Leukocyte migration into sites of inflammation involves multiple molecular interactions between leukocytes and vascular endothelial cells, mediating sequential leukocyte capture, rolling, and firm adhesion. In this study, we tested the role of molecular interactions between fractalkine (FKN), a transmembrane mucin-chemokine hybrid molecule expressed on activated endothelium, and its receptor (CX3CR1) in leukocyte capture, firm adhesion, and activation under physiologic flow conditions. Immobilized FKN fusion proteins captured resting peripheral blood mononuclear cells at physiologic wall shear stresses and induced firm adhesion of resting monocytes, resting and interleukin (IL)-2–activated CD8+ T lymphocytes and IL-2–activated NK cells. FKN also induced cell shape change in firmly adherent monocytes and IL-2–activated lymphocytes. CX3CR1-transfected K562 cells, but not control K562 cells, firmly adhered to FKN-expressing ECV-304 cells (ECV-FKN) and tumor necrosis factor α–activated human umbilical vein endothelial cells. This firm adhesion was not inhibited by pertussis toxin, EDTA/EGTA, or antiintegrin antibodies, indicating that the firm adhesion was integrin independent. In summary, FKN mediated the rapid capture, integrin-independent firm adhesion, and activation of circulating leukocytes under flow. Thus, FKN and CX3CR1 mediate a novel pathway for leukocyte trafficking.

Weight-neutral treatment with a glucagon-like peptide-1 analog activates several cardioprotective pathways, prevents HFD-induced insulin resistance and inflammation, reduces monocyte vascular adhesion, and improves cardiac function in vivo by activating AMP-activated protein kinase. These data support a role for glucagon-like peptide-1 analogs in limiting the cardiovascular risks of obesity.

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 videomicroscopic 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 (PϽ0.01). 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.

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX3CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-α-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX3CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX3CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX3CR1 Ab. Treatment with the anti-CX3CR1 Ab significantly prolonged allograft survival from 7 ± 1 to 49 ± 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.

Hypothermic preservation is known to cause renal graft injury, especially in donation after circulatory death (DCD) kidney transplantation. We investigated the impact of cold storage (SCS) versus short periods of normothermic ex vivo kidney perfusion (NEVKP) after SCS versus prolonged, continuous NEVKP with near avoidance of SCS on kidney function after transplantation. Following 30 min of warm ischemia, kidneys were removed from 30-kg Yorkshire pigs and preserved for 16 h with (A) 16 h SCS, (B) 15 h SCS + 1 h NEVKP, (C) 8 h SCS + 8 h NEVKP, and (D) 16 h NEVKP. After contralateral kidney resection, grafts were autotransplanted and pigs followed up for 8 days. Perfusate injury markers such as aspartate aminotransferase and lactate dehydrogenase remained low; lactate decreased significantly until end of perfusion in groups C and D (p < 0.001 and p = 0.002). Grafts in group D demonstrated significantly lower serum creatinine peak when compared to all other groups (p < 0.001) and 24-h creatinine clearance at day 3 after surgery was significantly higher (63.4 ± 19.0 mL/min) versus all other groups (p < 0.001). Histological assessment on day 8 demonstrated fewer apoptotic cells in group D (p = 0.008). In conclusion, prolonged, continuous NEVKP provides superior short-term outcomes following DCD kidney transplantation versus SCS or short additional NEVKP following SCS.

Kidney transplantation is an optimal therapy for pediatric patients with end-stage kidney disease. This pilot study sought to examine multidimensional QOL outcomes after kidney transplant using VAQOL and General Health, the PedsQL 4.0, PedsQL End Stage Renal Disease Module, and Impact on Family Module. Sample included 12 adolescents aged 13-18 yr and their parent; three children aged eight to 12 yr and their parent; and six parents of children aged two to seven yr. All were 73 months post transplant. The median age at transplant was 9.3 yr and median time since transplant was 3.2 yr. VAQOL mean was 7.7/10 (child report) and 7.3/10 (parent report); the mean general health was 7.4/10. High levels of fatigue (> or =5/10) were reported in 43%. PedsQL subscale mean values were lower than healthy reference scores. PedsQL Renal Module demonstrated great concern with physical appearance and physical symptoms (thirst and headaches), difficulty with peer and family interaction, and school disruption. Low scores on parental emotional function depict the negative impact of transplant on family functioning. Discordance exists between child and parental reports of QOL. Prospective studies are needed to explore multidimensional QOL to improve long-term outcomes after pediatric kidney transplant.

Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastro-intestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell-derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.

CX 3 CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX 3 CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX 3 CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX 3 CL1 in human ECV-304 cells stably expressing untagged or green fluorescent proteintagged forms of the chemokine. CX 3 CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX 3 CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX 3 CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX 3 CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX 3 CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli.