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International Immunology Advance Access originally published online on January 12, 2006
International Immunology 2006 18(2):301-311; doi:10.1093/intimm/dxh369
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A transmembrane chemokine, CXC chemokine ligand 16, expressed by lymph node fibroblastic reticular cells has the potential to regulate T cell migration and adhesion

Takahiro Hara1,2, Tomoya Katakai1,4, Jong-Hwan Lee1,2, Yukiko Nambu1,2, Natsuki Nakajima-Nagata1, Hiroyuki Gonda1,3, Manabu Sugai1 and Akira Shimizu1,2,3

1 Center for Genomic Medicine, Graduate School of Medicine and 2 Division of Systemic Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
3 Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan
4 Present address: 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan

Correspondence to: T. Katakai; E-mail: tkatakai{at}virus.kyoto-u.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Stromal cells in lymphoid tissues provide microenvironmental fields required for the triggering of efficient immune responses. Fibroblastic reticular cells (FRCs) are one of the integral constituents of such stromal fields; they construct the reticular network and are considered to regulate immune cells' behavior. However, the factors that mediate the interaction between lymphocytes and FRCs are poorly understood. Here we show that a mouse lymph node (LN)-derived FRC cell line, BLS4, expresses a transmembrane chemokine, CXC chemokine ligand (CXCL) 16, in response to tumor necrosis factor {alpha} (TNF{alpha}) and IFN{gamma}. TNF{alpha}-induced expression of CXCL16 depends on NF{kappa}B, p38 MAPK and PKA. Matrix metalloproteinase activity is required for producing soluble CXCL16 in the culture supernatant, likely via shedding at the juxtamembrane region of the extracellular domain. IL-12 enhances the expression of CXCR6 in anti-CD3/CD28-stimulated CD8+ T cells and their adhesion to the BLS4 cell surface in a TNF{alpha}-dependent fashion. The adherence is significantly inhibited in the presence of both anti-CXCL16 and anti-vascular cell adhesion molecule 1 (VCAM-1) antibodies. CXCL16 expression is also detected in the FRCs in LN sections and in gp38+VCAM-1+ FRCs isolated from LNs. Taken together, these findings suggest that CXCL16 is an important mediator of lymphocyte–stromal interaction within lymphoid tissues.

Keywords: chemotaxis, CXCR6, inflammatory cytokines, lymphocytes, stromal cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Lymphocytes require specific environmental fields to accomplish their functions efficiently. Secondary lymphoid tissues, including lymph nodes (LNs), spleen and Peyer's patches, are such specialized sites for inducing adaptive immune responses (1, 2). In particular, LNs are located at key positions in the lymphatic system, filtering and monitoring the tissue fluid exudate drained from peripheral tissues and carrying information about those tissues (1–3). The functions of the LNs are carried out via a unique microarchitecture in which distinct subsets of immunohematopoietic cells are strategically compartmentalized (3, 4). Dendritic cells (DCs) carrying information about peripheral tissues function as messengers that arrive at the paracortex (T zone) of the LN (in which the majority of T cells is accumulated), where they prime antigen-specific responses (3, 5).

The non-hematopoietic stromal population supports such microarchitecture in the lymphoid tissues. Atleast two types of stromal cells of mesenchymal origin play crucial roles in the immune cells' distribution and movement within the LN as a structural backbone (6, 7). Follicular DCs are a well-documented type of lymphoid stromal cells that are distributed in B cell follicles or germinal centers, retain antigens for a prolonged period and play crucial roles in B cell attraction, activation and maturation (6). In contrast, fibroblastic reticular cells (FRCs) are a poorly characterized stromal population that constructs an intricate three-dimensional meshwork called the reticular network (RN) by producing fibrous extracellular matrix (ECM) bundles and supports the overall tissue architecture of the LN (7–11). Especially, the RN system in the T zone is an orderly lattice-like network structure, which functions as transport machinery for small molecules and supports T cell–DC interactions (3, 9, 10). The characteristic network scaffold made by these stromal cells appears to be suitable for simultaneously providing mechanical strength to the tissue and making spaces for the active motility of immune cells. The movement of immune cells within the lymphoid tissue is likely to depend on various kinds of chemoattractants and adhesion molecules expressed by stromal cells.

Chemokines are a family of low-molecular weight factors that are involved in the chemotaxis of various cell types and trigger motility signals via seven-transmembrane G-protein-coupled receptors (GPCRs) (12, 13). Most chemokines are secreted from cells as soluble forms, some of which are afterward immobilized via binding to various ECM components to produce a chemokine gradient (13). In contrast, CXCL16 and CX3CL1 are membrane-anchored chemokines, and have been shown to possess an extracellular chemokine domain, mucin-like stalk, transmembrane-spanning region and intracellular part (14–16). These transmembrane chemokines potentially function not only as chemokines but also as adhesion molecules (17, 18). CXCR6 has been identified as a specific receptor for CXCL16 (16). Activated T cells and NKT cells have been shown to express CXCR6, implying the importance of the CXCL16/CXCR6-mediated directional cue both in innate and adaptive immunity (16, 19). However, the details of the function of CXCL16 in lymphocyte behavior within the lymphoid tissues remain poorly understood.

