Lymphotoxin-β Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes This information is current as Christoph Huber, Caroline Thielen, Harald Seeger, Petra of September 28, 2021. Schwarz, Fabio Montrasio, Mark R. Wilson, Ernst Heinen, Yang-Xin Fu, Gino Miele and Adriano Aguzzi J Immunol 2005; 174:5526-5536; ; doi: 10.4049/jimmunol.174.9.5526 http://www.jimmunol.org/content/174/9/5526 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2005/04/18/174.9.5526.DC1 Material http://www.jimmunol.org/ References This article cites 52 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/174/9/5526.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Lymphotoxin-␤ Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes1 Christoph Huber,* Caroline Thielen,† Harald Seeger,* Petra Schwarz,* Fabio Montrasio,* Mark R. Wilson,‡ Ernst Heinen,† Yang-Xin Fu,§ Gino Miele,2* and Adriano Aguzzi2* Affinity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly defined. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT ␤ receptor (LT␤R) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LT␤R signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells. Hence, the set of genes presented in this study includes markers emanating from LT␤R signaling and transcripts relevant to GC Downloaded from and FDC function. The Journal of Immunology, 2005, 174: 5526–5536. ignaling through the lymphotoxin (LT)3 ␤ receptor 4, 7, 8), impaired affinity maturation (LT␤RϪ/Ϫ) (1), and compro- (LT␤R) and the TNFR1 is crucial for organogenesis and mised B cell memory (LT␣Ϫ/Ϫ,LT␤RϪ/Ϫ, and TNFR1Ϫ/Ϫ) (1, 8, S maintenance of the structural integrity of secondary lym- 9). FDCs may represent a key player in promoting and regulating phoid organs (1, 2). These organs provide the compartmentalized these events occurring during the GC reaction. In vitro experi- http://www.jimmunol.org/ microenvironment essential for mounting efficient humoral im- ments have shown that FDCs and FDC-conditioned medium exert mune responses. chemotaxis on B cells and CD4ϩ T cells (10). FDCs can promote Mice deficient for LT␤R, TNFR1, or their ligands suffer from survival and proliferation of GC B cells (11, 12), and coculture complex and partially overlapping pathologic phenotypes of the with FDCs can enhance Ig secretion by B cells (13). lymphoreticular system. TNF-␣ mice and TNFR1-deficient Given the complex nature of GCs, the identification of mole- (TNFR1Ϫ/Ϫ) mice develop lymph nodes, but TNFR1Ϫ/Ϫ mice cules that act downstream of LT and TNF signaling and that may show hypoplastic Peyer’s patches (3–5). LT␣-deficient (LT␣Ϫ/Ϫ) control these phenomena has proven difficult. Also, the specific and LT␤-deficient (LT␤Ϫ/Ϫ) mice lack Peyer’s patches and most molecular contribution of FDCs to these processes has been elu- lymph nodes, except for mesenteric and cervical lymph nodes (6, sive, as it has proven technically impossible to isolate mature by guest on September 28, 2021 7). LT␤RϪ/Ϫ mice display the severest phenotype: they lack Pey- FDCs to purity without the concomitant loss of markers that au- er’s patches and all lymph nodes (1). thenticate their mature state (FDC-M1) and functionality (CD21/ All these mutant mice develop a spleen, but show varying de- CD35 and FDC-M2). grees of disturbance in white pulp compartmentalization, as well as To gain insights into FDC-associated functions controlled by impaired formation of germinal center (GC) and follicular den- LT␤R signaling, many of which may contribute to the GC reac- dritic cell (FDC) networks after challenge with antigenic sub- tion, we pursued two complementary strategies aimed at defining stances. These deficits are reflected in reduced isotype switching the transcriptional profile associated with the presence of GC and (LT␣Ϫ/Ϫ,LT␤Ϫ/Ϫ,LT␤RϪ/Ϫ, TNF-␣Ϫ/Ϫ, and TNFR1Ϫ/Ϫ) (1, 3, FDC networks. First, we used high-density oligonucleotide mi- croarrays to identify transcripts affected in vivo by administration of a soluble LT␤R-Ig, which blocks LT␤R signaling. Second, we *Institute of Neuropathology, University Hospital of Zu¨rich, Zu¨rich, Switzerland; screened for transcripts overrepresented in cell preparations en- † ‡ Institute of Human Histology, University of Lie`ge, Lie`ge, Belgium; School of Bi- riched for FDCs relative to non-FDC