Originally published online as doi:10.1189/jlb.0104037 on June 14, 2004

Published online before print June 14, 2004

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(Journal of Leukocyte Biology. 2004;76:472-476.)
© 2004 by Society for Leukocyte Biology

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

Uwe Ritter^*,1, Florian Wiede^*, Dirk Mielenz, Ziba Kiafard, Jörg Zwirner and Heinrich Körner^*,,2

^* IZKF Nachwuchsgruppe 1, Nikolaus-Fiebiger Zentrum für Molekulare Medizin, and
Abteilung für Immunologie, Nikolaus-Fiebiger Zentrum für Molekulare Medizin, Universität Erlangen, Germany;
Abteilung für Immunologie, Georg-Augustus-Universität, Göttingen, Germany; and
Comparative Genomics Centre, James Cook University, Townsville, Australia

¹Correspondence: Nikolaus-Fiebiger-Zentrum für Molekulare Medizin, IZKF, Nachwuchsgruppe 1, Glückstrasse 6, D-91054 Erlangen, Germany. E-mail: uritter{at}molmed.uni-erlangen.de

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About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF^–/–) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF^–/– BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7⁺ splenic DCs only marginally.

Key Words: tumor necrosis factor • mouse • knockout • bone marrow precursor cells • chemokine receptor

Dendritic cells (DCs) are central for the induction of an immune response to pathogens. An insult such as the inoculation of a pathogen causes the tissue resident DCs to leave the tissue and transport antigen to the draining lymph node (LN) and to induce an antigen-specific immunity [¹ ]. After their encounter with the antigen and during migration to the LN, these cells undergo radical changes from immature phagocytic precursor cells, which express low levels of major histocompatibility complex (MHC) class II and costimulatory molecules CD80 and CD86, to antigen-presenting cells (APC), able to prime naïve T cells [¹ ]. As this priming event takes place mainly at a specific localization—the T cell compartment of the LN—and as the adaptive immune response has to be initiated rapidly, it follows that there have to be specific mechanisms supporting the quick transit of DCs. The chemokine receptor (CCR)7 constitutes one of these molecular mechanisms. It has been demonstrated that the transcription rate and the surface expression of CCR7 are induced during differentiation of human DCs [^{2 3 4 5 6} ]. Therefore, cells at a certain stage of their development start to exhibit this receptor and become responsive to its ligands CCL19 and CCL21 [⁷ ]. The further analysis of these ligands and their receptor in gene-deficient mice has shown that both are important components of the navigation system that guides maturing DCs from the periphery to the secondary lymphoid organs during inflammation [^{8 9 10} ].

Recently, we could demonstrate that tumor necrosis factor (TNF) is crucial for the effective generation of mature murine bone marrow-derived DCs (BM-DCs) in vitro [¹¹ ]. These BM-DCs mature spontaneously in the sole presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and are characterized by a high expression of costimulatory molecules such as CD40 and CD86. As a result of the lack of a mouse CCR7-specific monoclonal antibody (mAb), the investigation of the CCR7 surface expression during DC maturation has been limited to human cells. As we were interested in the expression kinetics of the murine receptor on DCs, we generated a specific mAb for mouse CCR7 and investigated the expression of CCR7 during BM-DC maturation in cultures of C57BL/6 (B6.WT) and TNF-negative C57BL/6 (B6.TNF^–/–) BM precursor cells. The analysis of CCR7 revealed a new TNF-dependent heterogeneity of this CCR on differentiating cells.

To verify the specificity of the anti-CCR7 mAb, a preparation of splenocytes and BM-DC surface molecules was used in an immunoprecipitation experiment. The anti-CCR7 mAb precipitated specifically a protein with a size of 43 kD (Fig. 1A ), which is close to the estimation of the receptor size that was based on the genomic sequence [¹² ]. Furthermore, the binding characteristics of the anti-CCR7 mAb to CCR7 on T cells [⁹ ] were tested. The mAb was added to the cells for 30 min and was allowed to bind to the receptor. Subsequently, chemotaxis in response to the CCR7 ligand CCL21 was analyzed in a transmigration assay. The addition of CCL21 caused the internalization of CCR7 (data not shown) and induced a strong migratory response toward the ligand (Fig. 1B) . This indicates that this newly generated mAb did not interfere with the binding of the ligand to the receptor. The media control (only mAb, no ligand) demonstrated that the mAb was not agonistic and did not trigger a response of the receptor by itself (Fig. 1B) . In a third experiment, the influence of the anti-CCR7 antibody on in vivo migration to the splenic T cell compartment was tested. Cells that had been incubated with anti-CCR7 mAb and controls were equally distributed within the T cell area of the periarteriolar lymphoid sheaths. The immunoprecipitation experiment confirms the specificity of the mAb for CCR7. The uninhibited migration of T cells in vitro and in vivo in response to CCL21 shows clearly that the anti-CCR7 mAb does not interfere with cell migration.