We recently established an FRC cell line, BLS4, from the adherent cell fraction of mouse LNs (11). BLS4 cells express several molecular markers characteristic of FRCs, such as ER-TR7, gp38 and vascular cell adhesion molecule 1 (VCAM-1). Notably, when co-cultured with lymphocytes in vitro, these cells construct an intricate meshwork of ECM fibers containing ER-TR7 antigen. Moreover, BLS4 cells express several chemokines in the steady state or when stimulated with cytokines such as tumor necrosis factor {alpha} (TNF{alpha}). Therefore, BLS4 cells are considered to be a unique model for studying the lymphocyte–stromal interactions occurring in lymphoid tissues. In this study, we characterized in detail the induction of CXCL16 by inflammatory cytokines in BLS4 cells. Our findings suggest that CXCL16 is an important mediator of FRC function.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Mice were maintained at the animal care facility in the Center for Genomic Medicine, Kyoto University. Procedures involving animals and their care were conducted according to the guidelines for animal treatment of the Institute of Laboratory Animals, Kyoto University.

Cells
BLS4 cells and stable transfectants for I{kappa}B{alpha}SR were established as described previously (11) and maintained in 10% FCS–DMEM medium. When necessary, BLS4 cells were stimulated with 10 ng ml–1 TNF{alpha}, 10 ng ml–1 IL-1ß, 5 ng ml–1 IFN{gamma}, 10 ng ml–1 IL-4 (Peprotech, London, UK) or 10 µg ml–1 LPS (Sigma, St Louis, MO, USA) for 48 h. For pharmacological study, BLS4 cells were treated with dimethyl sulfoxide, 25 µM PD98059, 10 µM SB203580, 1 µM KT5720 (Calbiochem, San Diego, CA, USA) or 0.5 µM wortmannin (Nakarai Tesque, Kyoto, Japan). CD8+ T cells were prepared from BALB/c mice LNs. In brief, single-cell suspension of pooled peripheral LNs was depleted of plastic plate-adherent cells and CD8+ cells were enriched as the bound fraction of magnetic cell sorter (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-CD8 antibody (culture supernatant of TIB105 hybridoma) followed by anti-rat IgG microbeads (Miltenyi Biotec). Purified CD8+ T cells were stimulated with immobilized anti-CD3 antibody (PharMingen) in 10% FCS–RPMI1640 medium containing anti-CD28 antibody (PharMingen) and 5 ng ml–1 IL-2 (Peprotech) for 2–4 days. On day 2, the culture was expanded in medium containing 5 ng ml–1 IL-2 with or without 10 ng ml–1 IL-12 (Peprotech). After removing dead cells with Lympholight-M (Cedalane, Hornby, Canada), the activated CD8+ T cells were used for experiments.

Reverse transcription–PCR and northern blotting
Total RNA was extracted from BLS4 cells or purified LN cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For semi-quantitative reverse transcription (RT)–PCR analysis, oligo(dT)12–18-primed cDNA was synthesized from the total RNA using Superscript II reverse transcriptase (Invitrogen). Four- or five-fold serial dilutions of cDNA were amplified by PCR with ExTaq DNA polymerase (Takara, Osaka, Japan) and the following specific primer pairs: GAPDH, 5'-CCATCACCATCTTCCAGGAG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'; CXCL16, 5'-CCTTGTCTCTTGCGTTCTTC-3' and 5'-GGTTGGGTGTGCTCTTTGTT-3', or 5'-GCAACCAGGGCAGTGTCGCTGGAAGTTGTT-3' and 5'-CTCCCAGGGTTGGGTGTGCTCTTTGTTTA-3'; CD11c, 5'-GCCTGTCCCTTGCTGCTGCCACCAA-3' and 5'-GCTCCACTTTGGGTGGTGAACAGTT-3'; Mac-1, 5'-GGAGGCAAAGGCTGTTAACCAGACA-3' and 5'-TCCGGAACTCGTCCGAGTACTGCAT-3'; ADAM10, 5'-GACTGGAGTAGAGGAAGGAGCCCGGGCAC-3' and 5'-CGGTCTGTGAAGACATAGGCCAGGCAGTAG-3'. For northern blot analysis, 20 µg of RNA was separated by electrophoresis on 1% denaturing agarose gels and transferred to Hybond-N+ membranes (Amersham, Piscataway, NJ, USA). The blotted membranes were hybridized with an [{alpha}-32P]deoxycytidine triphosphate-labeled probe for CXCL16 corresponding to the coding sequence of the chemokine domain and subjected to washing under stringent conditions. Northern blots were analyzed by exposure to film followed by densitometry using a BAS2000 Bio-image analyzer (Fuji Film, Tokyo, Japan).

Flowcytometry
BLS4 cells were harvested from culture dishes with 0.02% EDTA–PBS. After blocking with PBS containing 1% BSA and 10% mouse serum, cells were stained with monoclonal anti-CXCL16 antibody (R&D Systems, Minneapolis, MN, USA) followed by biotin–anti-rat IgG (Caltag, Burlingame, CA, USA) and allophycocyanin (APC)–streptavidin (Molecular Probes, Eugene, OR, USA). CXCR6 expressed on T cell surface was detected with CXCL16–hIgG1-Fc fusion protein (CXCL16-Fc, kindly provided by S. Yonehara, Kyoto University) followed by PE–anti-human IgG (Southern Biotech, Birmingham, AL, USA). The cells were then analyzed using a FACScalibur flowcytometer and Cell Quest software (Becton Dickinson, Mountain View, CA, USA).

