The Journal of Immunology ●

Cutting Edge: IFN-Producing Cells Respond to CXCR3 Ligands in the Presence of CXCL12 and Secrete Inflammatory upon Activation1

Anne Krug,* Ravindra Uppaluri,† Fabio Facchetti,‡ Brigitte G. Dorner,* Kathleen C. F. Sheehan,* Robert D. Schreiber,* Marina Cella,* and Marco Colonna2*

from the blood to inflamed lymph nodes through HEV. Consistent Human natural IFN-producing cells (IPC) circulate in the with this hypothesis, human blood IPC express L- and blood and cluster in chronically inflamed lymph nodes around CXCR3, the receptor for the inflammatory chemokines CXCL9 high endothelial venules (HEV). Although L-selectin, CXCR4, (monokine induced by IFN-␥), CXCL10 (IFN-␥-inducible and CCR7 are recognized as critical IPC homing mediators, 10), and CXCL11 (IFN-␥-inducible ␣ chemoattractant) (4). the role of CXCR3 is unclear, since IPC do not respond to However, it has been shown that human IPC do not migrate in CXCR3 ligands in vitro. In this study, we show that migration vitro in response to CXCR3 ligands (5). Similarly, IPC do not of murine and human IPC to CXCR3 ligands in vitro requires respond in vitro to CCL3 (macrophage-inflammatory protein 1 engagement of CXCR4 by CXCL12. We also demonstrate that (MIP-1␣)), CCL4 (MIP-1␤), and CCL5 (RANTES) despite the CXCL12 is present in human HEV in vivo. Moreover, after expression of CCR5 (5). Human IPC also express CXCR4 and interaction with pathogenic stimuli, murine and human IPC respond to the CXCR4 ligand CXCL12 in vitro (5, 6). Importantly, secrete high levels of inflammatory chemokines. Thus, IPC CXCL12 secreted by some tumors attracts IPC and protects them migration into inflamed lymph nodes may be initially me- from IL-10-induced apoptosis in vivo (6). Upon activation with diated by L-selectin, CXCL12, and CXCR3 ligands. Upon pathogenic stimuli, IPC up-regulate CCR7 and migrate in re- pathogen encounter, IPC positioning within the lymph node sponse to the CCR7 ligands CCL19 (EBV-induced molecule 1 may be further directed by CCR7 and IPC secretion of in- ligand /MIP-3␤) and CCL21 (secondary lymphoid flammatory chemokines may attract other IPC, promoting tissue chemokine) (5, 7, 8). cluster formation in lymph nodes. The Journal of Immu- Murine IPC have been recently identified (9–12) and found to nology, 2002, 169: 6079Ð6083. be significantly reduced in number in the spleen of L-selectin- deficient mice (9), corroborating a role of L-selectin in IPC mi- atural IFN-producing cells (IPC)3 are a small population gration through HEV. Murine IPC can be cultured in vitro in rel- of leukocytes with a plasma cell-like morphology that atively large numbers (13), providing the opportunity to further N are specialized in the secretion of type I IFN (i.e., IFN-␣, investigate the mechanisms of IPC homing. In this study, we show IFN-␤, and IFN-␻) in response to certain viruses (1–4). Human that murine IPC cultured from (BM) express L-se- IPC have been found in the blood and in lymph nodes affected by lectin, CXCR4, and CXCR3 and migrate poorly in vitro in re- inflammation, clustered around high endothelial venules (HEV) sponse to CXCR3 ligands, like human IPC. Remarkably, we find (4). This anatomical localization has suggested that IPC migrate that CXCL12 restores responsiveness of murine and human IPC to CXCR3 ligands in vitro and that CXCL12 is present in HEV in

