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The Journal of Immunology, 2006, 177: 7665-7672.
Copyright © 2006 by The American Association of Immunologists, Inc.

Oncostatin M Enhances CCL21 Expression by Microvascular Endothelial Cells and Increases the Efficiency of Dendritic Cell Trafficking to Lymph Nodes1

Makoto Sugaya2,3, Lei Fang3, Adela R. Cardones3, Takashi Kakinuma, Samer H. Jaber, Andrew Blauvelt4 and Sam T. Hwang5

Dermatology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-{alpha}) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-{alpha} did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by ~10-fold and in mouse skin by ~4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by ~3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CCL21 (also known as secondary lymphoid tissue chemokine/SLC) and its receptor, CCR7, are critically involved in the migration of Ag-presenting dendritic cells (DC)6 (1, 2, 3) and tumor cells (4, 5) from peripheral tissues to draining lymph nodes (LN) via afferent lymphatic vessels. The expression of CCL21 is restricted to high endothelial venules, stromal cells in secondary lymphoid organs (6), and lymphatic vessels in multiple organs (6), including skin (1). DC from CCR7-deficient mice are strikingly impaired in their ability to enter lymphatic vessels and fail to migrate normally to secondary lymphoid organs (3, 7). Moreover, there is a marked reduction of naive T cells and disorganization of the DC network within LN of mice homozygous for the paucity of LN T cell (plt) mutation, which is characterized by the lack of CCL21 expression in LN (2, 8, 9). Collectively, these data suggest that CCL21 and CCR7 are vital for normal migration and/or retention of specific T cells and DC within secondary lymphoid organs.

Recent evidence suggests that CCL21 expression by lymphatic vessels in the periphery can be enhanced in vivo by factors produced by activated DC or by treatment with proinflammatory cytokines such as TNF-{alpha} (10), raising the possibility that CCL21 expression is regulated via cytokines present within inflamed tissues. Furthermore, treatment of skin with TNF-{alpha} increases the efficiency of migration of DC from the skin to regional draining LN, suggesting that these cytokines may have a role in promoting egress of DC from peripheral tissues (10).

Lymphatic (EC) endothelial cells (LEC) differ from blood vascular EC (BVEC) by expressing a characteristic set of proteins, including lymphatic vascular EC-1 (LYVE-1), podoplanin, vascular endothelial growth factor (VEGF) receptor 3, and Prox-1 (11). Interestingly, human dermal microvascular EC (DMEC) isolated and cultured from neonatal (12) and adult skin (13, 14) are heterogeneous, with a significant population of cells expressing lymphatic-associated proteins, including podoplanin and LYVE-1. Separation of dermal EC by selection with podoplanin Abs revealed that the EC with LEC-like characteristics preferentially expressed CCL21 (13).

Oncostatin M (OSM) is a member of the IL-6 family of cytokines with pleiotropic biologic effects (reviewed in Ref. 15). Produced by a number of inflammatory cells, including activated T cells, monocytes, and DC (15, 16), OSM can exert both pro- and anti-inflammatory effects, depending on the model system examined (17). EC express high numbers of OSM receptors, and OSM has been shown to up-regulate the expression of several cytokines and chemokines, including CXCL1 and CXCL2, in HUVECs (18). Furthermore, OSM is a potent growth factor for AIDS-associated Kaposi’s sarcoma (19, 20, 21), a tumor comprised morphologically of cells with the characteristics of LEC (22). Infection of primary or immortalized BVEC with Kaposi’s sarcoma-associated herpesvirus, also known as human herpesvirus-8, the etiologic agent of Kaposi’s sarcoma, initiates enhanced expression of established LEC markers, including LYVE-1, podoplanin, Prox-1, and to a lesser extent, CCL21 (23, 24). The prominent role of OSM in stimulating growth of Kaposi sarcoma-associated herpesvirus-infected cells led us to ask whether or not this cytokine could alter the expression of CCL21 by LEC and BVEC.

In this study, we show that OSM increases expression of CCL21 by EC in vitro and enhances expression of CCL21 mRNA within the skin and LN in vivo. Interestingly, the topical application of a contact-sensitizing hapten resulted in markedly enhanced OSM expression in skin. In addition, we uncover additional potential mechanisms by which OSM may increase the efficiency of DC trafficking from the periphery to draining LN. Collectively, our results suggest that OSM may play a distinct role in DC trafficking to secondary lymphoid organs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, cell lines, and reagents

Female C57BL/6 mice and BALB/C mice (6–8 wk old) were purchased from The Jackson Laboratory. The National Cancer Institute Animal Care and Use Committee approved all experiments involving animals. Human DMEC and human dermal fibroblasts (HDF) were purchased from the Endothelial Cell Core Facility (Department of Dermatology, Emory University School of Medicine, Atlanta, GA) and cultured as described previously (25). Human lung microvascular EC (LMEC) were purchased from Cambrex and cultured in EGM-2 MV (Cambrex) in tissue-culture-treated plastic flasks (Corning). Recombinant mouse and human OSM, TNF-{alpha}, IL-1beta, IFN-{gamma}, and IL-6 were obtained from R&D Systems. U0126 and PD98059 were purchased from Cell Signaling Technology and were dissolved in DMSO before use. CCL19-human IgG fusion protein (CCL19-Ig) was a gift from Dr. U. von Andrian (Harvard Medical School, Boston, MA). TC2031-2, a human melanoma cell line, was a gift from Dr. J. Wunderlich (Surgery Branch, National Cancer Institute, Bethesda, MD). The Jurkat T cell leukemia line was described previously (26). The H9 T cell lymphoma line is a variant of the HUT 78 cell line and was obtained from the American Type Culture Collection.

