow cytometry curves of spleen lymph showing cellular events distributed relative to size and granularity in control situation (A) and after LPS exposure for 120 min (B). The lower panels show speciﬁc staining and http://www.jimmunol.org/ distribution of CD3+ total T cells and CD45+ B cells in control situation (C) and after LPS exposure for 120 min (D).

FIGURE 4. Concentration of proinflammatoy cytokines after splenectomy. Concentration of the proinflammatory cytokines IL-6 (A) and TNF-a In vivo perturbation of phosphosignaling in spleen (B) in serum of control animals with intact spleen receiving vehicle alone lymphocytes after LPS treatment d ( )(n = 5) and splenectomized (o) (n = 5) rats in experimental endo- LPS induced a specific cytokine secretion pattern in spleen lymph toxemia induced by i.v. injection of LPS (3.0 mg/kg). The experiment and resulted in an increase in lymphocyte cell flux. We therefore ended 180 min after LPS administration. Intact animals received vehicle examined if the LPS response resulted in specific perturbations in alone and were kept for 180 min. ANOVA, pp , 0.05 compared with by guest on October 6, 2021 serum from splenectomized rats. Values are mean 6 SEM. lymphocyte signaling compared with peripheral blood and LPS treated full blood. Cells were harvested from isolated spleen lymph before and 60 and 120 min after LPS treatment in eight rats. We 48.2 6 3.5% and 50.6 6 2.0%. In control situation, the expression determined the level of phosphorylated STAT3 (Y705), CREB of B cells did not differ in spleen lymph and blood, averaging (S133), ERK1/2 (T202/Y204), and p38 (T180/Y182) by protein- 34.2 6 6.0 and 33.5 6 1.9 of the total number of cells in the modification specific Abs and flow cytometry (see Supplemental respective fluids (Fig. 6B). After LPS, however, there was a rapid Fig. 1 for verification of Abs in human and rat leukocytes). The significant drop in B cells in lymph 60 min after LPS exposure to phosphorylation levels of STAT3 and CREB were significantly 21.0 6 3.2%, with a concomitant rise in B cell fraction in blood upregulated after 60 and 120 min of LPS treatment, respectively, to 44.5 6 4.4%. The flow cytometric analysis suggests that the whereas ERK1/2 and p38 demonstrated a weaker increase in systemic inflammation affected the distribution of lymphocytes phosphorylation (Fig. 8A,8B). Peripheral lymphocytes in LPS- exiting through lymphatics draining the spleen. treated animals showed an increased phosphorylation of STAT3 We wanted to determine the flux of cells exiting via spleen and a weaker CREB phosphorylation compared with spleen lymphatics and thus entering the systemic circulation during the lymph leukocytes (Fig. 8C). In addition, peripheral lymphocytes initial phase of an inflammation because the mechanism and demonstrated a more striking phosphorylation of ERK1/2 and quantity of this transport is unknown (4). Because of a variable p38. To exclude some of the systemic effects of LPS, we com- lymph flow and concentration of cells between animals, that may pared with in vitro full blood leukocytes treated with LPS or IL-6. at least partly be due to variable anatomy of the lymph vessels, we In vitro treated lymphocytes demonstrated a striking STAT3 chose to normalize relative to the corresponding control flux. As phosphorylation induced by IL-6 only (Fig. 8E). Based on these evident from Fig. 7A, there was a significant increase of 60% in experiments, we conclude that the signaling profile in lympho- the flux of T cells already after 60 min of LPS exposure, in- cytes harvested from lymph reflect the cytokine stimulation, and creasing slightly further the following hour. The opposite pattern not LPS itself. As judged from Fig. 8B and 8C, it may appear as was observed for B cells (Fig. 7B), having ∼40% of its initial flux if the STAT3 phosphorylation in spleen lymphocytes was weaker in the same period. Whereas both CD4+ (Fig.7C) and CD8+ flux compared with PBLs, and the CREB phosphorylation more pro- (Fig. 7D) increased significantly after LPS exposure, the increase longed. The results from the stimulated samples were therefore was most pronounced for the latter that was 220% of control at compared with its own control. When comparing basal/constitu- 120 min after injection. These experiments showed that a LPS- tive phosphorylation of STAT3 and CREB we observed a signifi- induced inflammation results in an early mobilization of T cells cantly higher phosphorylation level in spleen lymph lymphocytes from the splenic lymphatic bed that may be of importance for the compared with PBLs (Fig. 8D). This may be explained by the immune response. cytokine concentration in the spleen microenvironment from The Journal of Immunology 4553

