No abstract

Fractalkine (FKN), a CX 3 C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin-and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX 3 CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX 3 C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.Leukocyte trafficking of cells out of the bloodstream and into sites of inflammation requires multiple steps (1, 2). In the classic pathway of leukocyte migration, the first step involves transient, selectin-mediated interactions between the rolling leukocytes and the endothelium (3, 4). In the next step, integrins are activated by locally produced chemokines to induce firm adhesion of the leukocyte to endothelial cells (5-7). Leukocytes then extravasate through the vascular wall and into the tissue.Recently, a new pathway by which leukocytes can be induced to firmly adhere to endothelium and traffic into inflamed tissues has been described. This process is mediated by the chemokine fractalkine (FKN), 1 expressed on endothelial cells, and its G-protein coupled receptor, CX 3 CR1, expressed on PBMC. Fractalkine (neurotactin in the mouse) is the first, and thus far only, member of the CX 3 C chemokine family, so named because the first two conserved cysteine residues are separated by three amino acids (8,9). Another unique characteristic of fractalkine is that it is the only chemokine that is expressed on the cell surface. By nucleotide sequence analysis, fractalkine consists of a chemokine head tethered to the cell surface by a mucin stalk, followed by a single transmembrane spanning domain and a short cytoplasmic tail (8, 9). In addition, fractalkine expression on endothelium is increased by proinflammatory cytokines interleukin-1 and tumor necrosis factor-␣. The receptor for fractalkine, CX 3 CR1 (V28), is expressed on T cells, monocytes, macrophages, and natural killer cells (10, 11). Fractalkine and CX 3 CR1 function as cell adhesion molecules under both st...