Generation of bone marrow-derived DC (BMDC)

BMDC were prepared as described previously (27). Briefly, femurs and tibia were obtained from 8- to 12-wk-old mice, soaked in alcohol for 4 min, and rinsed with PBS three times. Bone marrow was flushed from the bones, filtered through a 45-µm filter (Falcon), and washed with PBS. Cells were treated with Ack lysis buffer for 2 min, washed two times, and maintained in culture for 7–8 days in complete RPMI 1640 containing 5% FBS that was supplemented with murine (m)GM-CSF (10 ng/ml) and mIL-4 (10 ng/ml) (PeproTech). On the last day, LPS (Sigma-Aldrich) 200 ng/ml was added to the culture to further activate DC. Cells were washed five times in PBS, and gradient centrifugation was performed using 14% Nycodenz (Sigma-Aldrich) at 1700 rpm for 10 min at 4°C to isolate DC.

Determination of OSM expression by BMDC using quantitative real-time RT-PCR and by ELISA

BMDC were prepared as described above except that no LPS was added into the culture before harvest. BMDC were isolated by gradient centrifugation and washed twice in PBS. BMDC were then seeded into a round-bottom 96-well plate at 5 x 105 cells/well with medium only, 20 ng/ml mTNF-{alpha} (PeproTech), or 200 ng/ml LPS for 24 h. Cell culture supernatants were collected for ELISA, and cells were harvested for quantitative real-time RT-PCR. Primers for mOSM and GAPDH were as follows: OSM for (forward), 5'-GCA GCT GTG GCT TTC TCT GG-3' and rev (reverse), 5'-TCG TCC CAT TCC CTG AAG AC-3'; GAPDH for, 5'-CGT GTT CCT ACC CCC AAT GT-3' and rev, 5'-TGT CAT CAT ACT TGG CAG GTT TCT-3'.

For mOSM ELISA, 1 µg/ml anti-mouse OSM (R&D Systems) was used to coat a 96-well ELISA plate at 4°C overnight. The plate was blocked with PBS containing 10% FBS (blocking buffer) at room temperature (RT) for 1 h. The plate was washed three times with PBS containing 0.05% Tween 20 (washing buffer) between each step. Recombinant murine (rm)OSM (R&D Systems) as standards and undiluted culture supernatants from BMDC were added to the plate and incubated at RT for 2 h. After washing three times, 100 µl of biotinylated anti-mouse OSM (1 µg/ml; R&D Systems) were added to wells at RT for 1 h. Lastly, streptavidin-HRP (1/1000 dilution) was added to wells at RT for 30 min before developing with ABTS substrate (Roche). For human OSM ELISA, a commercial ELISA kit (R&D Systems) was used according to the manufacturer’s instructions.

In vitro OSM stimulation

DMEC or LMEC at passages 3–6 were cultured in EGM-2 MV with 5% serum (except as noted in the text) in the absence of gelatin or fibronectin substrate and stimulated for 24, 48, or 72 h with recombinant human (rh) OSM (100 ng/ml) or other cytokines as indicated. Total RNA was isolated using RNeasy (Qiagen). cDNA was synthesized using Superscript II (Invitrogen Life Technologies). Quantitative RT-PCR was performed as described previously (25). Primers for human CCL21 were as follows: for, 5'-GGT TCT GGC CTT TGG CAT C-3'; rev, 5'-AGG CAA CAG TCC TGA GCC C-3'. Primers for human GAPDH were as follows: for, 3'-ACC CAC TCC TCC ACC TTT GA-3' and rev, 5'-CAT ACC AGG AAA TGA GCT TGA CAA-3'.

Western blotting and flow cytometry

EC were washed with PBS, and whole cell lysates were harvested on ice with a cell scraper in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing proteinase mixture (Roche) and phosphatase inhibitors (1 mM PMSF, 10 mM NaF, and 1 mM Na3VO4). After centrifugation, whole cell lysates were separated by 14% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked and soaked with a 1/1000 dilution of primary Ab at 4°C overnight. Membranes were then washed and incubated with 1:1000 of HRP-labeled secondary Abs for 1 h. After washing, membranes were incubated with working solution (SuperSignal West Pico Substrate; Pierce Scientific) for 5 min at RT and exposed to film. The Abs used in this study were goat anti-human CCL21 polyclonal Ab (R&D Systems), mouse anti-rabbit GAPDH mAb (Research Diagnostics), rabbit anti-ERK1/2 polyclonal Ab, and rabbit anti-phospho-ERK1/2 polyclonal Ab.

