A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes

Sequence Analysis.

Pattern searches of the National Center for Biotechnology Information (NCBI) sequence database with tfasta (36) retrieved related SLC expressed sequence tags (ESTs) from human and mouse. I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory) cDNA clones 332031, 389013, and 503192 (37) were obtained from Genome Systems (St. Louis) as EcoRI-NotI inserts in the pT7T3-Pac vector and sequenced. The Institute for Genomic Research clone EST113564 (38) was obtained from American Type Culture Collection and processed similarly.

RNA Expression Studies.

For Northern analysis, mRNA from mouse tissues or purified cells was subjected to gel electrophoresis, transferred to Hybond membranes, and probed by using the randomly primed mouse SLC EST cDNA. A human multi-tissue dot blot (CLONTECH) was probed with the human SLC EST. For in situ hybridizations, paraffin sections (5 μm) from C57BL6 mice were deparaffinized, fixed in 4% paraformaldehyde, and treated with proteinase K. After washing in 0.5× standard saline citrate (1× = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7), the sections were covered with hybridization solution, prehybridized for 1–3 hr at 55°C, and hybridized overnight with sense or antisense ³⁵S-labeled riboprobe transcribed from the full-length mouse SLC EST. After hybridization, sections were washed at high stringency, dehydrated, dipped in photographic emulsion NTB₂ (Kodak), stored at 4°C for 2–8 weeks, developed, and counterstained with hematoxylin and eosin.

Production of Recombinant Proteins.

The full-length mouse and human SLC ESTs were cloned into the pVL1393 baculovirus transfer vector and cotransfected with BaculoGold (PharMingen) into SF9 cells according to the manufacturer’s instructions. For protein production, SF21 cells were infected at a multiplicity of infection of 10–20 and cultured in serum-free media for 60 hr. Conditioned media was cleared, loaded onto a HiTrap heparin affinity column (Pharmacia), and eluted with a 0.2–1 M NaCl gradient in 50 mM Hepes (pH 7.9). Fractions containing only SLC were pooled, concentrated on Centriplus-10 concentrators (Amicon), and dialyzed against PBS.

Chemotaxis.

Mouse lymphocytes, obtained by passing teased spleen and lymph node cells through a 70-μm screen, were resuspended in RPMI 1640 medium containing 5% fetal calf serum, incubated for 1 hr at 37°C, and subjected to a chemotactic assay (25). For the assay, 10⁶ cells in 100 μl medium were added to the top chamber of a 6.5-mm diameter, 5 μm pore polycarbonate Transwell insert (Costar, Cambridge, MA) and incubated in triplicate with the indicated concentrations of SLC in the bottom chamber for 3 hr. Cells migrating to the bottom chamber were resuspended, stained with antibodies to cell surface markers (CD4, CD8, B220, CD44, and L-selectin) and subjected to flow cytometric analysis and counted with a FACScan (Becton Dickinson). A 1:20 dilution of input cells was similarly analyzed. In some studies, cells were pre incubated with 100 ng/ml pertussis toxin (List Biological Laboratories, Campbell, CA) for 2 hr at 37°C. Mouse peripheral blood lymphocytes were obtained by erythrocyte lysis of whole blood, and assayed as above.

Controlled Detachment Adhesion Assay.

Recombinant soluble ICAM-1-Fc (5–10 μg/ml) (produced with a plasmid provided by D. L. Simmons, Imperial Cancer Research Fund, Oxford, U.K.) diluted in Tris-buffered saline pH 9.0 were coated onto polystyrene plates that were assembled as the lower wall in a parallel plate flow chamber and mounted on an inverted phase-contrast microscope (Diaphot TMD). Human naive and unfractionated T lymphocytes were isolated as described (29). The cells (at 1 × 10⁶ cells/ml in Hanks’ balanced salt solution supplemented with 0.2% BSA) were incubated with SLC for 6 min at room temperature, perfused into the flow chamber, and incubated under static conditions for 6 min. Then flow was initiated and increased in 2-fold increments every 10 sec. The experiments were videotaped for analysis, and the number of cells remaining bound was determined at each interval by using National Institutes of Health’s image 1.6 analysis package. The same field of view was used for each experiment to ensure that the results reflected uniform site density and distribution of the immobilized protein. All adhesion experiments were performed a minimum of three times. Phorbol 12-myristate 13-acetate or Mn served as a control treatment to induce strong adhesion to the ICAM-1 substratum. For inhibition studies, the cells were pre incubated for 20 min with 10 μg/ml R3.1 (anti-αL, gift from S. Simon, Baylor University, Waco, TX) on ice.