FIGURE 6. Distribution of leukocyte populations in spleen lymph. Distribution of CD3+ (A), and B cells (B) in spleen lymph (L) (ﬁlled symbols) and blood (B) (open symbols) as percent of total number of leukocytes in control situation (t = 0) and after experimental endotoxemia induced by i.v. injection of LPS (3.0 mg/ kg) (o) (n = 5). Experiment ended 180 min after LPS administration. Controls (n = 5) received vehicle alone and were kept for 180 min. Values are means 6 SEM. ANOVA, pp , 0.05 compared with corresponding control value in blood; xp , 0.05 when compared with respective control values in lymph. L, lymph; B, blood.

where lymphocytes are recruited, but a signaling signature re- was postnodal, a fact that has to be taken into consideration when flecting differences in lymphocyte subsets cannot be ruled out. interpreting the results. Previous data, however, suggest minimal Collectively, these experiments suggest that lymphocytes in node influence. In the kidney, we found that albumin injected spleen lymph acquire a unique signature as response to a systemic prenodally in a hilar lymph vessel was completely recovered in the Downloaded from LPS-induced cytokine increase. thoracic duct (16). Since lymph node protein and fluid exchange decrease with increased flow (17), and the spleen lymph flow rates Discussion were on average ∼four times higher than that of the kidney, our Lymph as a representative for the fluid part of the kidney data suggest minimal exchange of fluid and proteins also in microenvironment the spleen nodes. One way to show that spleen lymph was the

In this study, we have isolated lymph from the spleen and thereby dominant component in the collected lymph might have been to http://www.jimmunol.org/ achieved access to the extracellular fluid phase of an organ of major compare with thoracic duct lymph, which derives from the entire importance for whole body fluid exchange as well as immunological abdominal area (18). We have sampled thoracic duct lymph in function of the organism. A central question in our study is whether a previous study (16), and preliminary experiments showed that its the lymph sampled derives from the spleen only, but the visual protein concentration was ∼65% of that in plasma, that is, lower verification in every experiment of the lymph vessel origin and the than in sampled spleen lymph, indicating that the origin of the dye injection experiments make us confident that we sampled lymph sampled lymph cannot be verified by such a comparison. originating from the spleen. To exclude an effect of the lymph node Another issue is the cellular composition of efferent lymph, on the composition of sampled lymph, we should ideally have which has been studied quantitatively in a sheep model (reviewed in cannulated efferent spleen tributaries (afferents to the splenic node), Ref. 19). These studies show that the majority of the lymphocytes by guest on October 6, 2021 but such cannulation was deemed impracticable. Because our found in efferent lymph draining the popliteal node originate from samples were from a lymphatic draining the splenic node, the lymph blood and enters the lymph node through specialized endothelium

FIGURE 7. Flux of leukocytes to spleen lymph. Flux (concentration multiplied with ﬂow) of T cells (A), B cells (B), and CD4+ (C) and CD8+ T cells (D) to spleen lymph relative to control ﬂux at t = 0 set to 100% after experimental endotoxemia induced by i.v. injection of LPS (3.0 mg/kg) (n = 5). Values are means 6 SEM. ANOVA, pp , 0.05 when compared with respective control values at t =0. 4554 SPLEEN MICROENVIRONMENTAL RESPONSE TO LPS