For flow cytometry analysis, microvascular EC were trypsinized, washed, resuspended in staining buffer (0.1% BSA/PBS), and then incubated with Abs against LYVE-1 for 45 min at 4°C (12).

Endothelial monolayer permeability assay

Permeability assays were performed using a Chemicon Vascular Permeability Kit (Chemicon International). DMEC and LMEC were grown on collagen-coated membrane inserts until confluence. For short-term permeability assays, upper chamber medium was replaced with serum-free medium containing 1 µM VEGF, 100 ng/ml human OSM (hOSM), or no treatment, together with 200 µl of FITC-dextran (50 µg/ml diluted from 2 mg/ml stock; Chemicon International) at 37°C. Medium from the lower chamber, which included any flow-through FITC-dextran, were removed at 15 min, 30 min, 1 h, and 2 h, and assayed using a CytoFluor fluorometer (Applied Biosystems). For long-term permeability assays, DMEC (grown on 1% collagen-coated, filter-based chambers) were treated overnight with OSM, other cytokines as indicated, or medium alone. FITC-dextran (per kit instructions) was placed in the upper chamber and allowed to diffuse for 1 h at 37°C before collection of buffer from the lower chamber for analysis.

In vivo permeability assay

mOSM (1 µg) or PBS was injected into the left hind footpads of C57BL/6J mice (n = 7/group). Twenty-four hours later, Evans blue dye (50 µl, 5 mg/ml; Sigma-Aldrich) was injected into the same footpads. Mice were sacrificed by cervical dislocation 1 min after dye injection, and the left popliteal LN were quickly harvested. Evans blue dye was then extracted from the draining LN by formamide (50 µl/LN) at 55°C for 2 h (28). Following measurement of OD at 630 nm in a spectrophotometer, the concentration of Evans blue dye (µg/ml) was calculated based on a standard curve of known amounts of Evans blue dye.

Chemotaxis assay

DMEC were grown in 24-well cell culture plates (Costar Transwell; Corning) and treated with rhOSM (100 ng/ml) for 48 h. Twenty minutes before the chemotaxis assay, wells were treated with 20 µg/ml neutralizing anti-CCL21 Ab (goat IgG; R&D Systems), control IgG, or medium alone. Calcein (Molecular Probes)-labeled BMDC (3 x 105) were placed in Transwell inserts (Corning) and allowed to migrate toward the DMEC for 4 h at 37°C. Cells were collected and counted by flow cytometry (FACSCalibur; BD Biosciences). rhCCL21 (500 ng/ml in fresh complete RPMI 1640) was used as a positive control.

Cytokine injections and application of trinitrochlorobenzene (TNCB) hapten

Anesthetized mice were injected in ear skin with PBS, 100 ng of rmOSM, or 50 ng of mTNF-{alpha} (R&D Systems) in 20 µl of sterile PBS. TNCB (1% final concentration; Sigma-Aldrich) was dissolved in olive oil:acetone (1:4 volume:volume ratio) and then applied on the dorsal and ventral sides of the mouse ear (10 µl/side) (29). Mice were euthanized 24 h later, and total RNA was isolated from the ear. Briefly, ears were soaked in ethanol for 5 min, rinsed, and split, and then incubated (dermal-side down) in serum-free DMEM containing Liberase (150 µg/ml; Roche) for 45 min at 37°C. Ears were then washed and minced, and total RNA was extracted using RNeasy (Qiagen). Quantitative RT-PCR was performed using the following primer pairs: mCCL21 for, 5'-ATCCCGGCAATCCTGTTCTT-3' and rev, 5'-GCCTTCCTCAGGGTTTGCA-3'; mCCR7 for, 5'-GGACACGCTGAGATGCTCACT-3' and rev, 5'-CCATCTGGGCCACTTGGAT-3'; and mIL-8 for, 5'-ACCACACTGCGCCAACACAGAAAT-3' and rev, 5'-TCC AGACAGAGCTCTCTTCCATCA-3'.

In vivo migration assay

BMDC (day 7 after culture) were treated with LPS for 24 h and then extensively washed before being resuspended in PBS. Mouse footpads were injected with PBS, 1 µg of rmOSM, or 500 ng of rmTNF-{alpha} 24 h before injection of 106 calcein-labeled BMDC into the footpads. After another 24 h, mice were euthanized, and the popliteal LN were collected, minced, and treated with 1 mg/ml collagenase D (Roche) and 0.05% DNase (Sigma-Aldrich) for 45 min at 37°C. Cells were then filtered, washed, and resuspended in 0.1% BSA/PBS. The highly fluorescent calcein-labeled cells were identified in the FITC channel and counted by flow cytometry.