Identification of SLC.

We searched the NCBI EST database by using human MCP-1 as a template and found several chemokine-like sequences. We then performed in situ hybridization on mouse tissues to identify those EST clones with expression patterns suggestive of novel activities. One mouse clone (I.M.A.G.E. Consortium clone ID 389013) hybridized strongly to secondary lymphoid tissues (as described below). Sequence analysis of this clone revealed a cDNA of 848 bp encoding a 133 amino acid protein (data not shown). Analysis of a closely related human EST (clone ID 503192) identified in the same screen revealed an 847 cDNA encoding a protein of 134 amino acids with 72% amino acid identity to the murine protein (data not shown). Both mouse and human predicted proteins contain a chemokine consensus pattern. Both proteins also contain a highly basic 34- (mouse) or 35- (human) amino acid C-terminal extension that includes two conserved cysteines. These proteins have recently been described by several reports in the literature (21, 23, 24). We refer to this chemokine by the name it was first given, SLC.

SLC Is Expressed in HEV, T Cell Areas, and Lymphatic Endothelium.

By in situ hybridization, we found SLC to be expressed at strikingly high levels in the HEV of lymph nodes (Fig. 1 A, E) and Peyer’s patches (data not shown). SLC was expressed at lower levels in the T-cell zones of lymph nodes (Fig. 1A), spleen (Fig. 1B), and Peyer’s patches (data not shown), but was undetectable in the B cell areas (follicles) of these organs. The distribution of signal in T cell zones was suggestive of expression by stromal cells rather than lymphocytes. SLC was also expressed in the endothelium of small vessels in multiple organs. These vessels were identified as lymphatics by their characteristic anatomic location in liver (Fig. 1F) and small intestine (Fig. 1G). SLC expression was not detected in brain (data not shown) or in arteries or veins. In control experiments, no hybridization was seen with an SLC sense probe (Fig. 1 C, D, H).

To corroborate the results of in situ hybridization, we performed Northern analysis on mRNA blots of human and mouse tissues. Hybridization of human SLC to a human multitissue dot blot revealed that this mRNA was most abundant in lymph nodes and appendix, with moderate amounts in spleen, small intestine, thyroid, pancreas, trachea, and salivary gland (Fig. 2A). Multiple tissues demonstrated low level hybridization. No signal was observed in any region of the brain (data not shown) or in peripheral blood lymphocytes. In mouse tissues, SLC was expressed most strongly in peripheral and mesenteric lymph nodes and at lower levels in Peyer’s patches, spleen, and thymus (Fig. 2B). Freshly isolated murine B cells, T cells, and macrophages were negative.

SLC Stimulates Chemotaxis of Naive T Lymphocytes.

Because SLC is expressed in areas of T lymphocyte entry and accumulation, we investigated its chemotactic activity toward lymphocyte subsets. We found that mouse SLC was a highly efficacious chemoattractant for mouse splenic T lymphocytes, able to stimulate the migration of over 70% of the input T cells (Fig. 3A). At submaximal concentrations SLC showed greater activity toward CD4⁺ than CD8⁺ T cells. It exhibited less activity toward B cells. SLC attracted more T cells and had greater T cell specificity than SDF-1α, described previously as the most efficacious chemokine for resting lymphocytes (25). SLC showed a preferential activity toward those CD4⁺ T lymphocytes that expressed high levels of L-selectin and low levels of CD44 (Fig. 3B). In mice, this pattern of cell surface markers is indicative of naive lymphocytes (26, 27). The chemotactic response to SLC was inhibited by preincubation of lymphocytes with pertussis toxin (Fig. 3C), consistent with signaling through a G_αi-coupled receptor. To examine the effect of SLC on those cells that are normally recruited to secondary lymphoid organs, we measured the chemotactic response of mouse peripheral blood lymphocytes. SLC was a strong chemoattractant for these cells, with greatest activity toward CD4⁺ and CD8⁺ T cells (Fig. 3D).

SLC Activates β2 Integrins on T Lymphocytes.

Because SLC is expressed in HEV and can stimulate the chemotaxis of naive lymphocytes, we predicted that it would also activate β2 integrins on T cells. To test this hypothesis, we measured the resistance of human lymphocytes to detachment by physiologic shear stress in a parallel plate flow chamber (19). SLC stimulated an increase in the percentage of T lymphocytes that remained attached to an ICAM-1 substrate as flow shear was increased (Fig. 4A). Naive T lymphocytes responded in a similar manner and this response could be blocked by an anti-α_L antibody (Fig. 4B).