FIGURE 8. Spleen lymph lymphocytes respond to LPS-induced IL-6 by phosphorylation of STAT3. A, In vivo lymphocyte response to LPS. Spleen lymph samples were collected before and at 60 and 120 min after i.v. injection of LPS and analyzed for phosphorylation of STAT3, CREB, p38 and Erk1/2, here repre- sented by pSTAT3 and pCREB. Phosphoprotein response presented as heat maps of lymph from 8 (B) and PBMC from 5 rats (C) treated with LPS for the indicated time. The control sample was used as a baseline for each phosphoprotein analysis. D,STAT3 and CREB phosphorylation in spleen lymphocytes relative to PBLs after i.v. LPS treatment. Values are Downloaded from mean 6 SE. xp , 0.05 when compared with spleen lymph; pp , 0.05 when compared with respective control values. E, Full blood samples were gated for lymphocytes, monocytes, and granulocytes by side scatter alone before analysis for phosphoprotein stimulation indicated change in phosphorylation of CREB and STAT3 as IL-6 speciﬁc signal response in lym- http://www.jimmunol.org/ phocytes. The data represents three separate experiments. See Materials and Methods for experimental details. by guest on October 6, 2021

in the so called high endothelial venules. It is thus likely that bathing the spleen cells and other cells residing in the splenic some of the lymphocytes isolated derived from venules in the lymphatic bed and to monitor the quantitative role of mediators lymph node and not from the spleen. Interestingly, whereas ef- released from the spleen during inflammation. Comparing the ferent lymph consists of lymphocytes only (19), our finding of cytokine ratio in lymph and plasma revealed a discrepancy between other leukocyte populations (Fig. 5) similar to that of afferent the two compartments, and demonstrated a variable response for lymph (19) may suggest that a significant proportion of the cells each cytokine. The concentrations of the proinflammatory cyto- isolated in our lymph samples derive from the spleen. The exact kines TNF-a and IL-6 and the anti-inflammatory cytokine IL-10 origin of the cells will, however, have to be sought in additional were significantly elevated in lymph compared with plasma after experiments. administration of LPS. Accordingly, these cytokines must be Extensive studies of splenic circulation and the role of the spleen produced and released from cells within the spleen (with a possi- in control of the intravascular fluid volume have been performed by ble contribution from splenic lymph nodes), because any solute Kaufman and coworkers. They have found that the spleen has being transported across the microvasculature from plasma to a very high blood flow (∼8 ml/min/g) (20), and also that there is interstitial fluid and lymph will be present in lower concentrations a significant arteriovenous volume difference that is filtered out of in lymph than in plasma (22). That the spleen production is the blood into the lymphatic system (2, 20). Such filtration is quantitatively important on an organism level was shown by exaggerated during volume loading (2) and LPS infusion (21), a drastically reduced IL-6 response in plasma after LPS exposure. favoring lymph formation even further in these conditions. In line IL-6 is a pleiotrophic mediator that is released “distally” to the with these observations, we found that spleen lymph accumulated “proximal” cytokines TNF-a and IL-1b during sepsis (23). avidly at a rate of ∼2 ml/min in control situation and increasing 8- Among its many functions, it may downregulate the proximal fold this value 3 h after LPS exposure. Because of anatomical cytokines thus limiting the inflammatory reaction. IL-6 further- variation in lymph vessels draining the spleen and the need to more appears to be a good indicator of activation of the cytokine ligate vessels during the preparation process, it is not possible to cascade in sepsis and to predict subsequent organ system dys- measure total lymph flow in the rat using our approach. function and death (23). The lack of IL-6 response after LPS- With the reservations discussed previously regarding the origin exposure in splenectomized animals shows the quantitative role of of the lymph, the present lymph vessel cannulation enabled us to the spleen in mounting an inflammatory response that may be of access the microenvironment of the spleen, namely, the fluid vital importance and predict survival. The Journal of Immunology 4555