Statistical calculations

Statistical calculations were performed using Student’s t test using the GraphPad Prism analysis software (GraphPad). Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OSM increases CCL21 mRNA and protein expression in vitro

To determine the effects of OSM treatment on EC in vitro, we treated DMEC rhOSM over several days and measured mRNA levels of CCL21 by real-time quantitative RT-PCR (Fig. 1A) and ELISA (Fig. 1B). The DMEC in our studies were devoid of contaminating cell populations as suggested by uniform expression of CD31 (12). Furthermore, DMEC exhibited 40–70% expression (12) of LYVE-1, a LEC-specific marker (30). Human LMEC were previously shown to be devoid of LYVE-1 expression (12) and were deemed representative of BVEC. In the presence of 5% serum, up-regulation of CCL21 mRNA was detected in DMEC at 24 as well as 48 h after addition of OSM, but not other cytokines (IL-1beta, IL-6, IFN-{gamma}, and TNF-{alpha}) (Fig. 1A). Under serum-free conditions, up-regulation of CCL21 mRNA was observed at 48 h (but not 24 h) in both LMEC (9.8-fold vs baseline) and DMEC (5.3-fold vs baseline) after treatment with OSM, but not TNF-{alpha} (data not shown). We also confirmed that OSM stimulated CCL21 protein synthesis and secretion into cell culture supernatants by DMEC within 48–72 h after stimulation (Fig. 1B).


Figure 1
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  		FIGURE 1. DMEC, but not other selected cell types, up-regulate protein and mRNA for CCL21 upon OSM stimulation. A, Following stimulation of DMEC in 5% serum for 24 or 48 h with IL-1beta (10 ng/ml), IFN-{gamma} (250 U/ml), TNF-{alpha} (10 ng/ml), IL-6 (10 ng/ml), or rhOSM (100 ng/ml), CCL21 mRNA expression was quantified by real-time RT-PCR. B, DMEC were stimulated with rhOSM at the indicated concentrations for 24, 48, and 72 h, after which supernatants were collected for ELISA-based measurement of CCL21 concentration. Values shown represent means and SDs of four separate experiments. C, HDF, DMEC, TC-2031-2, Jurkat, and H9 cells were treated with 100 ng/ml rhOSM (or left untreated) for 48 h. Total RNA was extracted, and quantitative RT-PCR was performed to measure CCL21 expression relative to GAPDH.

 
TNF-{alpha}, which has been reported to stimulate CCL21 expression in vivo by LEC following intradermal injection (10), did not stimulate CCL21 mRNA expression in either DMEC or LMEC after 24 h or 48 h of exposure at 10 ng/ml in the presence of serum (Fig. 1A). Because of greatly increased death when EC were exposed to TNF-{alpha} at higher concentrations (100 ng/ml) for >24 h, assessment of CCL21 mRNA expression under these specific conditions was not possible (data not shown). In contrast to its effect on CCL21 expression, OSM did not up-regulate expression of IL-8 after either 24 or 48 h of treatment, whereas TNF-{alpha} increased expression of IL-8 in DMEC and LMEC by ~150-fold after 24 h, suggesting that TNF-{alpha} was indeed active (data not shown). Lastly, whereas OSM stimulated CCL21 expression by DMEC and LMEC, it did not stimulate CCL21 expression in human melanoma cells (TC2031-2), T cell leukemia cells (Jurkat), lymphoblastic T cell lymphoma (H9) cells, or primary HDF, suggesting that OSM stimulated CCL21 mRNA in limited cell types (Fig. 1C). Thus, rhOSM treatment specifically increased transcriptional and translational expression of CCL21 in human microvascular EC.

OSM induces phosphorylation of ERK1/2 in DMEC, which is required for expression of CCL21

OSM has previously been shown to stimulate MAPK signaling in a variety of cells (31, 32). To determine whether this pathway was involved in expression of CCL21 in OSM-stimulated EC, we used phosphorylation of ERK1/2 as a sensitive indicator of MAPK activation. Using DMEC, we found no detectable ERK1/2 phosphorylation in unstimulated cells (Fig. 2A). By contrast, incubation with 100 ng/ml OSM for 30 min markedly induced ERK1/2 phosphorylation in these cells (Fig. 2A). U0126, a MAPK inhibitor, blocked phosphorylation of ERK1/2 in DMEC in a dose-dependent manner (0.05–5 mM). Because CCL21 production (Fig. 2B) appeared to be more sensitive to U0126 than did ERK1/2 phosphorylation (Fig. 2A), CCL21 production may be dependent on other signaling pathways (besides ERK1/2) that are inhibited by U0126. Similar results were obtained using PD98059, another MAPK inhibitor (data not shown). Interestingly, both U0126 and PD98059 also blocked CCL21 production in cell-free culture supernatants of DMEC stimulated with OSM (Fig. 2B and data not shown), suggesting that CCL21 protein expression in EC is, in part, dependent upon MAPK signaling.


