Thomas J. Schall

Identification and Molecular Characterization of Fractalkine Receptor CX3CR1, which Mediates Both Leukocyte Migration and Adhesion

Leukocyte trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. Fractalkine, a novel transmembrane molecule with a CX3C-motif chemokine domain atop a mucin stalk, induces both adhesion and migration of leukocytes. Here we identify a seven-transmembrane high-affinity receptor for fractalkine and show that it mediates both the adhesive and migratory functions of fractalkine. The receptor, now termed CX3CR1, requires pertussis toxin-sensitive G protein signaling to induce migration but not to support adhesion, which also occurs without other adhesion molecules but requires the architecture of a chemokine domain atop the mucin stalk. Natural killer cells predominantly express CX3CR1 and respond to fractalkine in both migration and adhesion. Thus, fractalkine and CX3CR1 represent new types of leukocyte trafficking regulators, performing both adhesive and chemotactic functions.

Molecular cloning and expression of a receptor for human tumor necrosis factor

A human tumor necrosis factor (TNF) binding protein from serum of cancer patients was purified to homogeneity and partially sequenced. Synthetic DNA probes based on amino acid sequence information were used to isolate cDNA clones encoding a receptor for TNF. The TNF receptor (TNF-R) is a 415 amino acid polypeptide with a single membrane-spanning region. The extracellular cysteine-rich domain of the TNF-R is homologous to the nerve growth factor receptor and the B cell activation protein Bp50. Human embryonic kidney cells transfected with a TNF-R expression vector specifically bind both 125I-labeled and biotinylated TNF-alpha. Unlabeled TNF-alpha and TNF-beta were equally effective at displacing the binding of labeled TNF-alpha to TNF-R expressing cells. Northern analysis indicates a single species of mRNA for the TNF-R in a variety of cell types. Therefore, the soluble TNF binding protein found in human serum is probably proteolytically derived from the TNF-R.

Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor

The immunoregulatory proteins C-C chemokines are potent chemoattractants of lymphocytes and monocytes, as well as activators and attractants of eosinophils and basophils. We have isolated a cDNA that encodes a seven transmembrane-spanning receptor, with homology to other chemoattractant receptors, that encodes a protein designated C-C CKR-1 that acts as a receptor for the C-C chemokines. Human and murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), human human monocyte chemotactic protein 1 (MCP-1), and RANTES all bind to the C-C CKR-1 with varying affinities. Chemokine binding affinity does not predict how well the ligand will transmit a signal through the receptor: RANTES and human MIP-1 alpha induce a similar intracellular calcium flux while binding with disparate affinities, while MCP-1 and human MIP-1 beta induce calcium mobilization only at high concentrations. Finally, C-C chemokines were shown to bind a C-C CKR-1-related gene product encoded by cytomegalovirus, suggesting a role for C-C chemokines in viral immunity.

Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis

OBJECTIVEnEndometriosis is a common gynecologic disorder in which the concentration and activation of peritoneal macrophages are increased. The goal of this study was to quantify pelvic fluid concentrations of two cytokines involved in macrophage recruitment and activation.nnnSTUDY DESIGNnA case-control study of women undergoing pelvic surgery was conducted by collecting peritoneal fluid from 12 women without evidence of endometriosis (controls), 12 with mild, and 12 with moderate to severe endometriosis. Concentrations of RANTES and interferon gamma, soluble cytokines known to recruit and activate macrophages, were quantified by enzyme-linked immunosorbent assays.nnnRESULTSnPelvic fluid concentrations of RANTES are elevated in women with endometriosis and the levels correlate with the severity of disease. By contrast, concentrations of interferon gamma appear unaffected by the presence of or severity of endometriosis.nnnCONCLUSIONnThe findings indicate that RANTES, a cytokine with potent chemotactic activity for human monocytes, may play an important role in the recruitment of peritoneal macrophages in endometriosis.

Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up‐regulated upon dendritic cell maturation

The lone CX3C chemokine, fractalkine (FK), is expressed in a membrane‐bound form on activated endothelial cells and mediates attachment and firm adhesion of T cells, monocytes and NK cells. We now show that FK is associated with dendritic cells (DC) in epidermis and lymphoid organs. In normal human skin, dual‐color fluorescence microscopy co‐localized FK expression with Langerhans cells expressing CD1a. In tonsil, FK‐positive DC expressed CD83, a marker for mature DC. Human and murine cultured DC up‐regulated FK mRNA expression with maturation. Furthermore, CD40 ligation, but not TNF‐α or lipopolysaccharide treatment, of activated, migratory DC that had migrated from skin explants resulted in a 2.5‐fold increase of surface expression of FK without significant alterations of expression of CD80, CD86, CD54 or MHC class II. Since FK mediates adhesion of T cells to activated endothelial cells, the increased expression of FK during DC maturation (and particularly by CD40 ligation) may play a role in the ability of T cells and mature DC to form conjugates and engage in cell‐cell communication.

Chemokine Class Differences in Binding to the Duffy Antigen-Erythrocyte Chemokine Receptor

The Duffy blood group antigen-erythrocyte chemokine receptor has been shown to bind to chemokines of both the C-X-C and C-C classes and to the malarial parasites Plasmodium vivax and Plasmodium knowlesi. We performed experiments to evaluate the binding properties of this receptor for the newly appreciated “C” and “non-ELR C-X-C” classes of chemokines. Binding to mouse erythrocytes was also evaluated for the first time. Whereas ELR C-X-C and C-C chemokines bound to human erythrocytes with high affinity, differences in the ability of non-ELR chemokines to act as competitive inhibitors were noted. While non-ELR chemokines were unable to displace C-X-C chemokines on human cells, they exhibited a low affinity interaction with the C-C chemokine binding site. The newly discovered C chemokine, lymphotactin, was unable to displace either C-X-C or C-C chemokines. On mouse erythrocytes, non-ELR chemokines exhibited a low affinity for both the C-X-C and C-C chemokines binding sites; again lymphotactin failed to bind. Binding competition studies using an anti-Duffy monoclonal antibody and chemokines suggested a common binding domain. These data show that the chemokine superfamily has at least four functional subdivisions, each interacting differently with the Duffy antigen-erythrocyte chemokine receptor. In addition the chemokine binding function is conserved between mouse and man. Unlike other proteins in the superfamily C and non-ELR C-X-C chemokines do not efficiently bind red blood cells, thus their role may not require clearance from circulation.

Receptor/ligand interactions in the C-C chemokine family.

The past few years have been particularly enriching for investigators interested in basic immunoregulatory and inflammatory processes. Receptors for such central regulators as IL-1, TNF, and IFN-g have been cloned by molecular techniques, and progress is being made in the understanding of their signal transduction mechanisms. In addition, recent immunological history has witnessed the appreciation of entirely new areas in immunobiology, some of which are beginning to fundamentally impact on our understanding of basic inflammatory processes. Nowhere is this better exemplified than in the area of the recently discovered superfamily of immune cytokines which we have come to call the chemokines