Immunoprecipitation
BLS4 cells were washed with PBS and lysed in 1% NP-40, 120 mM NaCl, 50 mM Tris, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, 10 µg ml–1 leupeptin and 10 µg ml–1 aprotinin. For preparing culture supernatants, after confluent BLS4 cells in 10-cm culture dishes were cultured with or without murine TNF{alpha} (10 ng ml–1, Peprotech) for 48 h, they were washed with PBS and further incubated in fresh medium for 48 h. Then, culture supernatants were collected, filtered and stored until experiments. To inhibit matrix metalloproteinase (MMP) activity, MMP inhibitor III or GM6001 (Calbiochem) was added to BLS4 cell cultures at various concentrations during the incubation before collecting the supernatants. Cell lysates and culture supernatants were incubated with monoclonal anti-CXCL16 antibody (R&D Systems) overnight at 4°C and precipitated with Protein G-Sepharose 4 Fast-flow (Amersham). The immunoprecipitates were separated by 10% SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore, Bedford, MA, USA). Specific bands were detected using polyclonal biotin–anti-CXCL16 antibody (R&D Systems) followed by streptavidin–HRP (Jackson, Bar Harbor, ME, USA) and chemiluminescent reaction using ECL Plus (Amersham).

Chemotaxis assay
Chemotaxis assays were performed using Transwell inserts (6.5-mm diameter, 5-µm pore, Corning Costar, NY, USA) (20). One hundred thousands CD8+ T cells in 100 µl of medium were added to the upper chamber and 0.6 ml of medium containing recombinant chemokines [mouse CXCL12 (100 ng ml–1) or soluble CXCL16 (20 ng ml–1), Peprotech] or 2-fold-diluted BLS4 culture supernatants were added to the lower chamber. After 2 h of incubation, cells that had migrated to the lower chamber were collected for counting using a FACScalibur flowcytometer with a constant time period (60 s). For blocking the chemokine signaling, T cells were pre-treated with 1 µg ml–1 pertussis toxin (PTx) (Calbiochem) for 30 min at 37°C. For inhibition of chemokines, neutralizing antibodies (10 µg ml–1 each) were added to the supernatant beforehand.

Adhesion assay
BLS4 cells grown on 24-well culture plates were incubated with or without TNF{alpha} for 48 h. CD8+ T cells were labeled with a fluorescent dye, CMFDA (Molecular Probes), for 20 min at 37°C and 3 x 104 cells in medium were added to the BLS4 monolayer. For inhibition of adhesion, antibodies against CXCL16, VCAM-1, ICAM-1 or their combinations (10 µg ml–1 each) were added to the medium. After 1 h of incubation, non-adherent cells were removed and cells bound to the BLS4 monolayer were harvested using 0.05% trypsin, 0.02% EDTA–PBS. CMFDA-labeled cells were counted using a FACScalibur flowcytometer with a constant time period.

Immunohistochemistry
LNs were isolated from mice, embedded in OTC compound (Sakura Finetechnical, Tokyo, Japan) and then frozen in liquid nitrogen (21). Cryosections (20 µm) were fixed with cold acetone and treated with 0.05% Tween 20 PBS containing 1% BSA and 10% goat and mouse sera. Sections were stained with antibodies by direct or indirect methods. Biotin–anti-CXCL16, biotin–goat IgG (R&D Systems), ER-TR7 (BMA, Augst, Switzerland), anti-gp38 (8.1.1) (22) or FITC–anti-CD11c (PharMingen) was used as the primary antibody, and PE–streptavidin (Molecular Probes), FITC–anti-hamster IgG antibody or APC–anti-rat IgG antibody (Caltag) was used as the secondary reagent. Stained sections were mounted with PermaFluor (Shandon, Pittsburgh, PA, USA) and examined using a confocal laser scanning microscope (TSC-SP2, Leica Microsystems, Tokyo, Japan). Digital images obtained were prepared using Adobe Photoshop software (Adobe, San Jose, CA, USA).

Analysis of isolated FRCs from mouse LNs
LN cell suspension containing stromal cells was obtained as described previously (23) with slight modifications. Cells were directly stained for gp38, VCAM-1 and CXCL16 or stained after fixation and permeabilization using Cytofix/Cytoperm solution (PharMingen), and then analyzed by flowcytometry. CXCL16 was detected with biotin–anti-CXCL16 antibody (R&D Systems) followed by streptavidin–APC (Molecular Probes). For the enzymatic digestion of BLS4 cells, cells were harvested from culture dishes with 0.02% EDTA–PBS and treated with trypsin (0.2%, 37°C, 10 min) or collagenase type I (1 mg ml–1, 37°C, 30 min). For further purification of FRCs, cells were labeled with anti-VCAM-1 antibody followed by anti-rat Ig{kappa} light chain particles-DM (BD Biosciences). After magnetic separation using BD IMagnet (BD Biosciences), collected cells were further stained with anti-gp38 followed by FITC–anti-hamster IgG and PE–anti-rat IgG antibodies. VCAM-1+gp38+ FRCs were sorted by BD FACSAria (BD Biosciences).

Frozen section adhesion assay
Adhesion of T cells to LN section was assessed as described previously (10). T cells were labeled with fluorescent dyes, CMFDA (5 µM) or CMTMR (15 µM) (Molecular Probes) at 37°C for 20 min, followed by treatment with control human IgG or CXCL16-Fc at 4°C for 30 min. The adhesion assay was performed at 37°C for 45 min. After removal of non-adherent cells, section was fixed with 5% PFA–PBS and stained for ER-TR7. Then, digital images were obtained by confocal microscopy and bound cells were counted. Statistical significance was determined using Student's t test. P-values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Inflammatory cytokines induce the expression of CXCL16 in BLS4 cells
During the course of searching for chemokines expressed in an LN FRC cell line, BLS4, we found that the expression of a membrane-type chemokine, CXCL16, is induced by inflammatory cytokines such as TNF{alpha}, IL-1 or IFN{gamma}, or with LPS (Fig. 1A) but not IL-4 (data not shown) as detected by semi-quantitative RT–PCR. By northern blot analysis, mRNAs for CXCL16 were also detected as two distinct bands of ~1.8 and ~2.5 kb. The transcripts appeared as early as 2 h and reached a maximum level by 18 h after the TNF{alpha} stimulation, indicating that this chemokine is an early-responding gene (Fig. 1B). Using a mAb recognizing the chemokine domain of CXCL16, we also detected TNF{alpha}- and IFN{gamma}-induced expression on the surface of BLS4 cells by flowcytometry (Fig. 1C). Moreover, these two cytokines showed a synergistic effect on the CXCL16 expression (Fig. 1C and D).


Figure 1
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  		Fig. 1. CXCL16 is induced in BLS4 cells by inflammatory cytokines. (A) BLS4 cells were stimulated with TNF{alpha}, IL-1ß, IFN{gamma} or LPS for 48 h and mRNAs for CXCL16 were detected by semi-quantitative RT–PCR analysis. Five-fold serial dilutions of cDNAs were amplified with specific primers and the number of PCR cycles was 21 for GAPDH and 30 for CXCL16. PCR products were standardized relative to GAPDH. (B) Time course of CXCL16 expression induced by TNF{alpha}. BLS4 cells were stimulated with TNF{alpha} for various periods and CXCL16 transcripts were detected by northern blot analysis. Ethidium bromide staining of total RNAs is also shown. (C) Cell-surface expression of CXCL16 in BLS4 cells. BLS4 cells were stimulated with TNF{alpha} and/or IFN{gamma} for 48 h and assessed by flowcytometry. Staining with control rat IgG (open) and anti-CXCL16 antibody (filled) is shown in each histogram. (D) TNF{alpha} and IFN{gamma} affect CXCL16 induction synergistically. BLS4 cells stimulated with TNF{alpha} and/or IFN{gamma} at various concentrations were assessed by flowcytometry. CXCL16 expression is shown as {Delta}MFI (the difference in mean fluorescent intensity values between control antibody and anti-CXCL16 antibody staining).

 
Since TNF{alpha}, IL-1 and LPS all activate the transcription factor NF{kappa}B (24), we next addressed the involvement of this transcription factor in CXCL16 expression by using a stable transfectant for I{kappa}B{alpha}SR, a dominant inhibitory mutant of NF{kappa}B activation (25). Strikingly, the CXCL16 induction was completely suppressed in the I{kappa}B{alpha}SR transfectant, while control cells (vector only) showed normal CXCL16 expression (Fig. 2A). Thus, NF{kappa}B activation is indispensable for the induction of CXCL16 expression in BLS4 cells by TNF{alpha}.


Figure 2
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  		Fig. 2. CXCL16 induction by TNF{alpha} is NF{kappa}B, p38 MAPK and PKA dependent. (A) Control (vector) or I{kappa}B{alpha}SR transfectant was stimulated with TNF{alpha} for 48 h and CXCL16 expression was detected by flowcytometry. Staining with control antibody (open) and anti-CXCL16 antibody (filled) is shown in each histogram. (B) BLS4 cells were stimulated with TNF{alpha} in the presence of pharmacological inhibitors for 48 h and CXCL16 expression was detected by flowcytometry (upper panels). {Delta}MFIs (the difference in mean fluorescent intensity values) were shown in (C).

 
To further know about the signaling pathways involved in the CXCL16 induction driven by TNF{alpha}, we tested the effects of various pharmacological inhibitors. Interestingly, p38 MAPK inhibitor SB203580 and PKA inhibitor KT5720 markedly suppressed the CXCL16 expression, whereas PI3K inhibitor wortmannin or MEK1/2 inhibitor PD98059 did not (Fig. 2B and C). In addition, simultaneous treatment of SB203580 and KT5720 completely blocked CXCL16 induction (Fig. 2B and C). These results indicate that, along with NF{kappa}B, signalings mediated by p38 MAPK and PKA also play crucial roles in the TNF{alpha}-triggered CXCL16 expression in BLS4 cells.