Departments of *Pathology and Immunology and †Otolaryngology, Washington Uni- vivo. Following stimulation with viruses, CpG oligonucleotides, or versity School of Medicine, St. Louis, MO 63110; and ‡Istituto di Anatomia Pato- cells expressing CD40 ligand (L), IPC secrete inflammatory che- logica, Universita´ di Brescia, Spedali Civili di Brescia, Brescia, Italy mokines, which may attract other IPC, promoting cluster forma- Received for publication July 8, 2002. Accepted for publication October 2, 2002. tion in secondary lymphoid organs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 A.K. was supported by the Deutsche Forschungsgemeinschaft (KR 2199/1-1). Materials and Methods R.I.U. and R.D.S. were supported by the National Cancer Institute (CA90403, CA76464). Preparation, culture, and stimulation of cells 2 Address correspondence and reprint requests to Dr. Marco Colonna, Department of Murine BM cells from 129 ϫ 1/SvJ mice (The Jackson Laboratory, Bar Pathology and Immunology, Washington University School of Medicine, 660 South Harbor, ME) were cultured in Flt3-L (10 ng/ml; R&D Systems, Minneap- Euclid, Box 8118, St. Louis, MO 63110. E-mail address: mcolonna@pathology. ϫ 6 wustl.edu olis, MN) at 2–4 10 cells/ml for 7–9 days and were sorted into CD11cϩ/CD11blow and CD11cϩ/CD11bhigh subpopulations on a MoFlo 3 Abbreviations used in this paper: IPC, natural IFN-producing cell; HEV, high en- cytometer (purity Ͼ95%; Cytomation, Fort Collins, CO). Human IPC were dothelial venule; IP-10, IFN-␥-inducible protein 10; I-TAC, IFN-␥-inducible T cell ␣ chemoattractant; MIP, macrophage-inflammatory protein; L, ligand; BM, bone mar- isolated and cultured as described previously (4, 7). Cells were stimulated row; Mo-DC, monocyte-derived dendritic cell; DC, dendritic cell; m, mouse; h, with CpG oligonucleotide 2216 (3 ␮g/ml) (14), influenza virus PR8 (0.1–1 human. multiplicity of infection), and CD40L-transfected J558L cells (4).

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00

● 6080 CUTTING EDGE: CXCL12 INDUCES IPC RESPONSIVENESS TO CXCR3 LIGANDS

Abs, flow cytometry, and ELISA Anti-mouse (m) CXCR3 mAb 173 was generated by immunizing male Armenian hamsters with a peptide containing the amino-terminal 37 resi- dues of mCXCR3. This peptide sequence is unique to mCXCR3. Anti- mCXCL9 mAb 2F.5.5.5 was generated by immunizing male Armenian hamsters with recombinant mCXCL9 (R&D Systems). Specificity of 2F.5.5.5 was confirmed by ELISA (data not shown). Anti-mCXCL10 1F11 was kindly provided by A. D. Luster (15). Anti-mCXCR3 was either con- jugated with FITC or detected by biotinylated goat anti-hamster Ab (Jack- son ImmunoResearch Laboratories, West Grove, PA) and streptavidin-al- lophycocyanin (Molecular Probes, Eugene, OR). Cells were counterstained with directly labeled rat anti-mouse CD11c, CD62L, CD11b, or CD86 (BD PharMingen, San Diego, CA). Goat anti-mCXCR4 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) was detected with biotinylated donkey anti-goat antiserum (Jackson ImmunoResearch) and streptavidin-allophy- cocyanin (Molecular Probes). Murine intracellular CCL3, CCL4, and CCL5 were detected as described elsewhere (16). Human intracellular che- mokines were detected with directly labeled Abs (BD PharMingen and R&D Systems). mCXCL9 and mCXCL10 were measured in the superna- tant by ELISA using monoclonal hamster anti-mCXCL9 (5 ␮g/ml) and anti-mCXCL10 (2.5 ␮g/ml), goat anti-mCXCL9 (1 ␮g/ml; R&D Systems), or anti-mCXCL10 (0.2 ␮g/ml; Santa Cruz Biotechnology), biotinylated anti-goat Ab (Vector Laboratories, Burlingame, CA), and avidin-HRP (Sigma-Aldrich, St. Louis, MO). Human CXCL10 and CCL3 were de- tected by ELISA using matched Ab pairs (BD PharMingen and R&D Sys- tems). ABTS (Sigma-Aldrich) was used as substrate.