Figure 2
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  		FIGURE 2. OSM induces phosphorylation of ERK1/2 in DMEC, which is required for expression of CCL21. A, Induction of ERK1/2 phosphorylation in DMEC stimulated for 30 min with 100 ng/ml OSM. Phosphorylation of ERK1/2 is blocked in a dose-dependent manner by U0126, a specific inhibitor of MAPK signaling. B, CCL21 protein in cell-free culture supernatants of DMEC stimulated for 24 h with OSM is also blocked by U0126 in a dose-dependent manner, implicating MAPK signaling as critical for CCL21 expression.

 
Conditioned medium from OSM-stimulated DMEC mediates CCL21-dependent chemotaxis of BMDC

To determine whether the increased CCL21 protein produced by DMEC could stimulate migration of CCR7-expressing BMDC, we performed a chemotaxis assay with mouse BMDC and conditioned medium from OSM-treated DMEC. Preliminary experiments indicated that rh CCL21 stimulated murine BMDC in chemotaxis assays with similar potency and efficacy as did rm CCL21 (data not shown). CCR7 surface expression was confirmed by flow cytometry in the BMDC, by binding the CCL19-Ig chimeric protein to BMDC (data not shown), and by >10-fold increased migration to recombinant CCL21 in chemotaxis assays (data not shown). Baseline migration of BMDC toward untreated DMEC could be inhibited by ~50% with neutralizing anti-CCL21 Abs (Fig. 3), suggesting that chemotactically active amounts of CCL21 were being produced by DMEC in the absence of exogenous OSM. Treatment of DMEC with OSM for 48 h resulted in enhanced migration of BMDC that was similar to that achieved by the addition of exogenous CCL21 to culture medium from nonstimulated DMEC (Fig. 3). The addition of anti-CCL21, but not control IgG, Abs to cell supernatants from OSM-treated DMEC resulted in a ~60% reduction in BMDC migration (Fig. 3), suggesting that OSM treatment functionally increases the capacity of DMEC to stimulate CCL21-dependent chemotaxis.


Figure 3
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  		FIGURE 3. Migration of BMDC toward OSM-treated DMEC. DMEC were grown to confluence and were either untreated or treated with rhOSM as noted in Materials and Methods. Certain wells were treated with either control IgG or neutralizing Ab against CCL21. BMDC were allowed to migrate toward the EC monolayers for 4 h at 37°C. As positive control, BMDC were allowed to migrate toward 500 ng/ml rhCCL21. Results are representative of three experiments with similar findings.

 
OSM treatment increases CCL21 mRNA expression in skin and draining LN in vivo

To assess the effects of OSM on CCL21 production in vivo, we injected the ears of C57BL/6 mice with PBS, 100 ng of rmOSM, or 50 ng of rmTNF-{alpha}, and extracted total skin RNA 24 h later (Fig. 4A). Both rmOSM and rmTNF-{alpha} treatments increased mRNA expression of CCL21 to a similar degree, but whereas TNF-{alpha} up-regulated CCR7 mRNA expression, rmOSM treatment did not. Peripheral LN are also a rich source of expression of CCL21 (6). To determine whether OSM could enhance CCL21 expression in the LN, we injected mouse footpads with OSM and then measured CCL21 and CCR7 mRNA expression in the draining popliteal LN. OSM did not change the size of the draining LN compared with PBS treatment (data not shown). Notably, CCL21 mRNA was up-regulated by ~10-fold in the draining LN, whereas CCR7 expression remained unchanged (Fig. 4B). Therefore, both skin and skin draining LN show strong up-regulation of CCL21 upon OSM treatment, possibly contributing to enhanced DC migration into lymphatic vessels and retention, respectively, of DC in the LN.


Figure 4
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  		FIGURE 4. Up-regulation of CCL21 in vivo in response to OSM treatment. Ears of C57BL/6 mice were given intradermal injections of PBS, OSM (100 ng), or TNF-{alpha} (50 ng) in a total volume of 20 µl of PBS. A, Total RNA was extracted from ears after 24 h, and quantitative RT-PCR was performed to measure CCL21 and CCR7 mRNA expression relative to GAPDH. Results are average of 2–3 mice per group and are representative of three experiments with similar findings. B, Mouse footpads were injected with PBS or 1 µg of rmOSM. Draining popliteal LN (n = 5 per treatment group) were harvested 24 h later and pooled for RNA isolation and real-time quantitative RT-PCR assessment of the indicated genes. Results are expressed relative to GAPDH and are representative of three experiments with similar findings.

 
OSM enhances migration of BMDC to draining LN in vivo

Based on the data above, we predicted that intradermal injection of skin with OSM would result in enhanced DC migration from peripheral tissues (i.e., skin) to draining LN in vivo, presumably by increasing expression of CCL21 by LEC. To test this hypothesis, mouse footpads were pretreated with PBS, OSM, or TNF-{alpha} before injection of BMDC. After 24 h, popliteal LN were collected, and migratory DC within LN were quantified by flow cytometry. Pretreatment with TNF-{alpha} or OSM increased BMDC migration to popliteal LN by ~3- or 2-fold, respectively, compared with PBS (Fig. 5), indicating that both cytokines were capable of enhancing BMDC migration to regional LN.


