A silent chemokine receptor regulates steady-state leukocyte homing in vivo

Abstract

The location of leukocytes in different microenvironments is intimately connected to their function and, in the case of leukocyte precursors, to the executed differentiation and maturation program. Leukocyte migration within lymphoid organs has been shown to be mediated by constitutively expressed chemokines, but how the bioavailability of these homeostatic chemokines is regulated remains unknown. Here, we report in vivo evidence for the role of a nonsignaling chemokine receptor in the migration of leukocytes under physiological, i.e., noninflammatory, conditions. We have studied the in vivo role of the silent chemokine receptor CCX-CKR1 by both loss- and gain-of-function approaches. CCX-CKR1 binds the constitutively expressed chemokines CC chemokine ligand (CCL)19, CCL21, and CCL25. We find that CCX-CKR1 is involved in the steady-state homing of CD11c(+)MHCII(high) dendritic cells to skin-draining lymph nodes, and it affects the homing of embryonic thymic precursors to the thymic anlage. These observations indicate that the silent chemokine receptor CCX-CKR1, which is exclusively expressed by stroma cells, but not hematopoietic cells themselves, regulates homeostatic leukocyte migration by controlling the availability of chemokines in the extracellular space. This finding adds another level of complexity to our understanding of leukocyte homeostatic migration.

Fig. 1.

CCX-CKR1 binds the chemokines CCL19, CCL21, and CCL25. (A) CCX-CKR1-transfected HEK293 cells (dark line) bind a CCL19-Fc fusion protein. Mock transfected HEK293 cells (filled gray population) were used as control. (B) CCL19 (filled circles), CCL25 (filled squares), and CCL21 (filled triangles), but not CXCL12 (open circles), CCL17 (open squares), or CCL22 (open triangles) compete with CCL19-Fc for binding on CCX-CKR1 transfectants. (C) CCX-CKR1 does not induce an increase in intracellular calcium upon the addition of the indicated chemokines. Chemokine receptor transfectants loaded with the calcium-sensitive dye fura2-AM were exposed to the indicated chemokines, and the ratio of relative fluorescence intensities was measured over time. (D) (Left) CCX-CKR1 transfectants internalize CCL19-Fc in a time dependent manner. CCX-CKR1-HEK293 cells incubated at 4°C with CCL19-Fc were either warmed to 37°C (thick line) or not warmed (thin line) before cooling the cells to 4°C and adding an anti-Fc secondary reagent. To rule out the possibility that incubation at 37°C by itself reduced receptor levels, CCX-CKR1-HEK293 cells were warmed to 37°C in the absence of CCL19-Fc, cooled to 4°C, and stained with CCL19-Fc (dashed line). (Right) The reduction of surface CCL19-Fc caused by warming the cells (Left, thick line) relative to the amount of surface staining without prior addition of ligand (Left, dotted line) is plotted over time.

Fig. 2.

CCX-CKR1 is expressed in stromal but not hematopoietic cells in thymus, intestine, lymph node and epidermis. (A–C) Sections from wild-type mice were negative. (A–F) Overview images. (G–J) Higher magnifications of images. For the thymus, EGFP^CCX-CKR1 expression is shown for the subcapsular zone (G) and the cortico-medullary junction (J). (Scale bars: in A–F, 200 μm; in G–J, 40 μm.)

Fig. 3.

The absence of CCX-CKR1 impairs the steady-state homing of DCs entering lymph nodes by way of afferent lymphatics but not the homing of DCs entering by way of the blood. (A) Flow cytometric analysis of DCs in skin-draining lymph nodes of wild-type and CCX-CKR1-deficient mice. Contour plots are gated on B220-negative Thy1-negative cells. Gates are set to distinguish CD11c⁺MHCII^high DCs that enter lymph nodes by way of afferent lymphatics from CD11c⁺MHCII^low DCs that enter by way of the blood. (B) The steady-state number of CD11c⁺MHCII^high, but not CD11c⁺MHCII^low DCs, in skin-draining lymph nodes is reduced in CCX-CKR1-deficient mice. At least 10 7-week-old mice were analyzed per genotype. (C) Enumeration of CD11c⁺MHCII^high DCs isolated from dermal and epidermal sheets of individual CCX-CKR1-deficient mice and wild-type littermates subjected to proteolytic digestion. Each dot represents one mouse, and the bars indicate the mean. (D) Epicutaneous application of FITC normalizes the numbers of CD11c⁺MHCII^high DCs in CCX-CKR1-deficient mice. The number of CD11c⁺MHCII^highFITC⁺ DCs 48 h after FITC treatment in six skin-draining lymph nodes per mouse was determined. At least seven 7-week-old mice were analyzed per genotype. (E) FACS analysis of wild-type (dashed line) and CCX-CKR1-deficient (thin line) CD11c⁺MHCII^high DCs 48 h after FITC treatment. Both histograms are gated on CD11c⁺MHCII^high DCs as shown in A. Filled gray populations are CD11c⁺MHCII^high DCs from a mouse 48 h after treatment with carrier alone (Left) and after FITC treatment without the CD86 stain (Right). The thick line indicates the levels of CD86 staining on CD11c⁺MHCII^high DCs without FITC treatment.

Fig. 4.

Overexpression of CCX-CKR1 impairs homing of hematopoietic thymic precursors to the embryonic thymic anlage. (A) In situ hybridization of E13.5 thymic anlagen of wild-type and Foxn1::CCX-CKR1 transgenic littermates. Sections stained with sense probes remained negative. (Scale bar: 200 μm.) (B) Immunohistochemical staining of E13.5 thymic anlagen of a Foxn1::CCX-CKR1 transgenic mouse for CCL25 expression. Staining is restricted to the thymic anlagen (circled in white) as in wild-type mice (our unpublished data). tr, trachea; oe, oesophagus. (Scale bar: 200 μm.) (C) The number of CD45-positive hematopoietic precursors that home to the E12.5 thymic anlage is reduced in Foxn1::CCX-CKR1 transgenic mice in comparison with wild-type littermates. (D) The fraction of BrdU-positive thymocytes was determined in E14.5 wild-type and Foxn1::CCX-CKR1 transgenic littermates. (E) The fraction of annexin V-positive thymocytes was determined in E14.5 littermates of the indicated genotypes. (F) The number of lineage marker-negative CD117-positive PIR-positive prethymic T lineage precursors in the fetal liver was determined for E14.5 littermates by FACS. (G) The fraction of lineage marker-negative CD117-positive PIR-positive thymic precursors was determined in the blood of E14.5 littermates by FACS. (H) Thymocyte numbers are normal in newborn Foxn1::CCX-CKR1 transgenic mice. (D–H) At least eight offspring of Foxn1::CCX-CKR1 founder no. 11 were analyzed per genotype.