Production of soluble CXCL16 by BLS4 cells depends on MMP activity
From its amino acid sequence, CXCL16 is estimated to be an ~27-kDa protein, but the full-length band detected in TNF{alpha}-stimulated BLS4 cell lysate as assessed by SDS-PAGE was ~50 kDa, indicating that the protein is highly glycosylated (Fig. 3A). In order to examine whether BLS4 cells can produce a soluble form of CXCL16, the culture supernatant was collected for the immunoprecipitation of CXCL16 using antibody recognizing the chemokine domain. A band significantly smaller (~33 kDa) than full-length CXCL16 was detected in the immunoprecipitate from BLS4 culture supernatant in a TNF{alpha}-dependent manner (Fig. 3A). This suggests that the soluble CXCL16 produced by BLS4 cells is an extracellularly truncated form. When BLS4 cells were treated with pharmacological inhibitors of MMP, MMP inhibitor III or GM6001, during TNF{alpha} stimulation, the amount of soluble CXCL16 produced in the culture supernatant was markedly reduced, while the amount of the full-length protein in the cell lysate was conversely increased (Fig. 3B). Therefore, MMP activity in the BLS4 cells is involved in shedding cell-surface CXCL16 to release the soluble form.


Figure 3
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  		Fig. 3. BLS4 cells produce soluble CXCL16 in a manner dependent on MMP activity. (A) Detection of soluble CXCL16 in the supernatant of BLS4 cells. BLS4 cells were pre-incubated with or without TNF{alpha}, and CXCL16 protein in cell lysates or supernatants (sup.) was immunoprecipitated and detected by western blotting. Full-length and soluble forms were detected as ~50- and ~33-kDa bands, respectively. Recombinant protein (rCXCL16: 6x histidine-tagged extracellular domain) was used as control (~40 kDa). Cross-reaction to Ig light chain was also observed (IgL). (B) Inhibition of MMP activity reduces the production of soluble CXCL16 by BLS4 cells. BLS4 cells pre-stimulated with TNF{alpha} were further treated with MMP inhibitor III for 48 h and supernatants or cell lysates were collected. CXCL16 was detected by immunoprecipitation followed by western blotting.

 
Soluble CXCL16 produced by BLS4 cells partially mediates the chemotaxis of activated CD8+ T cells
Activated T cells have been reported to express CXCL16-specific receptor, CXCR6 (16). We also confirmed that the expression of CXCR6 was induced in CD8+ T cells isolated from mouse LNs and activated with anti-CD3 and anti-CD28 antibodies (Fig. 4A). In addition, the presence of IL-12 during the stimulation significantly enhanced the expression of CXCR6 in the activated CD8+ T cells (Fig. 4A), while neither IFN{gamma} nor IL-4 showed any remarkable effect (data not shown). The induction of CXCR6 was also observed to a lesser extent in CD4+ T cells stimulated with anti-CD3, anti-CD28 antibodies and IL-12 (data not shown). We next checked the in vitro chemotactic activity in IL-12-stimulated, activated CD8+ T cells. As expected, the chemotaxis of the IL-12-stimulated cells toward recombinant CXCL16 was elevated compared with that of cells in the absence of this cytokine, whereas the responsiveness toward CXCL12 was only slightly augmented (Fig. 4B). Blocking GPCR-mediated chemotactic signals with PTx completely abolished the responsiveness to CXCL16 (Fig. 4B). Therefore, CXCR6 expression and chemotaxis in response to CXCL16 are well correlated in our assay system.


Figure 4
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  		Fig. 4. IL-12 augments CXCR6 expression in activated CD8+ T cells and increases chemotaxis toward CXCL16. (A) The presence of IL-12 during activation enhances CXCR6 expression in CD8+ T cells. CD8+ T cells isolated from LNs were stimulated with anti-CD3, anti-CD28 antibodies and IL-2 in the presence or absence of IL-12. CXCR6 expression was detected by CXCL16-Fc binding and analyzed by flowcytometry. Staining with control human IgG (open) and CXCL16-Fc (filled) and the percentages of CXCR6-positive fractions are shown in each histogram. (B) IL-12-stimulated, activated CD8+ T cells show chemotaxis toward recombinant soluble CXCL16. Chemotactic activities of cells were determined by a conventional transwell assay and the results are shown as fold increment relative to the control (medium). For blocking chemokine receptor signals, cells were pre-treated with PTx. CXCL12 was used as a positive control.

 
Culture supernatant from unstimulated BLS4 cells induced little chemotactic activity in the IL-12-activated CD8+ T cells (Fig. 5A). In marked contrast, culture supernatant from TNF{alpha}-stimulated BLS4 cells dramatically elevated the chemotaxis of the same cells (Fig. 5A), suggesting that the TNF{alpha}-induced chemokines from BLS4 cells induce the attraction of T cells. We found that neutralizing antibody to CXCL16 constantly inhibited ~20% of the migration of IL-12-activated CD8+ T cells (Fig. 5A), indicating that this fraction of the reduction in chemotaxis is likely to be due to the contribution of soluble CXCL16 in the mixture of chemokines produced by BLS4 cells. Besides CXCL16, we previously have shown that many other chemokines are induced in BLS4 cells by TNF{alpha} stimulation (11). Thus, as expectedly, from chemotaxis assay using neutralizing antibodies, we observed that CCL5 plays a relatively major role in T cell chemotaxis toward the supernatant of activated BLS4 cells, while CCL4 and CXCL12 have some minor contributions similar to CXCL16 (Fig. 5A). Therefore, CXCL16 is one of the chemoattractants for activated T cells produced by BLS4 cells.