Considering the substantial increase in IL-10 in spleen lymph tion of STAT3 and CREB (Fig. 8A–C). The implication of STAT3 after LPS exposure, it was somewhat surprising that there was no activation of spleen-released lymphocytes may be to increase difference between the serum concentrations of IL-10 in sple- survival (32). We suggest that in vivo, lymphocytes in spleen nectomized and intact animals after treatment. The explanation is lymph are activated by the locally produced spleen cytokines IL-6 probably a difference in sympathetic nervous system activation in and IL-10. In vitro, the signaling responses to LPS stimulation in the two experimental situations. Lymph vessel cannulation requires CREB, ERK1/2, and p38 were practically absent (Fig. 8E). more extensive surgery than a simple removal of the spleen. It is To summarize, we have gained access to the microenvironment thus likely that there is a stronger activation of the sympathetic in the spleen by cannulating an efferent lymphatic, and thus been nervous system and thus a drive for IL-10 secretion similar to what able to monitor circulatory fluid balance as well as cellular function has been observed in acute brain injury (24) in the former situation. in the splenic lymphatic bed. The spleen has a relatively high lymph Whereas we are not aware of any previous studies on cytokine flow, a permeable microvasculature exhibiting a low sieving of secretion to spleen lymph invivo, Straub and coworkers have studied plasma proteins. During an LPS-induced systemic inflammation, the regulation of spleen cytokine secretion in vitro using a micro- the spleen produces inflammatory mediators that may actually superfusion chamber (25). With this model they found that stimu- determine the systemic level and thus the ensuing inflammatory lation by bacteria (similar to LPS in our experiments) increased response. A significant amount of lymphocytes exit the spleen via secretion of TNF-a, that after a delay period further stimulated the draining lymphatics, and exposure to the spleen microenvironment secretion of IL-6 (26). Although the timing of their response did not affects their signaling status. The lymphatic path is accordingly match ours exactly, the overall response pattern was similar. important in lymphocyte recirculation and thus immune function.

Function of cells secreted to lymph Downloaded from Disclosures In addition to what has been discussed previously, the LPS stimulus The authors have no financial conflicts of interest. will also elicit a cellular response. The spleen has a role in innate as well as adaptive immunity (4, 27), linked by DCs that mature and differentiate in the spleen microenvironment (28, 29). The precise References mechanisms by which lymphocytes leave the white pulp and the 1. Deng, Y., and S. Kaufman. 1996. Influence of atrial natriuretic factor on fluid J. Physiol. efflux from the splenic circulation of the rat. 491: 225–230. http://www.jimmunol.org/ exact anatomical route they use are, however, unknown (4). We 2. Kaufman, S., and Y. Deng. 1993. Splenic control of intravascular volume in the clearly showed that a significant amount of CD4+, CD8+ T cells, rat. J. Physiol. 468: 557–565. and other immune cells leave the spleen via efferent lymphatics 3. Sultanian, R., Y. Deng, and S. Kaufman. 2001. Atrial natriuretic factor increases splenic microvascular pressure and fluid extravasation in the rat. J. Physiol. 533: and eventually enter the systemic circulation. Although the vol- 273–280. ume of lymph and thus clearance of cells from the spleen may not 4. Mebius, R. E., and G. Kraal. 2005. Structure and function of the spleen. Nat. Rev. Immunol. 5: 606–616. reflect the total drainage due to variable lymphatic anatomy and 5. Hansen, K., and D. B. Singer. 2001. Asplenic-hyposplenic overwhelming sepsis: sampling efficiency, the relative distribution between the various postsplenectomy sepsis revisited. Pediatr. Dev. Pathol. 4: 105–121. fractions of cells should be representative for cells drained. We 6. Kiel, M. J., and S. J. Morrison. 2008. Uncertainty in the niches that maintain haematopoietic stem cells. Nat. Rev. Immunol. 8: 290–301. notice that compared with blood, CD4+ and CD8+ T cell fractions

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