Chemotaxis assay was measured in a 2-h transwell migration assay using 24-well Costar Transwell chambers (5-␮m pore size; Corning, Cambridge, MA) as described previously (5). Recombinant mCXCL12, mCXCL10, mCXCL11, and human (h) CXCL11 (PeproTech, Rocky Hill, NJ) were added to the lower wells in chemotaxis medium (RPMI 1640/1% human serum albumin), and 1 ϫ 105 cells were added to the Transwell insert. Migrated cells were counted by microscopy and flow cytometry. Where indicated, cells were pretreated with pertussis toxin (100 ng/ml; Sigma- Aldrich) for1hat37°C. FIGURE 1. Expression of L-selectin, CXCR3, and CXCR4 on murine BM-derived IPC. A, Expression of CXCR3 and CD62L on CD11blow IPC. Expression of CXCR3 and L-selectin on CD11bhigh DC was minimal. Gate Immunohistochemistry is set on CD11cϩ cells from Flt3-L-cultured murine BM cells. B, Expres- sion of CXCR3 and CXCR4 on CD11cϩ/CD11blow IPC. C, Expression of Immunohistochemical analysis of Formalin-fixed, paraffin-embedded ϩ ϩ lymph node sections was done as described elsewhere (17) using mono- CXCR3 on Ly-6G/C cells within CD11c DC purified from spleen. D, ϩ clonal anti-hCXCL12 Ab K15C (kindly provided by F. Arenzana-Seis- Down-regulation of CXCR3 and CD62L on gated CD11c cells from Flt3- dedos (18)). L-cultured murine BM cells after incubation for 24 h with influenza virus. E, Kinetics of CXCR3 (f, F) and CD86 (, E) expression in unstimu- lated (F, E)orinfluenza virus-stimulated (f, ) Flt3-L-cultured BM cells Results (gated on CD11cϩ/CD11blow IPC). F, Migratory response to CCL19 (f)or Murine IPC express L-selectin, CXCR3, and CXCR4 control medium () of CD11blow IPC stimulated with influenza virus. G, Dose-dependent down-regulation of CXCR3 on Flt3L-cultured BM cells Murine BM cells cultured in vitro with Flt3-L were highly en- by treatment with CXCL11 and CXCL10 (30 min, 37°C). riched in CD11cϩcells that included CD11blow and CD11bhigh populations. CD11blow cells expressed high levels of B220, Ly6- G/C and secreted type I IFN upon incubation with influenza virus and CpG oligonucleotides (Ref. 13 and data not shown). There- fore, CD11blow cells correspond to IPC in phenotype and function. CXCL12 induces responsiveness of IPC to CXCR3 ligands in As shown in Fig. 1A, CD11blow IPC expressed CXCR3 and L- vitro and is present in HEV in vivo selectin (CD62L). In addition, CXCR3ϩ IPC expressed CXCR4 It has been previously shown that human IPC migrate poorly in (Fig. 1B). CXCR3 was also present on primary IPC from mouse vitro in response to CXCR3 ligands despite the expression of spleen (Fig. 1C). Upon incubation in vitro with influenza virus or CXCR3 (4, 5). Similarly, murine BM-derived IPC showed a min- CpG oligonucleotides, IPC reduced the expression of both L-se- imal migration in response to CXCL11 in chemotaxis assays (Fig. lectin and CXCR3 (Fig. 1D). CXCR3 down-regulation on IPC 2A). In contrast, murine IPC responded to the CXCR4 ligand occurred within 3 h after stimulation of Flt3-L BM cells with in- CXCL12 and, remarkably, became responsive to CXCL11 in the fluenza virus and was paralleled by increased expression of the T presence of CXCL12 (Fig. 2A). The capacity of CXCL12 to induce cell costimulatory molecule CD86 (Fig. 1E). In addition, IPC ac- IPC responsiveness to CXCR3 ligands was also confirmed in hu- quired responsiveness to CCL19 within6hofstimulation with man IPC (Fig. 2B). Migration of murine IPC to CXCL12 was dose influenza virus (Fig. 1F), indicating up-regulation of CCR7 ex- dependent (Fig. 2C). A minimal CXCL12 concentration of 25 pression. CXCR3 expression was also lost after incubation of IPC ng/ml was required for detection of IPC migration to CXCL11 with the CXCR3 ligands CXCL10 and CXCL11 within 30 min at (Fig. 2C). In the presence of CXCL12, chemotaxis to CXCL11 37°C (but not at 4°C), demonstrating the specificity of the anti- showed also dose dependency (Fig. 2D). Pretreatment of IPC with CXCR3 Ab staining (Fig. 1G). pertussis toxin abrogated the migratory response to CXCL12 and The Journal of Immunology 6081

FIGURE 3. CXCL12 expression in human lymph node HEV. Paraffin sections of human lymph nodes were stained with anti-hCXCL12 (brown). GC, Germinal center.