Figure 5
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  		Fig. 5. IL-12-activated CD8+ T cells efficiently migrate toward TNF{alpha}-stimulated supernatant and adhere to the cell surface of BLS4 cells. (A) Supernatant of TNF{alpha}-stimulated BLS4 cells supports chemotaxis of IL-12-activated CD8+ T cells and CXCL16 accounts for a fraction of this activity. The chemotaxis assay was performed using diluted supernatants from BLS4 cultures as attractants. Neutralizing antibodies to the indicated chemokines were added to the supernatant for inhibition of each activity. (B) IL-12-activated CD8+ T cells adhere to TNF{alpha}-stimulated BLS4 cells via CXCL16 and VCAM-1. The adhesion assay was performed using BLS4 monolayers pre-stimulated or not pre-stimulated with TNF{alpha} and the results are shown as fold increment relative to the control (stimulation, medium). Several neutralizing antibodies were added to the medium during the assay.

 
CXCL16 is involved in the adhesion of activated CD8+ T cells to BLS4 cells
FRCs are considered to function as a foothold for lymphocyte movement within the LN, prompting us to address whether CXCL16 is involved in the direct interaction of T cells and BLS4 cells. IL-12-stimulated, activated CD8+ T cells were placed on BLS4 monolayers and the adhesion was assessed. In contrast to unstimulated BLS4 monolayers, to which only a weak background adhesion of T cells was observed, monolayers stimulated with TNF{alpha} showed dramatically enhanced binding of T cells (Fig. 5B). Interestingly, neutralizing anti-CXCL16 antibody markedly diminished the T cell adherence to the TNF{alpha}-stimulated monolayers (Fig. 5B). This inhibitory effect on the adhesion was apparently more effective than that elicited by the antibody against VCAM-1, which also caused a significant inhibition, whereas anti-ICAM-1 antibody had no effect. Moreover, simultaneous blocking of both CXCL16 and VCAM-1 suppressed a large fraction of the T cell adhesion onto the BLS4 monolayer. Taken together, these findings suggest that, in concert with VCAM-1, CXCL16 plays a crucial role in the intimate interaction of T cells and FRCs.

CXCL16 is expressed in LN FRCs in vivo
By means of immunohistochemical analysis using antibodies against FRC markers such as ER-TR7 and gp38, the RN constructed by FRCs can be clearly stained as an intricate network structure in LN sections (Fig. 6A). Importantly, we were able to detect substantial signals for CXCL16, which is well co-localized with the FRC markers, compared with background non-specific signals (Fig. 6A). Matloubian et al. (16) have previously shown that DCs in lymphoid tissues express CXCL16. Consistent with their report, we also detected relatively strong expression of CXCL16 by CD11c+ DCs in LN sections; however, the pattern of this expression was clearly distinct from the pattern detected on the RN (Fig. 6B). Therefore, it is suggested that FRCs, in addition to DCs, actually produce CXCL16, which function in lymphocyte migration and adhesion within the LN microenvironment.


Figure 6
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  		Fig. 6. Immunohistochemical detection of CXCL16 expressed by FRCs in LN sections. (A) LN cryosections were stained with antibodies against ER-TR7, gp38 and CXCL16 (lower) or goat IgG (upper) and analyzed using a confocal microscope. CXCL16 is detected in ER-TR7+gp38+ FRCs (arrows). (B) LN sections were stained for ER-TR7, CD11c and CXCL16. CD11c+ DCs show strong CXCL16 signals (arrows). Size bars: 40 µm.

 
We also tried to detect the production of CXCL16 protein in the LN FRCs through a different approach. To analyze the features of ex vivo FRCs directly, we prepared cells from mouse LNs by enzymatic digestion, which contain stromal cells as described elsewhere (23). This approach enabled us to analyze a single-celled and distinctly separated gp38+VCAM-1+ population within the cell suspension by flowcytometry (Fig. 7A). Unfortunately, the treatment with trypsin or collagenases employed in these experiments seemed to digest the cell-surface CXCL16 on BLS4 cells in pilot experiments even though gp38 and VCAM-1 were both intact (Fig. 7B, only the case of trypsin is shown). Therefore, it is reasonable that the cell-surface CXCL16 staining of the FRCs in LN cell suspension was also undetectable (Fig. 7A). For this reason, we performed the intracellular detection of residual CXCL16 molecules after the LN cells were fixed and permeabilized. Strikingly, anti-CXCL16 antibody clearly stained intracellular CXCL16 in FRCs as well as in BLS4 cells regardless of the enzymatic digestion, while control antibody or the specific antibody neutralized with recombinant CXCL16 did not stain the cells (Fig. 7A and B).


Figure 7
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  		Fig. 7. Intracellular CXCL16 is detected in gp38+VCAM-1+ FRCs isolated from LNs. (A) Cell suspension prepared from pooled peripheral LNs was stained for CXCL16 in addition to gp38 and VCAM-1 for flowcytometry. In both the stromal (R1) and gp38+VCAM-1+ (R2) gates (upper panels), cell-surface CXCL16 was undetectable, possibly due to enzymatic digestion during the procedure (lower left). For intracellular staining, cells were fixed and permeabilized, followed by staining with anti-CXCL16 antibody (lower middle). To confirm the antibody specificity, recombinant CXCL16 was added during the staining for neutralization (lower right). Staining with control goat IgG (open) and anti-CXCL16 antibody (filled) is shown in each histogram. (B) Cell-surface CXCL16 on BLS4 cells is also lost upon enzymatic digestion (upper panels, the case of trypsin treatment is shown). Detection of intracellular CXCL16 in BLS4 cells, which is blocked by the addition of rCXCL16 (lower panels).