Figure 5
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  		FIGURE 5. In vivo migration of BMDC in response to OSM treatment. Calcein-labeled BMDC (1 x 106) were injected into the footpads of C57BL/6 mice that had been pretreated with PBS, 1 µg of OSM, or 500 ng of TNF-{alpha} 24 h before BMDC injection. Migrated BMDC were quantified 24 h later by flow cytometry. n = 8 per group. Results are representative of two experiments with similar findings.

 
Others have observed that BMDC produce factors that facilitate their own migration (10). To determine whether OSM could be one of these factors, we analyzed BMDC for the expression of OSM by both RT-PCR (for mRNA) and ELISA (for protein). OSM mRNA was readily detected in BMDC in contrast to B16 melanoma cells (Fig. 6A). Moreover, OSM protein was significantly elevated in the culture medium of BMDC after 24 h in vitro, which was further enhanced by treating BMDC with LPS, but not TNF-{alpha} (Fig. 6B). These results were consistent with the increased transcription of OSM mRNA by BMDC following LPS treatment (Fig. 6A). Thus, activated DC may be a source for OSM under inflammatory conditions.


Figure 6
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  		FIGURE 6. Expression of OSM by BMDC (in vitro) and in skin (in vivo). BMDC were cultured in round-bottom 96-well plates with medium only, TNF-{alpha} (20 ng/ml), or LPS (200 ng/ml) for 24 h. A, Total RNA was extracted from BMDC, and quantitative RT-PCR was performed to measure mOSM expression relative to GAPDH. B16 melanoma cell RNA was collected as a negative control for mRNA expression of OSM. B, Cell culture supernatants were collected for ELISA to determine OSM concentration. C, Ears were injected with TNF-{alpha} as described above (see Fig. 4 legend) or treated with 1% TNCB in olive oil:acetone (D) (see Materials and Methods) and assessed by RT-PCR for expression of OSM mRNA. Results are representative of three experiments with similar findings.

 
Topical application of a contact-sensitizing hapten increases OSM expression in skin

To determine whether TNF-{alpha} could directly stimulate OSM expression in vivo, we injected TNF-{alpha} into mouse ear skin and assessed OSM expression 24 h later. In contrast to its ability to induce CCL21 and CCR7 mRNA (Fig. 4A), TNF-{alpha} alone did not result in enhanced expression of OSM mRNA (Fig. 6C). TNF-{alpha} had no effect on OSM expression under either of these conditions or with lower doses of TNF-{alpha} at early time points (data not shown). This prompted us to ask whether topical application of a contact-sensitizing hapten may affect the expression of OSM. Under these conditions, skin DC would be expected to carry hapten-bearing Ag from the skin to LN via afferent lymphatic vessels, thus sensitizing mice to future antigenic challenge (33). One application of the commonly used contact dermatitis-sensitizing hapten, trinitrochlorobenzene (34), in olive oil:acetone vehicle resulted in a marked increase in OSM expression (Fig. 6D). These results suggest that TNF-{alpha} does not directly stimulate OSM production. The up-regulation of OSM after application of the hapten, however, suggests that OSM is up-regulated in skin under conditions in which DC are known to become activated and to later traffic to draining LN.

OSM increases EC monolayer permeability

OSM may also increase DC migration by increasing lymphatic flow and, thus, facilitating the passive transport of DC via the lymphatic vessels to regional LN. In a manner analogous to VEGF (previously known as vascular permeability factor; Ref. 35), OSM may increase the permeability of BVEC, resulting in the leakage of fluid from the intravascular space into the tissue space. This extracellular fluid must eventually be reabsorbed by the lymphatics, leading transiently to increased lymphatic flow that may carry along DC and other leukocytes to the draining LN.

To determine whether OSM altered vascular permeability, DMEC and LMEC were treated with 100 ng/ml rhOSM, 1 µM rhVEGF, or medium alone at 37°C. Permeability of EC monolayers to FITC-dextran was measured at 30 min, 1 h, and 2 h. As expected based on other studies (36), exposure of LMEC to VEGF resulted in significantly increased permeability to FITC-dextran (37, 38) as early as 30 min after treatment (Fig. 7A). Of note, OSM clearly increased permeability of LMEC monolayers beginning as early as 30 min following treatment, nearly to the same extent as VEGF, one of the most potent vascular permeability-enhancing cytokines known (35). In contrast, DMEC showed far less responsiveness to VEGF and OSM compared with LMEC (Fig. 7A). DMEC that were treated with rhOSM for 18 h, however, showed a >10-fold increase in permeability compared with untreated EC (Fig. 7B) without compromising viability (data not shown). TNF-{alpha} and IL-6 had no apparent effect on permeability. Of note, addition of hOSM to the lower chamber did not result in enhanced permeability of FITC-dextran (Fig. 7B), suggesting that receptors for OSM were located on the apical membrane of the DMEC. Thus, exposure of DMEC and LMEC to OSM results in increased permeability of EC monolayers to FITC-dextran.