Activated IPC secrete high levels of CXCR3 ligands and other inflammatory chemokines Since CXCR3 expression on IPC was rapidly lost upon viral stim- ulation (Fig. 1, D and E), we asked whether this is an intrinsic feature of IPC activation or whether it is due to autocrine secretion of CXCR3 agonists which down-regulate the cognate receptor. As shown in Fig. 4A, sorted CD11blow IPC secreted high levels of CXCL10 when activated with influenza virus or CpG oligonucle- otides, whereas LPS had no effect. Stimulated IPC also produced the inflammatory chemokines CCL3, CCL4, and CCL5 (Fig. 4B). CD11bhigh cells were also capable of producing inflammatory che- mokines upon stimulation with LPS (data not shown). The capac- ity of IPC to produce high levels of CXCR3 ligands as well as other chemokines was confirmed in human cells. Activation of human IPC with influenza virus, CpG, and CD40L induced pro- FIGURE 2. CXCL12 induces responsiveness of murine BM-derived duction of CXCL10 and CXCL9 as well as CCL3, CCL4, and IPC and human IPC to CXCR3 ligands. A, Chemotactic response to CCL5 (Fig. 5, A and B). IPC chemokine production was compa- CXCL11 (300 ng/ml) and/or CXCL12 (100 ng/ml) of murine CD11blow rable to that of monocyte-derived dendritic cells (Mo-DC) stimu- IPC sorted from Flt3L-cultured BM cells. The percentage of migrated cells lated with LPS and/or CD40L (data not shown) with only two is shown (mean ϩ SEM, n ϭ 3). B, Chemotactic response of purified exceptions: CCL22 was preferentially produced by Mo-DC human IPC to human CXCL11 (300 ng/ml) and/or CXCL12 (100 ng/ml). whereas CCL3 was mainly produced by IPC (Fig. 5C). Thus, IPC low C, Migration of CD11b IPC in response to increasing concentrations of and Mo-DC produce a similar spectrum of chemokines, although CXCL12 in the presence or absence of CXCL11 (300 ng/ml). D, Migration in response to distinct stimuli and, most likely, at different ana- of CD11blow IPC in response to increasing concentrations of CXCL11 in tomical locations. the presence or absence of CXCL12 (100 ng/ml). E, Migration of CD11blow IPC in response to CXCL12 (100 ng/ml) alone or in combination with CXCL11 (300 ng/ml) with or without pretreatment with pertussin Discussion toxin (100 ng/ml). We show that murine BM-derived IPC, like human blood IPC, express L-selectin, CXCR3, and CXCR4 and migrate in response to CXCL12. Most importantly, both murine and human IPC re- quire CXCL12 to respond to CXCR3 ligands in vitro. We also CXCR3 ligands (Fig. 2E), indicating that responsiveness to show that CXCL12 protein is present in lymph node HEV in vivo. CXCR3 ligands induced by CXCL12 requires signaling through

G␣i subunits of heterotrimeric G . To understand whether the cooperation between CXCL12 and CXCR3 ligands observed in vitro has any physiological relevance in vivo for the migration of IPC to secondary lymphoid organs, we investigated which cells express CXCL12 in human lymph nodes and tonsils. As shown in Fig. 3A, a CXCL12-specific mAb strongly stained the area surrounding germinal centers, which was previously shown to be a major source of CXCL12 in tonsils by in situ hybridization (19). In addition, the anti-CXCL12 Ab brightly stained HEV (Fig. 3B), indicating that HEV produce CXCL12 or acquire it by transcytosis (20). Thus, CXCL12 on HEV may me- FIGURE 4. Production of proinflammatory chemokines by murine BM- IPC. A, mCXCL10 was measured by ELISA in the supernatant of diate constitutive migration of IPC into the lymph nodes. The si- CD11blow IPC sorted from Flt3L-cultured BM cells and stimulated with multaneous action of CXCL12 and CXCR3 ligands that are pro- medium, influenza virus, CpG, or LPS for 48 h. B, mCCL3, mCCL4, and duced by HEV during inflammation (21) may boost IPC mCCL5 production by CD11blow IPC was determined by intracellular recruitment to inflamed lymph nodes. staining after stimulation for 18 h with CpG. 6082 CUTTING EDGE: CXCL12 INDUCES IPC RESPONSIVENESS TO CXCR3 LIGANDS

FIGURE 5. Production of proinflammatory chemokines by human IPC. A, hCXCL10 and hCCL3 were measured by ELISA in the superna- tant of purified human IPC (1–5 ϫ 105 cells/ml) stimulated with influenza virus, CpG, or CD40L- transfected cells for 48 h. B, Chemokine production by purified human IPC stimulated for 18 h with CpG, CD40L-transfected cells, CpG plus CD40L, and influenza virus was measured by intracellular staining. C, Production of CCL2, CCL3, CCL4, and CCL22 in Mo-DC stimulated for 18 h with LPS plus CD40L.

Thus, CXCL12 mediates constitutive migration of IPC and capaci- rapidly reach lymph nodes via HEV. Thus, IPC may be important tates IPC to migrate in response to inflammatory chemokines. for the early production of inflammatory chemokines in secondary These results are consistent with the previous demonstration that lymphoid organs, contributing to attraction and retention of Th1- IPC infiltrate tumors secreting CXCL12 in vivo and migrate to prone T cells (30). CXCR3 ligands in vitro through a monolayer of endothelial cells (6). CXCL12 has been previously detected in cultured HEV cells Acknowledgments and shown to support transmigration in vitro (22). We thank William Eades (Siteman Cancer Center Sorting Core Facility, Furthermore, CXCL12 is strongly expressed in endothelial cells Washington University School of Medicine, St. Louis, MO) for expert cell and perivascular cells of the skin (18) and in rheumatoid synovium sorting. (23). 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