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Figure 1. Characterization of the anti-CCR7 mAb. B6.WT splenocytes and BM-DCs harvested at day 10 of tissue culture were metabolically labeled with 35S-methionine. Cells were lysed, and immunoprecipitation was performed following a standard protocol [13 ] with our immunoglobulin G (IgG)2a CCR7-specific antibody (CCR7) or an unspecific IgG2a control (IgG). Immunoprecipitates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was dried and exposed to a phosphoimager plate. The molecular weight is indicated on the left. The mAb with specificity for the murine CCR7 was generated in the laboratory of J. Zwirner by repeatedly immunizing Lou/c rats intraperitoneally with 2–4 x 107 CCR7 rat basophilic leukemia (RBL)-2H3 transfectants. Cell fusions were performed according to standard techniques using the myeloma cell line X63-Ag8.653. Culture supernatants were screened by indirect immunofluorescence using CCR7-expressing RBL-2H3 transfectants. The murine CCR7 cDNA was obtained by polymerase chain reaction (PCR) amplification using oligonucleotide primers designed according to the published murine CCR7 DNA sequence [12 ]. (B) Splenic T cells from B6.WT mice were purified by depleting CD19-positive cells with magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions. These purified T cells were incubated with or without 40 µg/ml anti-CCR7 mAb. After 30 min of incubation, T cells were transferred to Transwell inserts (5 µm), separating the upper and lower chambers in 24-well plates (Costar, Cambridge, MA) and were incubated for 2 h. The lower chamber contained 1 µg/ml CCL21. Subsequently, migrated T cells were harvested from the lower chamber and stained with fluorescein isothiocyanate (FITC)-conjugated anti-T cell receptor-/ß chain in the presence of a set number of beads to quantify the migrated T cells [14 ]. (C) Carboxy fluorescein diacetate succinimidyl ester (CFSE)-labeled T cells were incubated with CCR7 mAb and injected intravenously. After 4 h, homing of CFSE-labeled T cells (green) to the T cell compartments (red) of the spleen was analyzed [15 ].

The lack of an anti-mouse CCR7 mAb has prevented an extensive investigation of the behavior of this receptor on mouse leukocytes. Although some data could be generated with a CCL19-IgG1-fusion protein [¹⁶ ], the obvious disadvantages of an agonistic chimeric molecule that competes with the ligand prevented an application in many models. The use of our mAb should allow us to study for the first time the surface expression of CCR7 on mouse DCs during normal, spontaneous differentiation in vitro and in response to proinflammatory stimuli.

We investigated the expression of CCR7 on BM-DCs from B6.WT and B6.TNF^–/– mice at different points of time by flow cytometry. The number of mature, CD11c-positive B6.WT BM-DCs expressing high levels of CD86 increased from 10% (day 6) to 43% (day 10; Fig. 2A and 2E ). In the B6.TNF^–/– genotype, the proportion of mature BM-DCs increased to maximal 18% (data not shown). This result was in accordance with earlier findings that TNF is important for the generation of mature BM-DCs [¹¹ ]. Furthermore, this analysis of BM-DCs cultured with GM-CSF over a period of 10 days showed that CCR7 could be detected on all mature B6.WT and B6.TNF^–/– BM-DCs (Fig. 2B 2D and 2F) . Therefore, we demonstrated that this receptor can be used in the mouse as an additional DC maturation marker together with CD40, -80, and -86 and MHC class II [⁶ ], as it has been described for human DCs [¹⁸ ].

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Figure 2. Analysis of the CCR7 surface expression on BM-DCs cultured with GM-CSF. BM-DCs were generated from B6.WT or B6.TNF–/– mice as described in detail elsewhere [11 , 17 ]. After 6 (A, B), 8 (C, D), and 10 days (d; E, F) of culture in GM-CSF, the cells were harvested and stained with anti-CD11c (clone HL3; PEI-conjugated) and anti-CD86 (clone GL1; FITC). The mAb were purchased from BD PharMingen (San Diego, CA). The rat anti-mouse CCR7 mAb (IgG2a) was purified and used unlabeled. To detect the CCR7 mAb, Cy5-conjugated goat anti-rat IgG was used as secondary reagent (Dianova, Hamburg, Germany). Multicolor flow cytometry was performed as described [15 ]. The cells were electronically gated according to light-scatter properties and the expression of the DC marker CD11c. Data were collected using a FACSCalibur flow cytometer (BD PharMingen) and analyzed with CellQuest software (BD PharMingen). Mature (R1) and immature (R2) BM-DCs were quantified by electronic gating. The mean fluorescent intensity of CCR7 on mature (R1; gray) or immature (R2; transparent) DCs is shown. (One representative experiment of two is shown.)