splenic cell types by both ological Sciences, University of Wollongong, Wollongong, New South Wales, Aus- tralia; and §Department of Pathology, University of Chicago, Chicago, IL 60637 microarrays and suppression subtractive hybridization. As might Received for publication October 27, 2004. Accepted for publication February be expected, some of the transcripts were found to overlap in both 14, 2005. populations. This allowed us to identify LT␤R signaling-depen- The costs of publication of this article were defrayed in part by the payment of page dent genes preferentially expressed in GCs in association charges. This article must therefore be hereby marked advertisement in accordance with FDCs. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants of the Bundesamt fu¨r Bildung und Wissenschaft (EU) and the Swiss National Foundation, the U.S. National Prion Research Program, Materials and Methods and the NCCR on Neural Plasticity and Repair (to A.A.), and by the Verein zur Enrichment of FDC clusters Fo¨rderung des Akademischen Nachwuchses and the Stiftung fu¨r Biomedizinische Forschung (to C.H.) and Functional Genomics Centre, Zu¨rich. FDCs were prepared by adapting a procedure of Thielen et al. (14). Eight- ␮ 2 Address correspondence and reprint requests to Drs. Adriano Aguzzi or Gino Miele, to 10-wk-old female C57BL/6 mice were injected with 200 g of OVA in Institute of Neuropathology, University Hospital of Zu¨rich, Schmelzbergstrasse 12, alum i.p. to increase the amount of FDCs present in secondary lymphoid CH-8091 Zu¨rich, Switzerland. E-mail address: [email protected] or tissues. Spleens were harvested 3 days after injection and cut in small [email protected] pieces using a tissue chopper. The spleen pieces were digested two times 3 Abbreviations used in this paper: LT, lymphotoxin; SAM, significance analysis of for 20 min at 37°C in RPMI 1640 medium containing 1 mg/ml collagenase microarray; PNA, peanut agglutinin; SSH, suppression subtractive hybridization; A (Boehringer Mannheim), 0.5 mg/ml dispase (type II; Boehringer Mann- DIG, digoxigenin; GC, germinal center; FDC, follicular dendritic cell; ECM, extra- heim), 0.04 mg/ml DNase (type I; Boehringer Mannheim), and 0.4% BSA cellular matrix; LPA, lysophosphatidic acid; MFG, milk fat globule. (Sigma-Aldrich). The supernatants of the two digestions were pooled and Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 5527 the cells were collected by centrifugation. The pelleted cells were resus- using the algorithms provided by the dChip software. For the correlation pended in PBS containing 0.4% BSA and layered over FCS for four re- analysis of the genes represented on the MOE430A chips, cluster A was peated sedimentations at1gat4°C. The cells contained in the supernatant divided into two smaller subclusters exhibiting expression minima at days of the first sedimentation were collected and remaining FDCs were re- 3 or 27 postinjection. The average expression pattern of the two subclusters moved using FDC-M1 coupled magnetic beads (Dynal Biotech). The re- was used to calculate the correlation coefficients. For genes represented on sulting cell fraction was frozen and used as the FDC-depleted sample for the MOE430B chips the entire cluster A of this chip set was used for RNA isolation. To remove contaminating macrophages, the FDC-enriched calculation of the correlation coefficients. Functional annotation of genes cell clusters obtained after the four repetitive sedimentations were incu- was performed using information provided by Affymetrix NetAffx (͗www. bated in a plastic culture dish for 60 min with RPMI 1640 containing 10% affymetrix.com/analysis/index.affx͘), LocusLink (͗www.ncbi.nlm.nih.gov/͘), ͗ ͘ FCS at 37°C, 5% CO2. Nonadherent cell clusters were frozen and used as and PubMed ( www.ncbi.nlm.nih.gov/entrez/query.fcgi ) databases. All the FDC-enriched cell fraction for immunofluorescence staining and RNA microarray data sets are available at ͗www.ncbi.nlm.nih.gov/geo͘. Acces- isolation. sion numbers are GSE2123 (FDC-E vs FDC-D) and GSE2124 (soluble LT␤R-Ig-treated mesenteric lymph nodes). Treatment of C57BL/6 mice with soluble LT␤R-Ig Immunohistochemistry of cytospin FDC clusters Ten-wk-old female C57BL/6 mice were injected with 100 ␮g of soluble LT␤R-Ig i.v.