Figure 7
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  		FIGURE 7. Permeability changes in DMEC and LMEC in response to OSM. A and B, DMEC and LMEC were cultured to confluence on cell membrane inserts and treated with 1 µM VEGF, 100 ng/ml OSM, or medium alone at 37°C. A, Short-term permeability to FITC-dextran was measured by quantifying fluorescence 30 min, 1 h, and 2 h after treatment as described in Materials and Methods. Results are an average from two wells and representative of two experiments with similar results. B, To measure permeability after longer exposure to OSM, IL-1beta (100 ng/ml), hOSM (100 ng/ml), IL-6 (10 ng/ml), or TNF-{alpha} (10 ng/ml) was added to the upper chamber (containing DMEC growing on top of a filter membrane) of the two-part permeability assay kit for 18 h at 37°C. FITC-dextran was then added to the upper chambers of all test kits and allowed to diffuse for 1 h before collection of medium from lower chambers for analysis. In one case (OSM lower), hOSM (100 ng/ml) was added to the lower chamber for 18 h at 37°C before adding FITC-dextran. Results are an average from three wells and representative of two experiments with similar results. Fluorescence is measured in arbitrary units. C, Evans blue dye was injected into mouse footpads that had been treated with either rmOSM (1 µg) or PBS 24 h earlier. One minute after injection of dye, mice were euthanized, and the Evans blue dye was extracted from draining LN for quantification of OD measurement at 630 nm (28 ). The y-axis indicates the concentration of dye in the LN as determined by comparison with a standard curve of known amounts of dye.

 
To determine whether enhanced vascular permeability observed in vitro resulted in enhanced lymphatic flow in vivo, we treated mouse footpads with rmOSM, waited 24 h, and then injected Evans blue dye into the footpad. Lymphatic transport of Evans blue dye to draining LN normally occurs within minutes, and this technique has been used to document defects in lymphatic drainage in skin (28). Compared with PBS mock treatment, injection with OSM significantly increased the accumulation of dye in the LN by ~3-fold (p = 0.0291) (Fig. 7C). Therefore, OSM increases the permeability of DMEC and LMEC, which results in increased lymphatic flow and faster accumulation of Evan’s dye in the draining LN.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CCL21 and its receptor, CCR7, are critical for migration of DC to secondary lymphoid organs (1, 2, 3, 39), yet little is known about the regulation of CCL21 expression by LEC. Despite useful information gleaned from the plt mouse, this mutation does not provide insight as to how CCL21 expression (particularly by LEC) is regulated in vivo. Sallusto and colleagues (10) have suggested that DC-derived cytokines such as TNF-{alpha} increase the efficiency of DC migration from peripheral tissues by increasing expression of CCL21 on LEC. Although treating skin with TNF-{alpha} enhanced both CCL21 staining in lymphatic vessels in skin as well as DC emigration, it was not shown that TNF-{alpha} acted directly on LEC (10).

The principal finding of this study is that OSM strongly stimulates the expression of CCL21 in vitro and in vivo. It was somewhat surprising that both DMEC and LMEC could be stimulated to produce CCL21 by OSM because CCL21 expression is associated with lymphatic vessels in vivo (6). This may reflect the plasticity of EC cultured in vitro, a phenomenon observed by others following infection with human Kaposi’s sarcoma-associated virus (24).

Our in vitro studies strongly suggest that TNF-{alpha} does not directly stimulate CCL21 production by DMEC, although it clearly can up-regulate CCL21 following injection in the skin. Because in vivo injection of TNF-{alpha} yielded enhanced CCL21 expression, but did not stimulate OSM production, we believe that TNF-{alpha} indirectly regulates CCL21 production through a mechanism independent of OSM (Figs. 4 and 6). Of note, a single topical application of TNCB, a contact sensitizer, induced dramatic up-regulation of OSM, suggesting that other factors present during the sensitization phase of contact dermatitis may elicit OSM production.

OSM stimulated CCL21 mRNA expression in skin-draining LN, which may facilitate the retention of DC once they traffic there from skin. Indeed, OSM pretreatment of the footpads resulted in enhanced trafficking of labeled-BMDC to the draining LN. DC have been reported to be a source of OSM production (16), and our results confirm that DC stimulated with LPS (a TLR-4 agonist) increase OSM expression at both the mRNA and protein levels, implicating TLR activation as a molecular trigger for OSM production (16). By contrast, we show that TNF-{alpha} had no direct effect on OSM production by BMDC. Lastly, we demonstrate that CCL21 protein synthesis is dependent upon signaling via the MAPK pathway, an integral component of OSM-mediated signaling (31, 32).