After 10 days of culture, the population of mature B6.WT CD11c^positive CD86^high CCR7^high BM-DCs reached 40% mature DCs (Fig. 3A ). In contrast, the proportion of mature BM-DCs from B6.TNF^–/– mice peaked at 18–20% (Fig. 3B) . During the process of maturation, the up-regulation of CD40 and CD86 seems to occur rapidly. In an analysis of the kinetics of these two surface molecules, there is no transient, intermediate population detectable [¹¹ ]. It is interesting that if CCR7 is used as a maturation marker, a small population of CD11c^positive CD86^intermediate CCR7^intermediate BM-DCs could be detected after 10 days of maturation (Fig. 3A and 3B , upper-left quadrant). To exclude a staining artifact and to confirm the CCR7 expression, these cells were isolated by flow cytometry and analyzed by PCR. We could demonstrate that CCR7 mRNA was indeed expressed within this population (data not shown). To test whether this small population could be increased by further activation, we stimulated BM-DCs after a maturation period of 10 days with TNF for 24 h and 48 h. After 24 h of stimulation with recombinant TNF, a population of CD11c^positive CD86^intermediate CCR7^intermediate BM-DCs is detectable. BM-DCs from B6.WT and B6.TNF^–/– mice show an accumulation of these cells (Fig. 3C and 3D) compared with cells without TNF stimulation (Fig. 3A and 3B) . After 48 h of TNF stimulation, a clear separation of CD11c^positive CD86^high CCR7^high mature BM-DCs as well as CD11c^positive CD86^low CCR7^negative could be detected (Fig. 3E and 3F) , and the intermediate population had disappeared. The characteristics of this CCR7^intermediate BM-DC population as well as the correlation of the CCR7 expression with the expression of other maturation markers such as CD40 and CD86 are not yet understood and have to be analyzed in more detail.

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Figure 3. Analysis of the CCR7 surface expression on BM-DCs after addition of TNF. Nonadherent BM cells were analyzed by flow cytometry at day 10 (d 10) of culture with GM-CSF (A, B). Aliquots of the cells were cultured for 24 h (C, D) or 48 h (E, F) in the presence of GM-CSF and TNF (16 ng/ml). CD11c-positive BM cells were electronically gated, and the proportion of subpopulations was determined. One representative experiment of two is shown.

To test whether CCR7 is induced by other inflammatory stimuli, we harvested BM-DCs at day 8 and incubated them with LPS to trigger DC maturation [¹¹ , ¹⁷ ]. After 24 h of LPS stimulation, 96% of BM-DCs expressed high levels of CD86 and CCR7 (Fig. 4A ). The transition from immature BM-DCs to mature BM-DCs is much quicker if LPS is used instead of TNF. The transient population of CD11c^positive CD86^intermediate CCR7^intermediate BM-DCs, which we could detect after TNF induction, does not appear after LPS stimulation (compare Figs. 4A and 3C ).

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Figure 4. LPS induced CCR7 expression on DC populations. (A) BM-DCs were generated from B6.WT mice in the presence of GM-CSF. After 8 days of culture, these cells were harvested and further incubated with LPS (1 µg/ml) for a further 24 h. The induction of CCR7 and CD86 on CD11c-positive BM-DCs was determined by fluorescein-activated cell sorter analysis as described above. (B) Splenocytes isolated from B6.WT mice were incubated for 24 h with medium or with LPS (1 µg/ml). The expression of CD8 and MHC class II on CD11c-positive DCs is shown. (C) The expression of MHC class II on subpopulations of CD11c-positive DCs was determined by electronically gating (R1=CD8positive CCR7high; R2=CD8negative CCR7high; R3=CD8negative CCR7low). One representative experiment is shown.

Furthermore, we isolated CD11c-positive cells from the spleen and characterized their CCR7 expression after 24 h of tissue culture. The CD8-positive DC subpopulation is almost entirely CCR7⁺, whereas more than 60% of the CD8-negative, splenic DCs present as CCR7^– cells. Maturity or immaturity of the analyzed cells, respectively, is confirmed by the correlation of MHC class II and CCR7 expression (Fig. 4C , R1–R3). The data presented are consistent with earlier findings that demonstrated that the splenic CD8⁺ DC population constitutes the most mature cell population in this lymphoid organ [¹⁹ ] and show that already, tissue culture without further stimulus causes strong DC maturation [²⁰ ]. Addition of LPS for 24 h to these cells leads only to a marginal, further induction of CCR7 on the investigated subpopulations (Fig. 4B and 4C) .

In conclusion, we have generated an anti-mouse CCR7 mAb that is neither agonistic nor antagonistic, and we could use this mAb to demonstrate that CCR7 is expressed on spontaneously differentiated as well as TNF-activated, mature B6.WT and B6.TNF^–/– BM-DCs and on splenic DC subpopulations. This antibody will now allow us to address different questions regarding the biological role of CCR7 during maturation of DCs and lymphocytes in vivo and migration of CCR7-positive cell populations in general.

 
The work was supported by the DFG (KO 1315/3-3 and 4-1 to H. K.; ZW 38/ 3-3 to J. Z.) and the Federal Ministry of Education and Research (BMBF) and the Inter-disciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nürnberg (HK NW1).

 
² Correspondence: Comparative Genomics Centre, James Cook University, Townsville, Qld 4811, Australia. E-mail: heinrich.korner@jcu.edu.au

Received January 23, 2004; revised April 4, 2004; accepted April 30, 2004.

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