6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7

Abstract

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

Figure 1

6CK/SLC is a chemotactic agonist for the lymphocyte chemokine receptor CCR7 (stable L1/2 transfectants). (A–C) Anti-FLAG staining of G_iα-linked receptor L1/2 transfectants. Solid line, M1 anti-FLAG mAb; dotted line, isotype control. Staining of (A) wild-type L1/2, (B) FLAGged CCR7 transfectants, and (C) FLAGged CCR1 transfectants. (D) Chemotaxis of L1/2 cells stably transfected with muCCR7 or hCCR1 to various chemokines or control medium. Stably transfected cells were selected to homogeneity by several rounds of sorting for the FLAG epitope tag. Mean and range of duplicate wells is shown. Representative of three individual experiments.

Figure 2

6CK/SLC is a chemotactic agonist for the lymphocyte chemokine receptor CCR7 (transient CCR7 transfectants). (A and B) GFP expression of transient CCR7 transfectants. (A) hCCR7/GFP transient transfectants before chemotaxis (solid line) overlaid on mock transfected control (dotted line). (B) hCCR7/GFP transient transfectants after chemotaxis to h6CK/ SLC (solid line) overlaid on mock transfected control (dotted line). (C) Chemotaxis of transient CCR7 transfectants. Cells were transfected with either human or mouse CCR7 cDNA constructs along with a GFP vector. 24 h after transfection, when the chemotaxis was performed, the muCCR7 population was ∼25% GFP(+), and the hCCR7 population was ∼42% GFP(+). For each receptor/chemokine combination, chemotaxis was performed in six replicate wells, and the number of migrated cells enumerated as described previously (10, 11). Migrated cells in the replicate wells of each receptor/chemokine combination were then pooled and analyzed by flow cytometry for the frequency of GFP-expressing (transfected) cells. The number of GFP(+) and GFP(−) cells was determined for the starting population and for each migrated population, and the percent migration was determined from these numbers. Maximum migration was 4.8% for hCCR7 transfectants and 2.6% for muCCR7 transfectants. Mean and range of duplicate wells are shown. Representative of two individual experiments.

Figure 3

MIP-3β, like 6CK/SLC, is a potent attractant for resting human and mouse lymphocytes. (A) Migration of freshly isolated human circulatory lymphocytes through 5-μm pores for 90 min. The optimal chemotactic dose was used for each chemokine (see Materials and Methods). Medium, the number of cells migrating into the bottom well in the absence of chemokine. Mean of two experiments (eight wells each) with individual healthy donors is shown; error bars indicate range. (B) Migration of freshly isolated mouse lymph node lymphocytes for 60 min through 5-μm pores. The optimal chemotactic dose was used. Mean of two experiments with different mouse strains (BALB/c and C57BL/6) is shown; error bars indicate range.

Figure 4

MIP-3β and 6CK/SLC attract similar lymphocyte subpopulations. (Top row) Percent migration of resting human peripheral blood lymphocytes. Subsets defined by multiparameter FACS^® analysis as follows: natural killer = CD3(−), CD56(+), CD16(+); naive CD4(+) = CD4(+), CD45RA(+), CD45RO(−); memory CD4(+) = CD4(+), CD45RA(−), CD45RO(+); CD8(+) T cells = CD3(+), CD8(+); B cells = CD3(−), CD19(+). Migration through 5-μm pores for 90 min. Mean of two experiments (eight wells each) with individual healthy donors is shown; error bars indicate range. (Bottom row) Percent migration of resting mouse lymphocytes defined as follows: naive CD4(+) = CD3(+), CD4(+), CD44(lo); memory CD4(+) = CD3(+), CD4(+), CD44(hi); CD8(+) T cells = TCRαβ(+), CD8(+); B cells = TCRαβ(−), B220(+). Migration through 5-μm pores for 60 min. Mean of two individual experiments (six wells each) with 20-wk C57BL/6 is shown; error bars indicate range.

Figure 5

MIP-3β desensitizes lymphocyte chemotaxis to 6CK/ SLC. Mouse lymph node cells were pretreated with 5× the optimal chemotactic dose of each chemokine (indicated under each column) and then washed and allowed to migrate towards the same or another chemokine (at the optimal chemotactic dose) through 5-μm pores for 15 min. This short chemotactic period was used because resensitization of pretreated cells was quite rapid: ∼50% of homologous desensitization was lost if chemotaxis was extended to 30 min. Less migration was achieved at 15 min than when the experiments were allowed to go to completion, but the number of migrated cells was sufficiently high for reliable reproducibility: mean migration of control pretreated cells was ∼25% of input for MIP-3β, ∼15% for 6CK/SLC, and ∼10% for SDF-1α. Mean of two experiments, each with duplicate wells is presented; error bars indicate range.