Constitutive endocytosis of the chemokine CX3CL1 prevents its degradation by cell surface metalloproteases.

CX(3)CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX(3)CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX(3)CL1 at the cell surface. We therefore investigated how transmembrane CX(3)CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX(3)CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX(3)CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX(3)CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX(3)CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX(3)CL1. Although CX(3)CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX(3)CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX(3)CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX(3)CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX(3)CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.

The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

Relative efficacy of intravenous immunoglobulin G in ameliorating thrombocytopenia induced by antiplatelet GPIIbIIIa versus GPIbalpha antibodies.

Intravenous immunoglobulin G (IVIG) is used to treat idiopathic thrombocytopenic purpura (ITP). Although many patients benefit from IVIG, some are refractory to this therapy. ITP is characterized by platelet clearance mediated primarily by antiplatelet antibodies against GPIIbIIIa and/or the GPIbalpha complex. These 2 groups of antibodies may induce ITP through different mechanisms. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by anti-GPIIbIIIa versus anti-GPIbalpha antibodies in mice. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against either mouse GPIIbIIIa (JON1, JON2, and JON3) or GPIbalpha (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with IVIG significantly ameliorated ITP in all anti-GPIIbIIIa-injected animals. Conversely, IVIG failed to prevent ITP in all anti-GPIbalpha-treated mice, except for p0p4. These results were repeated in C57BL/6 mice, and with different IVIG preparations. These data in mice suggest that patients with ITP mediated by anti-GPIbalpha antibodies may be less responsive to IVIG treatment.