 
In order to confirm the expression of CXCL16 in LN FRCs, we next tried purifying gp38+VCAM-1+ cells from LN cell suspension. After enzymatic digestion (content of gp38+VCAM-1+ cells, 0.3%), VCAM-1+ cells were roughly collected using IMagnet (43.9%). Then gp38+VCAM-1+ FRCs were sorted by FACS for further purification (88.3%) and we could detect substantial amount of CXCL16 message in this sorted population by RT–PCR (Fig. 8A and C). T cells (CD3+), B cells (B220+), macrophages (Mac-1+) and DCs (CD11c+) were also isolated individually and we observed that macrophages or DCs express CXCL16 as expected (Fig. 8B and C). Importantly, contamination of macrophages and DCs was undetectable in the purified FRCs. We also detected the expression of a MMP ADAM10, which has shown to mediate the cleavage of CXCL16 (26, 27), in FRCs from the LN (Fig. 8C) as well as BLS4 cells (data not shown). Therefore, these findings lead us to conclude that FRCs constructing the RN in LNs actually produce CXCL16 as a characteristic trait.


Figure 8
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  		Fig. 8. CXCL16 mRNA is detected in purified gp38+VCAM-1+ FRCs from LNs. (A) Purification of gp38+VCAM-1+ FRCs. After enzymatic digestion (left panel), VCAM-1+ cells were roughly separated by IMagnet (middle panel). Gated population was further purified by FACS as gp38+VCAM-1+ cells and the sorted cells were re-analyzed as shown in the right panel. (B) CD3+, B220+, Mac-1+ and CD11c+ cells were isolated from LN cell suspension by MACS. (C) Semi-quantitative RT–PCR analysis of isolated cells. Four-fold serial dilutions of cDNA were amplified with specific primer pairs. The number of PCR cycles was 35 for CXCL16, 35 for CD11c, 33 for Mac-1, 38 for ADAM10 and 33 for GAPDH.

 
To address whether CXCL16 actually involves in the adhesion of activated T cells to LN FRCs, we performed an in vitro binding assay on frozen section. When IL-12-activated CD8+ T cells were labeled with a fluorescent dye and placed onto LN sections, we observed that the cells preferentially bound on the RN in the paracortex (Fig. 9A). If equal number of T cells were labeled with two different dyes (green and red) and mixed, binding ratio between green and red cells to the section was nearly 1:1, indicating that the adhesion activity was uninfluenced by these dye treatments (Fig. 9B, left panel, and C). We next prepared a 1:1 mixture of two T cell groups; one is cells pre-treated with control human IgG and the other is cells pre-treated with CXCL16-Fc for CXCR6 blockage. When the cell mixture was applied to the binding assay, significant reduction of binding was observed in the CXCL16-Fc-treated group compared with the control group (Fig. 9B, right panel, and C). Similar result was obtained if the dye treatment was inverted (Fig. 9C). These findings suggest that the CXCL16–CXCR6 system is a part of mediators for the adhesion of activated T cells to FRCs within the LN.


Figure 9
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  		Fig. 9. CXCL16 partially involves in the adhesion of IL-12-activated CD8+ T cells to LN section. (A) Activated T cells (red) preferentially adhered to the ER-TR7+ RN (blue) in the paracortical region of the LN section [T, paracortex (T zone); B, follicle (B zone)]. (B) CXCR6 blockage significantly reduced the T cell adhesion to LN FRCs. Activated T cells were pre-treated with control human IgG or CXCL16-Fc, labeled with fluorescence dyes and mixed by 1:1 as indicated combination for adhesion assay. Ratios of bound cell numbers (green/red or red/green) in each region are plotted in (C). Mean adhesion of CXCL16-Fc-treated cells was significantly reduced compared with the control cells. *P < 0.01, **P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
One of the two transmembrane-type chemokines identified so far, CX3CL1 (fractalkine), has been well documented as to its unique feature of contributing to leukocyte trafficking via simultaneously acting as a chemokine and an adhesion molecule (14, 15, 17, 28). Similarly, it was recently shown that CXCL16 can also function in both ways (18). A metalloproteinase, ADAM10, seems to be responsible for the shedding to make soluble CXCL16 in endothelial cells, vascular smooth muscle cells and macrophages (26, 27). CXCL16 has also been identified as a scavenger receptor, SR-PSOX, for oxidized LDL, phosphatidylserine and gram-positive and -negative bacteria, implying the versatile function of this chemokine (29). It has been shown that CXCL16 is expressed by several cell types, including DCs, macrophages, endothelial cells and vascular smooth muscle cells, and is suggested to be involved in immune regulation and several types of disease pathology (16, 27, 29, 30).

