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/ Supplementary http://www.jimmunol.org/content/suppl/2010/03/15/jimmunol.090204 Material 9.DC1 References This article cites 35 articles, 2 of which you can access for free at: http://www.jimmunol.org/content/184/8/4547.full#ref-list-1 by guest on October 6, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 experi- ments 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 micro- environment. 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 lympho- cytes 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