Up-regulation of CCL21 at the transcriptional level occurred at a much slower rate (24–48 h) compared with the up-regulation of other chemokines following EC activation. For example, we reported >50-fold up-regulation of CCL20 mRNA by DMEC within 6 h of exposure to TNF-{alpha} (25). Recent evidence, however, suggests that the migration of Ag-bearing Langerhans cells and dermal DC from skin occurs over days, not hours. Specifically, tetramethylrhodamine isothiocyanate-bearing dermal DC migration was first observed in the draining LN 24 h after topical application and peaked after 2 days, and that Langerhans cells were even slower to accumulate in the LN (40). Our kinetic data are consistent with these observations of DC migration and raise the possibility that the up-regulation of CCL21 may be a rate-limiting factor in DC transport in vivo. Thus, we provide the first evidence that OSM regulates the in vitro and in vivo expression of CCL21 and that it alters the trafficking of DC in vivo.

Besides stimulating CCL21 expression by LEC, there are several other mechanisms by which OSM may facilitate DC migration. First, OSM has been reported to have other physiological effects on EC and transendothelial migration that may be relevant to DC trafficking. These effects include the up-regulation of ICAM-1 (41) and chemotactic cytokines besides CCL21 by EC (18). Second, others have reported that OSM stimulates neutrophil transmigration across human EC (18), so it is conceivable that OSM has similar effects on DC. Third, we demonstrated that OSM increases vascular permeability in both LEC and BVEC. Of note, OSM, but not several other cytokines tested, was effective in increasing permeability of DMEC to FITC-dextran when it was added to the top chamber (corresponding to the apical side of the DMEC), but not when it was added to the bottom chamber (corresponding to the basal side of the DMEC). These results suggested that OSM acts through receptors present on the apical membrane. Synovial as well as serum levels of OSM have been detected in humans with rheumatoid arthritis (42), and it is possible that OSM released in the local environment acts primarily through apical receptors on blood vessels. Lastly, injection of recombinant OSM in the footpad leads to enhanced draining LN accumulation of Evan’s blue dye (see Fig. 7C), suggesting that OSM had functionally enhanced afferent lymphatic flow. The increased lymphatic flow would serve to passively carry DC and other cells in the lymphatic vessel to the draining LN. It should also be noted that VEGF has been reported to increase the transendothelial migration of breast cancer cells (36). Thus, cytokines that modulate vascular permeability may share an ability to facilitate the transmigration of leukocyte and other cells.

Current models of DC activation and subsequent migration to LN (43) suggest that many inflammatory cytokines, bacterial products (e.g., LPS), and TLR ligands are capable of initiating the activation of DC (44). As activation occurs, DC lose adhesive connections such as E-cadherin (45) and up-regulate chemokine receptors such as CCR7, which are vital for successful entry into afferent lymphatic vessels that express CCL21 (1, 7). We hypothesize that a variety of immune cells at the site of inflammation, including activated DC, release OSM. As we have demonstrated in this work, activated BMDC, especially those treated with LPS, produce OSM at the mRNA and protein levels. Based on our current experiments, OSM acts to (1) increase expression of CCL21 in skin and draining LN, which would lead to greater attraction of DC into lymphatics and their enhanced retention in draining LN, and (2) alter the permeability of lymphatic vessels (or increase hydrodynamic lymphatic flow), which could conceivably facilitate DC entry into lymphatic vessels (or their passive transport toward the draining LN).

In summary, our results suggest a new role for OSM in regulating DC trafficking to lymphatic vessels via enhanced expression of CCL21 and alterations in the permeability of EC.


   Acknowledgment
 
We thank Dr. Mark C. Udey (National Cancer Institute, Dermatology Branch, Bethesda, MD) for helpful suggestions and comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. S.H.J. was supported by the National Institutes of Health Clinical Training Research Program. Back

2 Current address: Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan. Back

3 M.S., L.F., and A.R.C. contributed equally to the execution of the study. Back

4 Current address: Departments of Dermatology and Molecular Microbiology and Immunology, Oregon Health & Science University and Dermatology Service, Veterans Affairs Medical Center, 3710 Southwest U.S. Veterans Hospital Road, R&D 55, Portland, OR 97239. Back

5 Address correspondence and reprint requests to Dr. Sam T. Hwang, Senior Investigator, 10 Center Drive, Room 12N258, Bethesda, MD, 20852. E-mail address: hwangs{at}mail.nih.gov Back

6 Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; EC, endothelial cell; LEC, lymphatic EC; BVEC, blood vascular EC; DMEC, dermal microvascular EC; LYVE-1, lymphatic vascular EC-1; VEGF, vascular endothelial growth factor; OSM, oncostatin M; HDF, human dermal fibroblast; LMEC, lung microvascular EC; BMDC, bone marrow-derived DC; m, murine; for, forward; rev, reverse; RT, room temperature; rm, recombinant murine; rh, recombinant human; hOSM, human OSM; TNCB, trinitrochlorobenzene. Back

Received for publication March 27, 2006. Accepted for publication September 25, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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