Using BLS4 cells as a model for lymphoid tissue stroma, we demonstrated that CXCL16 expression is induced by inflammatory factors via NF{kappa}B-, p38 MAPK- and PKA-dependent pathways in these cells. A soluble form of CXCL16 is released from the cell surface due to MMP activity and partially contributes in the attraction of activated T cells. Moreover, CXCL16 present on the cell surface has a significant role in the adhesion of activated CD8+ T cells to BLS4 cells. Although it remains to be determined whether CXCL16 functions as chemokine or adhesion molecule, CXCL16 seems to be an important mediator for T cell–BLS cell interaction in collaboration with other canonical adhesion molecules such as VCAM-1. From frozen section binding assay, we also suggest that CXCL16 may participate in the adhesion of activated T cells to FRCs in vivo. These findings extend our insight into the physiological roles of CXCL16 and support the notion that FRC/RN in lymphoid tissues has the potential ability to regulate lymphocyte movement utilizing this unique chemokine within the LN (Fig. 10).


Figure 10
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  		Fig. 10. Model of CXCL16-mediated T cell–FRC interplay occurring in the LN. CXCL16 expression is maintained or enhanced in FRCs by inflammatory cytokines such as TNF{alpha} and IFN{gamma} via NF{kappa}B-, p38 MAPK- and PKA-mediated signaling pathways. For regulating T cell movement within the LN, CXCL16 potentially functions as a membrane-anchored chemokine/adhesion molecule in concert with other adhesion receptors as well as a canonical chemokine produced by shedding via MMP activity.

 
FRC/RN in the LN is also considered to be crucial for the recruitment and anchoring of DCs derived from peripheral tissue. Actually, in vitro differentiated DCs efficiently adhere to BLS4 cells, which suggests that FRCs could provide a foothold for DCs and support T cell–DC contact as the ‘immunoplatform’ (11). Antigen-bearing mature DCs are known to produce IL-12 (31), which induces T cells to secrete IFN{gamma} and augments CXCR6 expression during their activation. Simultaneous exposure to IFN{gamma} from activated T cells as well as TNF{alpha} from T cells and DCs presumably enhances the production of CXCL16 by FRCs. Such a putative factor circuit involving IL-12, IFN{gamma}, TNF{alpha} and CXCL16 is consistent with the observation that CXCR6 expression is correlated with Th1-type responses in humans (19). Recently, it has also been shown that CXCL16 plays important roles in experimental autoimmune encephalomyelitis, a typical Th1-mediated autoimmune disease model in mice (32). In addition to providing the field for immune responses, FRCs may secrete other soluble factors for T cell survival. We detected the expression of IL-7 and IL-15 in BLS4 cells (our unpublished results). When activated T cells were placed on BLS4 monolayers, activation-induced cell death of the T cells was markedly reduced particularly on TNF{alpha}-stimulated BLS4 cells, although CXCL16 seems not to involve in this phenomenon (our unpublished results). Taking all these observations into consideration, we can speculate that there is a positive feedback loop involving the factors and the close interactions between T cells, DCs and FRCs during the triggering of immune responses.

We previously showed that BLS4 cells construct a characteristic RN-like ECM network when co-cultured with lymphocytes or exposed to TNFR + LTßR signals in vitro (11). The direct contact between BLS4 cells and lymphocytes seems to be crucial. However, the molecular mediators involved in this lymphocyte–stromal interaction are still largely unknown, and therefore whether CXCL16 contributes to the process of constructing an appropriate microenvironment through the direct cell–cell interaction between different types of cells is an important issue to be addressed in the future.

In this study, we established an assay system for detecting intracellular protein and mRNA expression of CXCL16 in gp38+VCAM-1+ FRCs isolated from LNs and we clearly showed the constitutive expression of CXCL16 in FRCs. Within the LN microenvironment, stromal cells are likely to be continually exposed to various inflammatory stimuli such as TNF{alpha} and other related factors. Therefore, it seems reasonable that FRCs prepared from the LN express CXCL16 constitutively. In contrast, established stromal cell lines such as BLS4 are maintained in a condition free from such inflammatory stimuli. As a matter of course, this condition lowers the expression of CXCL16 to a basal level. Although we have checked the CXCL16 expression in FRCs or in total LN cells prepared from mice after the immunization, the enhancement of the expression was insignificant (data not shown). This suggests that the CXCL16 expression is already near maximum level in the in vivo FRCs and thus T cell motility mediated by the CXCL16–CXCR6 system might be dependent on the degree of the receptor expression on the lymphocyte side.

Comprehensive understanding of the microenvironment supported by lymphoid stromal cells, where the immune response is carried out, will be helpful in analyzing various infections and immune disorders. It is surmised that the stromal cells in lymphoid tissues possess powerful immune regulatory functions. Therefore, the molecular mediators participating in the complicated events mediated by the stromal cells must be identified and characterized. CXCL16 is one such stromal effector molecule.


   Acknowledgements
 
We thank S. Yonehara and T. Shimaoka for CXCL16-Fc, A. G. Farr for 8.1.1 antibody and T. Ofuji for technical assistance. This work was supported in part by Grants-In-Aid for Science Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Abbreviations
 
APC   allophycocyanin
DC   dendritic cell
ECM   extracellular matrix
FRC   fibroblastic reticular cell
GPCR   G-protein-coupled receptor
LN   lymph node
MACS   magnetic cell sorter
MMP   matrix metalloproteinase
PTx   pertussis toxin
RN   reticular network
RT   reverse transcription
TNF   tumor necrosis factor
VCAM   vascular cell adhesion molecule

   Notes
 
Transmitting editor: T. Watanabe

Received 24 August 2005, accepted 16 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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