Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells found as sentinels in peripheral tissues and lymphoid organs. DCs are classified into 2 populations, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) with distinct expression patterns of costimulatory molecules and Toll-like receptors.^1,2  Although both mDCs and pDCs are produced in the bone marrow (BM) and migrate into lymphoid tissues to control immune response, the migratory pathways of these DC subsets are different.³  As the migratory properties of DCs are of fundamental importance for their function, chemokines and their receptors have been extensively analyzed. However, the downstream signaling molecules critical for DC migration are largely unknown.

Chemokine receptors are coupled with heterodimeric G_i proteins that activate a variety of signaling pathways including Rac. DOCK2 is a novel member of the CDM family proteins, Caenorhabditis elegansCED-5, mammalian DOCK180, and Drosophila melanogasterMyoblast City, that are known to regulate the actin cytoskeleton by functioning upstream of Rac.^4,5  Although DOCK2 plays an important role in migration of lymphocytes and neutrophils,^6-10  the role of DOCK2 in DC migration remains unknown. In this study, we examined whether and how DOCK2-deficiency affects migration of mDCs and pDCs.

DOCK2-deficient (DOCK2^−/−) mice were backcrossed with C57BL/6 (B6) mice for more than 8 generations before use. All experiments were done in accordance with the guidelines of the committee of Ethics of Animal Experiments, Kyushu University.

To generate BM-derived pDCs and mDCs, BM cells were cultured for 7 days with Flt3 ligand (50 ng/mL; R&D Systems, Minneapolis, MN) or granulocyte-macrophage colony-stimulating factor (20 ng/mL; PeproTech, Rocky Hill, NJ), respectively. Cells were then purified with either anti-B220 or anti-CD11c microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). In some experiments, pDCs and mDCs were purified with a FACS Aria (BD Biosciences, Mountain View, CA) after staining the cells with anti-B220 and anti-CD11c antibodies (BD Biosciences).

Cells were stained with anti-B220, anti-CD11c, anti-mPDCA1 (Miltenyi Biotec), anti-CXCR3 (R&D Systems), anti-CXCR4¹¹  and/or anti-CCR7¹¹  antibodies, and analyzed on a FACS Calibur (BD Biosciences). For tissue staining, frozen sections were fixed in acetone or 4% paraformaldehyde, and incubated with anti-mPDCA1, anti-B220 and/or anti-MOMA1 (BMA Biomedicals, Augst, Switzerland) antibodies.

Transwell chemotaxis assay was performed as described,^6,9  with primary BM cells. EZ-Taxiscan chemotaxis assay was performed according to the manufacturer's protocol (GE Healthcare, Chalfont St Giles, United Kingdom) using BM-derived pDCs and mDCs.

BM-derived pDCs were labeled with PKH fluorescent kit (Sigma-Aldrich, St Louis, MO) and injected intravenously into mice.

Total RNA samples treated with RNase-free DNase I were reverse transcribed using oligo(dT) and subjected to polymerase chain reaction (PCR) with specific primers (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

Although both mDCs and pDCs were normally generated in the DOCK2^−/− BM, DOCK2^−/− mice exhibited a severe reduction of pDCs, but not mDCs, in the spleen (Figure 1A,B). Similar results were obtained when peripheral and mesenteric lymph node (LN) cells were analyzed (Figure 1B). Immunohistochemical analysis of the spleen and peripheral LN tissue sections revealed that, while wild-type pDCs were present mostly in the T cell area, such pDCs were scarcely found in DOCK2^−/− mice (Figure 1C).

This finding led us to examine whether homing of pDCs to secondary lymphoid organs is impaired in DOCK2^−/− mice. For this, we labeled BM-derived pDCs from B6 and DOCK2^−/− mice with different fluorescent dyes and injected intravenously equal numbers of each into wild-type mice. At 24 hours after transfer, the frequencies of DOCK2^−/− pDCs migrated to the spleen and LNs were reduced to approximately 50% of the wild-type levels (Figure 1D). Moreover, unlike B6 pDCs, DOCK2^−/− pDCs failed to populate the periarteriolar lymphoid sheaths of the spleen (Figure 1E). These results indicate that DOCK2^−/− pDCs are impaired in their homing to and localization within secondary lymphoid organs because of an intrinsic defect in pDCs. This mislocalization may affect survival or turnover of pDCs in vivo, as the ratio of DOCK2^−/− pDCs to B6 pDCs in the spleen gradually decreased with time (Figure 1F).

The chemokine receptors CXCR4 and CCR7 are known to mediate migration of pDCs and mDCs.^2,3,12-16  To examine whether DOCK2 functions downstream of chemokine receptors, we first analyzed chemotactic response of primary BM DCs to CXCR4 ligand CXCL12. In the transwell chemotaxis assay, mDCs from both B6 and DOCK2^−/− mice efficiently migrated (Figure 2A), indicating that DOCK2 is dispensable for mDC migration. However, unlike B6 pDCs, DOCK2^−/− pDCs did not show detectable responses to CXCL12 (Figure 2A). Moreover, no additive effect was found when DOCK2^−/− pDCs were stimulated with CXCL12 plus CXCL9, a CXCR3 ligand that is known to synergize with CXCL12^14,16  (Figure 2B). This defect does not result from the chemokine receptor expression, as BM pDCs from B6 and DOCK2^−/− mice comparably expressed CXCR4 and CXCR3 (Figure 2C).

To determine more precisely the role of DOCK2 in pDC migration, we analyzed BM-derived pDCs chemotaxing toward CCR7 ligand CCL21 in real-time with an EZ-Taxiscan (GE Healthcare). The expression of CCR7 was unchanged between B6 pDCs and DOCK2^−/− pDCs (Figure 2D). At first glance, however, DOCK2^−/− pDCs were less motile than B6 pDCs (Figure 2E). Detailed analysis revealed that DOCK2-deficiency not only reduced the migration speed, but also affected the directionality during pDC chemotaxis (Figure 2F).

Having found that DOCK2 is essential for migration of pDCs, but not mDCs, we then examined chemokine-induced Rac activation in these DC subsets. Although BM-derived B6 pDCs exhibited Rac activation in response to CCL21, such Rac activation was almost totally abolished in DOCK2^−/− pDCs (Figure 2G). Consistent with this finding, CCL21-induced actin polymerization and polarized F-actin accumulation were both impaired in DOCK2^−/− pDCs (Figure 2H,I). These results indicate that DOCK2 is a Rac activator indispensable for migration of pDCs. However, DOCK2-deficiency did not affect CCL21-mediated Rac activation in mDCs (Figure 2G). We found that unlike pDCs, mDCs express both DOCK2 and DOCK180 (Figure 2J). Therefore, if CDM family proteins are also important for Rac activation during mDC migration, this process may be coordinately regulated by DOCK2 and DOCK180.

In conclusion, we have shown that DOCK2 controls pDC migration by functioning downstream of chemokine receptors and activating Rac. While pDCs regulate activation of mDCs and NK cells through the direct interaction in secondary lymphoid organs,^17-19  LN homing of pDCs has been suggested to be critical for regulatory T-cell development and tolerance induction in a cardiac allograft transplant model.²⁰  How defective pDC migration affects immune response in DOCK2^−/− mice is an important issue that should be investigated in future studies.

This work was supported by the Genome Network Project, the Target Protein Project, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Contribution: K.G. and Y.T. performed research, analyzed data, and drafted the paper; A.N. and A.I. performed research; M.E., R.T., and T.S. interpreted data; and Y.F. designed research, analyzed and interpreted data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Yoshinori Fukui, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan; e-mail: fukui@bioreg.kyushu-u.ac.jp.